Latent Membrane Protein 1 Expression Research Articles

Egr-1, a new downstream molecule of Epstein-Barr virus latent membrane protein 1

To investigate the effect of Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) on human cancer cells, we sought to identify and analyze potential target genes that were differentially expressed in the presence and absence of LMP1. Our cDNA microarray analysis revealed that expression of early growth response gene-1 (Egr-1) was increased by LMP1 expression in MCF7 and Jurkat cells. An NFκB inhibitor (SN50) antagonized LMP1-induced enhancement of Egr-1 expression, indicating that LMP1 induced Egr-1 via NFκB. Furthermore, three lines of evidence indicated that Egr-1 was required for LMP1-induced cancer cell survival. First, Egr-1 expression enhanced the survival of doxorubicin-treated MCF7 cells. Second, inhibition of Egr-1 expression by siRNA (siEgr-1) effectively suppressed LMP-1-induced survival of MCF7 cells. Third, Egr-1 knockdown decreased LMP1-induced expression of Bfl-1. Similar relationships among EBV infection, Egr-1 and drug resistance were also observed in tissues of peripheral T-cell lymphoma-unspecified (PTCL-u) patients.

Activation of DNA Methyltransferase 1 by EBV LMP1 Involves c-Jun NH2-Terminal Kinase Signaling

EBV latent membrane protein 1 (LMP1) activates cellular DNA methyltransferases, resulting in hypermethylation and silencing of E-cadherin. However, the underlying mechanism remains to be elucidated. In this study, we show that LMP1 directly induces the dnmt1 promoter activity through its COOH-terminal activation region-2 YYD domain. Using (i) LMP1 mutants, (ii) dominant negative mutants c-jun NH(2)-terminal kinase (JNK)-DN, p38-DN, and constitutive active mutant IkappaB, as well as (iii) dsRNAs targeting c-Jun, JNK, and tumor necrosis factor receptor-associated death domain protein, and (iv) signal transduction inhibitors, we show that LMP1-mediated DNA methyltransferase-1 (DNMT1) activation involves JNK but not nuclear factor kappaB and p38/mitogen-activated protein kinase signaling. In addition, LMP1 is unable to activate dnmt1-P1 promoter with activator protein-1 (AP-1) site mutation. Chromatin immunoprecipitation assay results also confirm that LMP1 activates P1 promoter via the JNK-AP-1 pathway. Furthermore, chromatin immunoprecipitation assay data in LMP1-inducible cells disclose that LMP1 induces formation of a transcriptional repression complex, composed of DNMT1 and histone deacetylase, which locates on E-cadherin gene promoter. Treatment with JNK inhibitor, SP600125, prevents the formation of this repression complex. Statistical analyses of the immunohistochemical staining of 32 nasopharyngeal carcinoma (NPC) biopsies show LMP1 expression (18 of 32, 56.25%), DNMT1 expression (31 of 32, 97%), and phospho-c-Jun (27 of 32, 84.38%), suggesting that overexpression of these proteins is observed in NPC tumor. Overall, these results support a mechanistic link between JNK-AP-1 signaling and DNA methylation induced by the EBV oncogene product LMP1.

MUC1 Induced by Epstein-Barr Virus Latent Membrane Protein 1 Causes Dissociation of the Cell-Matrix Interaction and Cellular Invasiveness via STAT Signaling

