EBV-positive Burkitt's Lymphoma Cell Research Articles

Chidamide Induces Epstein-Barr Virus (EBV) Lytic Infection and Acts Synergistically with Tenofovir to Eliminate EBV-Positive Burkitt Lymphoma.

Epstein-Barr virus (EBV) is a type of human γ-herpesvirus, and its reactivation plays an important role in the development of EBV-driven Burkitt lymphoma (BL). Despite intensive chemotherapy, the prognosis of relapsed/refractory BL patients remains unfavorable, and a definitive method to completely eliminate latent EBV infection is lacking. Previous studies have demonstrated that histone deacetylase (HDAC) inhibitors can induce the transition of EBV from latency to the lytic phase. The lytic activation of EBV can be inhibited by tenofovir, a potent inhibitor of DNA replication. Herein, we explored the antitumor effect and EBV clearance potential of a novel HDAC inhibitor called chidamide, combined with tenofovir, in the treatment of EBV-positive BL. In the study, chidamide exhibited inhibitory activity against HDAC. Moreover, chidamide inhibited BL cell proliferation, arrested cell cycle progression, and induced BL cell apoptosis primarily by regulating the MAPK pathways. Additionally, chidamide promoted the transcription of lytic genes, including BZLF1, BMRF1, and BMLF1 Compared with chidamide alone, the addition of tenofovir further induced growth arrest and apoptosis in EBV-positive BL cells and inhibited the transcriptions of EBV lytic genes induced by chidamide alone. Furthermore, our in vivo data demonstrated that the combination of chidamide and tenofovir had superior tumor-suppressive effects in a mouse model of BL cell tumors. The aforementioned findings confirm the synergistic effect of chidamide combined with tenofovir in inducing growth inhibition and apoptosis in EBV-positive BL cells and provide an effective strategy for eliminating EBV and EBV-associated malignancies. SIGNIFICANCE STATEMENT: High levels of Epstein-Barr virus (EBV)-DNA have consistently been associated with unfavorable progression-free survival and overall survival in EBV-associated lymphomas. Therefore, identifying novel strategies to effectively eradicate tumor cells and eliminate EBV is crucial for lymphoma patients. This study confirmed, for the first time, the synergistic effect of chidamide combined with tenofovir in the treatment of Burkitt lymphoma and the eradication of EBV virus.

4C Analysis of EBV-Host DNA Interactome.

EBV persist as multicopy episomes in latently infected cells and alter transcriptional program of host systems. Knowledge of EBV tethering site helps us understand how EBV attaches to and regulates the host chromosome. Here, we introduce a step-by-step protocol for 4C-seq analysis, including cell fixation, 4C-DNA construction, and sequencing library preparation performed with EBV-positive Burkitt's lymphoma cells. The method can be applied in a variety of studies and cell-types to identify target loci associated with bait positions, such as viral episomes.

L-Asparaginase synergizes with etoposide via the PI3K/Akt/mTOR pathway in Epstein-Barr virus-positive Burkitt lymphoma.

Burkitt lymphoma (BL) is an aggressive Epstein-Barr virus (EBV)-driven B-cell lymphoma characterized by the translocation and rearrangement of the c-Myc proto-oncogene. High-intensity multidrug chemotherapy regimens have a limited effect on the survival of refractory or relapsed BL patients, mainly owing to the high EBV load and drug resistance. l-asparaginase ( l-Asp) and etoposide (VP-16) play a beneficial role in EBV-related lymphoproliferative diseases; however, their roles and mechanisms in BL remain unclear. In this study, we found that VP-16 inhibited BL cell proliferation and arrested the cell cycle at the G2 /M phase. It also induced autophagy and activated the extrinsic and intrinsic apoptotic signaling pathways in BL cells. Mechanistically, VP-16 inhibited c-Myc expression and regulated the PI3K/Akt/mTOR signaling pathway. Notably, VP-16 also showed a specific synergistic effect with l-Asp to induce apoptosis in EBV-positive BL cells but not in EBV-negative BL cells. VP-16 combined with l-Asp further inhibited c-Myc expression and downregulated the PI3K/Akt/mTOR signaling pathway. Additionally, we found that VP-16 inhibited the expression of latent membrane protein 1 (LMP1), and in combination with l-Asp further decreased LMP1 expression in Raji cells. Our in vivo data also showed that the dual-drug combination significantly inhibited the growth of BL tumors and prolonged the survival of mice compared to VP-16 alone. In conclusion, this study provides new evidence that l-Asp may enhance the antitumor effect of VP-16 by inhibiting the PI3K/Akt/mTOR signaling pathway in EBV-positive BL cells.

The JAK2 inhibitor TG101209 exhibits anti-tumor and chemotherapeutic sensitizing effects on Burkitt lymphoma cells by inhibiting the JAK2/STAT3/c-MYB signaling axis

Constitutive activation of JAK2/STAT3 is a major oncogenic signaling event involved in the development of Burkitt lymphoma (BL). In the present study, we investigated the antilymphoma activity of TG101209, a specific JAK2 inhibitor, on EBV-positive and EBV-negative Burkitt lymphoma cell lines and primary BL cells. The results showed that TG101209 had a significant antilymphoma effect by inhibiting BL cell growth and inducing apoptosis along with cell differentiation toward mature B cells in vitro. We also found that TG101209 displayed significant synergistic action and a sensitizing effect on the anti-Burkitt lymphoma activity of doxorubicin. In vivo experiments indicated that TG101209 could suppress tumor growth and prolong the overall survival of BL cell-bearing mice. The mechanistic study indicated that TG101209, by suppressing the JAK2/STAT3/c-MYB signaling axis and crosstalk between the downstream signaling pathways, plays an antilymphoma role. These data suggested that TG101209 may be a promising agent or alternative choice for the treatment of BL.

Pharmacologic Activation of Lytic Epstein-Barr Virus Gene Expression without Virion Production.

Several therapeutic strategies targeting Epstein-Barr virus (EBV