Disruption of cellular adhesion is an essential pathobiologic step leading to tumor dissemination. Mucin 1 (MUC1) is a mucinous glycoprotein expressed at the surfaces of epithelial cells in many tissues and their carcinomas. MUC1 plays crucial roles in tumor invasion and metastasis, especially in opposing cell adhesion. We have shown that virus infection, specifically by the human tumor virus Epstein-Barr virus (EBV) induces a spectrum of cellular invasiveness and metastasis factors. Here we show that expression of MUC1 is increased in diverse latently EBV-infected cell lines that express latent membrane protein 1 (LMP1), the main viral oncoprotein, and that the level of MUC1 was suppressed by expression of a dominant-negative mutant of LMP1. Expression of LMP1 in EBV-negative nasopharyngeal cell lines induces expression of MUC1 through activation of the MUC1 promoter via binding of STAT1 and STAT3. Finally, LMP1 reduces cell adhesion ability, which is restored by inhibition of MUC1 expression with MUC1 small interfering RNA (siRNA). In addition, LMP1 increases cell invasiveness, which is suppressed by MUC1 siRNA. Thus, LMP1 induces MUC1, a factor important in an early step of detachment and release of tumor cells, which along with induction of other invasiveness and angiogenic factors may combine to act in a complex sequential process that culminates in metastasis of EBV-infected tumor cells.

Latent membrane protein 1 suppresses RASSF1A expression, disrupts microtubule structures and induces chromosomal aberrations in human epithelial cells

Epstein-Barr virus (EBV) infection is closely associated with nasopharyngeal carcinoma (NPC) and can be detected in early premalignant lesions of nasopharyngeal epithelium. The latent membrane protein 1 (LMP1) is an oncoprotein encoded by the EBV and is believed to play a role in transforming premalignant nasopharyngeal epithelial cells into cancer cells. RASSF1A is a tumor-suppressor gene commonly inactivated in many types of human cancer including NPC. In this study, we report a novel function of LMP1, in down-regulating RASSF1A expression in human epithelial cells. Downregulation of RASSF1A expression by LMP1 is dependent on the activation of intracellular signaling of NF-kappaB involving the C-terminal activating regions (CTARs) of LMP1. LMP1 expression also suppresses the transcriptional activity of the RASSF1A core promoter. RASSF1A stabilizes microtubules and regulates mitotic events. Aberrant mitotic spindles and chromosome aberrations are reported phenotypes in RASSF1A inactivated cells. In this study, we observed that LMP1 expression in human epithelial cells could induce aberrant mitotic spindles, disorganized interphase microtubules and aneuploidy. LMP1 expression could also suppress microtubule dynamics as exemplified by tracking movements of the growing tips of microtubules in live cells by transfecting EGFP-tagged EB1 into cells. The aberrant mitotic spindles and interphase microtubule organization induced by LMP1 could be rescued by transfecting RASSF1A expression plasmid into cells. Downregulation of RASSF1A expression by LMP1 may facilitate its role in transformation of premalignant nasopharyngeal epithelial cells into cancer cells.

Recombinant adeno-associated virus mediated RNA interference inhibits metastasis of nasopharyngeal cancer cells in vivo and in vitro by suppression of Epstein-Barr virus encoded LMP-1

Nasopharyngeal carcinoma (NPC) is a highly metastatic carcinoma characterized by consistent association with Epstein-Barr virus (EBV). Of the EBV-encoded product, latent membrane protein-1 (LMP-1) is considered to be an onco-protein playing an essential role in cell transformation and metastasis. In this study, we used a recombinant adeno-associated virus type 2 vector (rAAV-2) to deliver small hairpin RNA (shRNA) targeting EBV LMP-1 into the EBV-positive human NPC C666-1 cells and evaluated the effect of long-term suppression of LMP-1 on NPC growth and metastasis in vivo and in vitro. An NPC metastasis nude mouse model with NPC xenograft transplanted in liver was established. The NPC C666-1 cells infected with rAAV-shRNA-LMP-1 or rAAV-EGFP were inoculated in the livers of nude mice. Formation of liver and lung metastasis was evaluated at day 14 after tumor inoculation. Our results demonstrate that rAAV-shRNA-LMP-1 effectively infected C666-1 cells and suppressed LMP-1 expression. Such suppression, in turn, did not significantly inhibit tumor growth, but prevented NPC metastasis in the liver as well as in the lung. Consistent with in vivo data, the in vitro studies in NPC C666-1 cell cultures showed that suppression of LMP-1 by rAAV-shRNA-LMP-1 significantly reduced cell mobility and transmembrane invasion ability. These results demonstrated for the first time that long-term suppression of EBV-encoded LMP-1 in vivo is an effective means for preventing NPC metastasis.

Expression of syndecan-1 (CD138) in nasopharyngeal carcinoma is correlated with advanced stage and poor prognosis

Nasopharyngeal carcinoma (NPC) is an important Epstein-Barr virus-associated head and neck malignancy in Taiwan. Syndecan-1 (CD138) is involved in growth, differentiation, invasiveness, and metastatic potential of certain tumors, but its expression in NPC has never been studied. In this study, detection of expression of syndecan-1 protein and Epstein-Barr virus-encoded latent membrane protein-1 (LMP-1) in primary, recurrent, and metastatic NPC specimens in paraffin sections was performed by immunohistochemistry. The quantity of syndecan-1 messenger RNA in tumor cells was investigated by real-time reverse transcriptase polymerase chain reaction using laser capture microdissection. The results of immunohistochemical staining of syndecan-1 and LMP-1 correlated with clinicopathologic features of NPC. Eighteen (20.9%) of 86 primary, 9 (24.3%) of 37 recurrent, and 15 (44.1%) of 34 metastatic NPC samples were positive for syndecan-1, and 37 (43.0%) primary, 18 (48.6%) recurrent, and 12 (35.3%) metastatic samples were positive for LMP-1 expression. Primary NPCs with syndecan-1 protein expression were more frequently associated with advanced clinical stages and worse 5-year survival rates than those without (P = .015 and P = .0021, respectively). Conversely, the LMP-1 expression did not correlate with tumor stage or prognosis but occurred more often in nonkeratinizing carcinoma than keratinizing squamous cell carcinoma (unpublished observation). The inverse expression of syndecan-1 and LMP-1 was noted in primary NPC specimens (total 4/18 versus 35/68, P = .05). The reverse transcriptase polymerase chain reaction revealed low syndecan-1 messenger RNA levels in both primary and metastatic NPC. In conclusion, the protein expression of syndecan-1 in 21% of primary NPC was associated with advanced disease and poor prognosis, and the protein expression correlated with transcription levels.

Autoactivation of the Epstein-Barr Virus Oncogenic Protein LMP1 during Type II Latency through Opposite Roles of the NF-κB and JNK Signaling Pathways

Epstein-Barr virus (EBV) is associated with several human malignancies where it expresses limited subsets of latent proteins. Of the latent proteins, latent membrane protein 1 (LMP1) is a potent transforming protein that constitutively induces multiple cell signaling pathways and contributes to EBV-associated oncogenesis. Regulation of LMP1 expression has been extensively described during the type III latency of EBV. Nevertheless, in the majority of EBV-associated tumors, the virus is commonly found to display a type II latency program in which it is still unknown which viral or cellular protein is really involved in maintaining LMP1 expression. Here, we demonstrate that LMP1 activates its own promoter pLMP1 through the JNK signaling pathway emerging from the TES2 domain. Our results also reveal that this activation is tightly controlled by LMP1, since pLMP1 is inhibited by LMP1-activated NF-kappaB signaling pathway. By using our physiological models of EBV-infected cells displaying type II latency as well as lymphoblastoid cell lines expressing a type III latency, we also demonstrate that this balanced autoregulation of LMP1 is shared by both latency programs. Finally, we show that this autoactivation is the most important mechanism to maintain LMP1 expression during the type II latency program of EBV.

Arsenic trioxide reduces the invasive and metastatic properties of nasopharyngeal carcinoma cells in vitro

Nasopharyngeal carcinoma (NPC) is notorious for the metastases, which are in close association with Epstein-Barr virus-encoded latent membrane protein 1 (LMP1). Arsenic trioxide (As2O3) has been shown to induce apoptosis and differentiation in NPC xenografts. Then, can it repress the cancer cells' metastasis potential? To elucidate this issue, the present study was performed. LMP1-negative cell line HNE1 and LMP1-positive cell line HNE1-LMP1 were used as in vitro model. Cells (1 x 10(5)/mL) were cultured with or without 3 microM As2O3 for 48 h. Then the survival cells were collected to investigate their potential of colony formation, attachment, invasion, and migration. Both confocal immunofluorescence staining and Western blot were used to detect the changes of LMP1 expression. The changes of MMP-9 were examined by RT-PCR assay and Western blot. The results were as follow: i) the colony formation inhibition rate (75.41 +/- 3.9% in HNE1-LMP1 cells vs 37.89 +/- 4.9% in HNE1 cells), the rate of attachment (HNE1-LMP1 vs HNE1: 56.40 +/- 3.5 vs 65.87 +/- 5.9%), the invasion inhibitory rate (HNE1-LMP1 vs HNE1: 56.50 +/- 3.7 and 27.91 +/- 2.1%), and the migration inhibitory rate (HNE1-LMP1 vs HNE1: 48.70 +/- 3.9 vs 29.19 +/- 6.27%) were all significantly different between the two cell lines (P < 0.01). ii) LMP1 was down-regulated in As2O3-treated HNE1-LMP1 cells. iii) The reduction of MMP-9 was found in As2O3-treated groups, more evident in HNE1-LMP1 cells. Thus, we conclude that As2O3 can reduce metastasis potential of NPC cells, involving inhibition of MMP-9 expression. LMP1 were also reduced in this process and seemed to enhance anti-metastasis activity of As2O3.

IL-10 can induce the expression of EBV-encoded latent membrane protein-1 (LMP-1) in the absence of EBNA-2 in B lymphocytes and in Burkitt lymphoma- and NK lymphoma-derived cell lines

AbstractEBV-positive nasopharyngeal carcinoma and Hodgkin, T, and natural killer (NK) lymphomas express EBNA-1 and the latent membrane proteins (LMP1-2; type II latency). In contrast to type III EBV-transformed lymphoblastoid cell lines, in these cells the LMPs are expressed in the absence of EBNA-2. We have previously reported that exposure to CD40 ligand and IL-4 could induce LMP-1 in an in vitro EBV-infected Hodgkin lymphoma-derived cell line, which expressed only EBNA-1. We show now that both human and EBV-encoded IL-10 can induce LMP-1 in the absence of EBNA-2 in the Daudi, P3HR1, and other BL cell lines. Interestingly, induction of LMP-1 was not accompanied by the downregulation of BCL-6. IL-10 could also induce LMP-1 in the conditional lymphoblastoid cell line ER/EB2-5 where EBNA-2 was downregulated in the absence of estrogen. Moreover, IL-10 could induce the expression of LMP-1 in tonsillar B cells infected with the nontransforming, EBNA-2-deficient EBV strain P3HR1 and enhance LMP-1 expression in 2 EBV-positive NK lymphoma lines. The demonstration that IL-10 can induce the expression of LMP-1 in an EBNA-2-independent manner shows that the major transforming EBV gene LMP-1 can be induced by extracellular signals in lymphoid cells, and IL-10 might contribute to the establishment of type II EBV latency.

The Latent Membrane Protein 1 of Epstein-Barr Virus (EBV) Primes EBV Latency Cells for Type I Interferon Production

Epstein-Barr virus (EBV) latency has been associated with a variety of human cancers. Latent membrane protein 1 (LMP-1) is one of the key viral proteins required for transformation of primary B cells in vitro and establishment of EBV latency. We have previously shown that LMP-1 induces the expression of several interferon (IFN)-stimulated genes and has antiviral effect (Zhang, J., Das, S. C., Kotalik, C., Pattnaik, A. K., and Zhang, L. (2004) J. Biol. Chem. 279, 46335-46342). In this report, a novel mechanism related to the antiviral effect of LMP-1 is identified. We show that EBV type III latency cells, in which LMP-1 is expressed, are primed to produce robust levels of endogenous IFNs upon infection of Sendai virus. The priming action is due to the expression of LMP-1 but not EBV nuclear antigen 2 (EBNA-2). The signaling events from the C-terminal activator regions of LMP-1 are essential to prime cells for high IFN production. LMP-1-mediated activation of NF-kappaB is apparently necessary and sufficient for LMP-1-mediated priming effect in DG75 cells, a human B cell line. IFN regulatory factor 7 (IRF-7) that can be activated by LMP-1 is also implicated in the priming action. Taken together, these data strongly suggest that LMP-1 may prime EBV latency cells for IFN production and that the antiviral property of LMP-1 may be an intrinsic part of EBV latency program, which may assist the establishment and/or maintenance of viral latency.

Downregulation of Epstein-barr Virus-Encoded Latent Membrane Protein-1 by Arsenic Trioxide in Nasopharyngeal Carcinoma Cells

It was documented that nasopharyngeal carcinoma (NPC) is associated with Epstein-Barr virus (EBV) and that EBV-encoded latent membrane protein-1 expression (LMP1) plays an important role in the pathogenesis of NPC. In preclinical studies, arsenic trioxide (As2O3) has been identified as a promising anticancer agent for treatment of NPC. The purpose of this study is to investigate if this agent can inhibit the expression of LMP1 and therefore lead to growth inhibition of NPC cells in vitro. LMP1-positive NPC cells, HNE1-LMP1, were treated with 3 micromol/L of As2O3 for 96 hours. The LMP1 protein expression and mRNA level in HNE1-LMP1 cells were determined by western blot, confocal immunofluorescence staining and semiquantitative reverse transcriptase reaction (RT-PCR). Apoptosis was determined by light microscopy and the TUNEL method. Alterations in the cell cycle distribution were also investigated by flow cytometry. MTT assay and colony formation assay were used to detect the proliferation of the cells. The LMP1-negative parental cell lines HNE1 and HNE2 were used as control in an attempt to elucidate the role of LMP1 in the anticancer effect of As2O3 on NPC cells. The expression of LMP1 at the protein and mRNA level was reduced after exposure to 3 micromol/L As2O3. This dose of As2O3 significantly induced apoptosis and growth retardation of HNE1-LMP1 cells. In addition, more HNE1-LMP1 cells were induced to G0/G1 and G2/M arrest. The same dose of As2O3 had a moderate effect on HNE1 and HNE2 cells. Arsenic trioxide can inhibit LMP1 expression and dictate apoptosis and alterations of cell cycle distribution as well as growth retardation. LMP1-positive NPC cells are more sensitive to As2O3 treatment than LMP1-negative NPC cells.

Stimulation of Epstein-Barr virus-infected human B cell growth by physiological concentrations of 4-hydroxynonenal

We had previously shown that cyclosporin A (CsA) directly promoted the immortalization of Epstein-Barr virus (EBV)-infected human B cells (EBV-B cells) via an oxidative stress mechanism. 4-Hydroxynonenal (HNE) is a reactive end-product of lipid peroxidation. We hypothesized that HNE may mediate a direct oxidative stress-promoting effect of CsA on EBV-B cells. HNE-protein adducts in CsA-treated EBV-B cell extracts were assayed immunochemically using a Slot-Blot method. Cell proliferation was assayed by [(3)H]-thymidine incorporation. EBV oncogene latent membrane protein-1 (LMP1) expression was assayed by using PE-conjugated anti-LMP1 antibody in flow cytometry. We found that CsA at 500 ng ml(-1) and 1000 ng ml(-1) significantly increased the level of HNE-protein adducts in EBV-B cells over the control (arbitrary units +/- SE) by 251.3 +/- 7.5 to 361.3 +/- 9.7 and 342.7 +/- 10.7, respectively (p < 0.05, n = 3). EBV-B cells treated with a physiological concentration of HNE (1 microM) for 0.5 and 1 h and cultured for 2 and 4 weeks showed significantly increased [(3)H]-thymidine incorporation. EBV-B cells treated with HNE (1 microM) for 1 h and subsequently cultured for 2 and 4 weeks had a significantly higher ( > 2.0 times) LMP1-positive cell population over the control. In conclusion, in accordance with our previous findings, we show that CsA treatment of EBV-B cells results in increased production of the lipid peroxidation reactive end-product HNE that directly promotes EBV-B cell proliferation and LMP1 expression. This observation provides evidence for further understanding the mechanism of CsA-induced oxidative stress on EBV-related post-transplant lymphoproliferative disorder (PTLD).

Detection of free circulating Epstein-Barr virus DNA in plasma of patients with Hodgkin’s disease

Free circulating Epstein-Barr virus (EBV) DNA is often present in the plasma of Hodgkins disease patients. The aim here was to evaluate the prevalence of this finding, its correlation with the immunohistochemical expression of LMP-1 (latent membrane protein 1) and the influence of other clinical factors. Prospective study in two public tertiary institutions: Hematology Service, Universidade Federal do Rio de Janeiro, and Oncology Service, Instituto Nacional do Câncer, Rio de Janeiro. A cohort of 30 patients with newly diagnosed Hodgkins disease was studied. The control group consisted of 13 healthy adult volunteers. EBV DNA was determined by conventional polymerase chain reaction (PCR). The median age was 28 years, and 16 patients were women. Advanced disease was present in 19 patients, and six were HIV-positive. EBV DNA was present in the plasma of 13 patients and one control (43% versus 8%, p = 0.03). EBV DNA prevalence was higher in HIV-positive patients (100% versus 29%, p = 0.0007) and those with advanced disease (63% versus 9%, p = 0.006). Among HIV-negative patients alone, EBV DNA prevalence remained higher in those with advanced disease. EBV DNA was found in 10/11 patients with LMP-1 expression in the lymph nodes, and in 3/19 without LMP-1 expression (kappa coefficient = 0.72). EBV DNA was present in 91% of patients with EBV-associated Hodgkins disease, and in all patients with HIV-associated Hodgkins disease. EBV DNA prevalence was higher in patients with advanced disease, irrespective of HIV status.

Modification of Cɛ mRNA Expression by EBV‐Encoded Latent Membrane Protein 1

To evaluate the effect of expression of latent membrane protein (LMP) 1 encoded by Epstein-Barr virus (EBV) on Cɛ mRNA expression, mRNA levels were examined by RT-PCR or Northern blot analysis upon transient transfection of LMP1 in the splenocytes derived from Brown-Norway rats with or without immunization with 2,4-dinitrophenyl-conjugated Ascaris suum antigen. Splenocytes were transfected with LMP1 expression vector, pSG5-LMP1, using lipofection method. Cɛ mRNA levels were considerably increased by transfection with pSG5-LMP1 in the splenocytes derived from the nonimmunized rats; however, Cɛ mRNA levels were decreased in the splenocytes derived from the immunized rats. Cɛ mRNA expression in IgE-producing cells are modulated by LMP1, which might depend on the differentiation status of B cells upon exposure to allergen.

The C-terminal Activating Region 2 of the Epstein-Barr Virus-encoded Latent Membrane Protein 1 Activates NF-κB through TRAF6 and TAK1

Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is oncogenic and indispensable for EBV-mediated B cell transformation. LMP1 is capable of activating several intracellular signaling pathways including the NF-kappaB pathway, which contributes to the EBV-mediated cell transformation. Two regions in the cytoplasmic carboxyl tail of LMP1, namely C-terminal activating regions 1 and 2 (CTAR1 and CTAR2), are responsible for NF-kappaB activation, with CTAR2 being the main NF-kappaB activator. Although the CTAR1-mediated NF-kappaB activation was previously shown to be TRAF3-dependent, we showed here that the CTAR2-mediated NF-kappaB activation is mainly TRAF6-dependent but TRAF2/5-independent. In contrast to the interleukin-1 receptor/toll-like receptor-mediated NF-kappaB pathways, the CTAR2-mediated NF-kappaB pathway does not require MyD88, IRAK1, or IRAK4 for TRAF6 engagement. Furthermore, we showed that TAK1 is required for NF-kappaB activation by LMP1. Thus, LMP1 utilizes two distinct pathways to activate NF-kappaB: a major one through CTAR2/TRAF6/TAK1/IKKbeta (canonical pathway) and a minor one through CTAR1/TRAF3/NIK/IKKalpha (noncanonical pathway).

Up-regulation of β-catenin by a viral oncogene correlates with inhibition of the seven in absentia homolog 1 in B lymphoma cells

The protein levels of beta-catenin are tightly regulated by the ubiquitin/proteasome system. We provide evidence that two distinct ubiquitin-dependent degradation pathways for beta-catenin are active in the same Burkitt's lymphoma cells: Along with the classical glycogen-synthase kinase 3beta-dependent destruction machinery, degradation of beta-catenin through seven in absentia homolog 1 (Siah-1) ubiquitin ligase is functional in these cells. We show that inhibition of endogenous Siah-1 stabilizes and activates beta-catenin in B cells. The principal Epstein-Barr virus oncoprotein, latent membrane protein 1, is involved in beta-catenin up-regulation, and expression of latent membrane protein 1 in B lymphoma cells is associated with decreased Siah-1 RNA and protein levels. Thus, we demonstrate the significance of the endogenous Siah-1-dependent ubiquitin/proteasome pathway for beta-catenin degradation in malignant human cells and its regulation by a viral oncogene.

Mosquito Salivary Gland Extracts Induce EBV-Infected NK Cell Oncogenesis Via CD4+ T Cells in Patients with Hypersensitivity to Mosquito Bites

Severe hypersensitivity to mosquito bites (HMB) is characterized by intense local skin reactions and systemic symptoms such as high fever, lymphadenopathy, and hepatosplenomegaly. Patients with HMB often have natural killer (NK) cell lymphocytosis associated with Epstein-Barr virus (EBV) infection. Here we investigated whether mosquito bites have any influence on the oncogenesis of EBV-infected NK cells. We examined six HMB patients with EBV-infected NK cell lymphocytosis. We first demonstrated that CD4+ T cells, but not NK cells, proliferated well in response to mosquito salivary gland extracts (SGE), especially to SGE of Aedes albopictus. When NK cells were cocultured with autologous CD4+ T cells stimulated by mosquito SGE, the expression of viral oncogene latent membrane protein 1 (LMP1) was remarkably enhanced. Next, we stimulated mononuclear cells of the patients with mosquito SGE, and NK cell counts were monitored for 28 d. The counts changed little from initial levels in the culture with mosquito SGE, whereas they decreased steadily in the culture without the extracts. Furthermore, we detected LMP1 mRNA in the skin lesion induced by mosquito SGE. These results suggest that mosquito bites can induce expression of the viral oncogene LMP1 in NK cells via mosquito antigen-specific CD4+ T cells, which is involved in the oncogenesis of NK cells in vivo.

LMP1 signaling and activation of NF-κB in LMP1 transgenic mice

Transgenic mice expressing Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) under the control of an immunoglobulin heavy-chain promoter and enhancer develop lymphoma at a threefold higher incidence than LMP1-negative mice. In vitro, LMP1 activates numerous signaling pathways including p38, c-Jun N terminal kinase (JNK), phosphatidylinositol 3 kinase (PI3K)/Akt, and NF-kappaB through interactions with tumor necrosis receptor-associated factors (TRAFs). These pathways are frequently activated in EBV-associated malignancies, although their activation cannot be definitively linked to LMP1 expression in vivo. In this study, interactions between LMP1 and TRAFs and the activation of PI3K/Akt, JNK, p38, and NF-kappaB were examined in LMP1 transgenic mice. LMP1 co-immunoprecipitated with TRAFs 1, 2, and 3. Akt, JNK, and p38 were activated in LMP1-positive and -negative splenocytes as well as LMP1-positive and -negative lymphomas. Multiple forms of NF-kappaB were activated in healthy splenocytes from LMP1 transgenic mice, in contrast to healthy splenocytes from LMP1-negative mice. However, in both LMP1-positive and -negative lymphomas, only the oncogenic NF-kappaB c-Rel, was specifically activated. Similarly to EBV-associated malignancies, p53 protein was detected at high levels in the transgenic lymphomas, although mutations were not detected in the p53 gene. These data indicate that NF-kappaB is activated in LMP1-positive healthy splenocytes; however, NF-kappaB c-Rel is specifically activated in both the transgenic lymphomas and in the rare lymphomas that develop in negative mice. The LMP1-mediated activation of NF-kappaB may contribute to the specific activation of c-Rel and lead to the increased development of lymphoma in the LMP1 transgenic mice.

Epstein-Barr Virus Latent Membrane Protein 1 (CAO) Up-regulates VEGF and TGFα Concomitant with Hyperlasia, with Subsequent Up-regulation of p16 and MMP9

EBV latent membrane protein 1 (LMP1) is an oncoprotein frequently expressed in nasopharyngeal carcinoma. We have generated transgenic mice expressing the nasopharyngeal carcinoma-derived CAO strain of LMP1 and LMP1 of the B95-8 strain, using the viral ED-L2 promoter for epithelial expression. LMP1(CAO) and LMP1(B95-8) induce transforming growth factor alpha expression and epidermal hyperplasia. However, levels of total epidermal growth factor receptor (EGFR) decline with the appearance of phosphorylated EGFR products, suggesting that the negative feedback loop upon EGFR expression is intact or that there is faster turnover at these early stages of carcinogenesis. In the L2LMP1(CAO) mice, increased levels of vascular endothelial growth factor are also seen at an early stage in the skin. As the phenotype worsens, with increasing hyperplasia and vascularization leading to keratoacanthoma, p16(INK4a) and matrix metalloproteinase 9 expression is induced. The lesions can progress spontaneously to carcinoma. Carcinoma cell lines developed from these mice show high levels of total and phosphorylated EGFR. These data show that the induction of signaling through EGFR by LMP1 is an early event in carcinogenesis and that any inhibition upon EGFR expression is lifted during progression. Furthermore, expression of LMP1 is not sufficient to inhibit induction of p16(INK4a) in response to abnormal proliferation. These data are consistent with the cooperative effects seen between LMP1 and loss of the INK4a locus in transgenic mice and with the frequency of loss of this locus in EBV-associated nasopharyngeal carcinoma.

AIDS‐related lymphomas: from pathogenesis to pathology

Human immunodeficiency virus (HIV)-associated lymphomas include: (1) lymphomas also occurring, although sporadically, in the absence of HIV infection. The vast majority of these lymphomas are high-grade B-cell lymphomas: Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL) with centroblastic (CB) features and DLBCL with immunoblastic (IBL) features; (2) unusual lymphomas occurring more specifically in HIV-positive patients and include two rare entities, namely 'primary effusion lymphoma' (PEL) and 'plasmablastic lymphoma' of the oral cavity. The pathological heterogeneity of acquired immunodeficiency syndrome-associated non-Hodgkin's lymphomas (AIDS-NHL) reflects the heterogeneity of their associated molecular lesions. In AIDS-BL, the molecular lesions involve activation of cMYC, inactivation of P53, and infection with Epstein-Barr virus (EBV). AIDS-IBL infected with EBV are characterised by frequent expression of latent membrane protein 1--an EBV oncoprotein. The biological heterogeneity of AIDS-NHL is highlighted by their histogenetic differences. Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/HHV8)-associated lymphomas, which often develop in persons with advanced AIDS, present predominantly as PEL. KSHV/HHV8 has also been recently detected in solid extracavitary-based lymphomas. The KSHV/HHV8-associated solid lymphomas are (1) unusual lymphomas that occur more specifically in HIV-positive patients; (2) extracavitary and arise in nodal and/or extranodal sites; and (3) histologically, they usually display a PEL-like morphology and plasma cell-related phenotype.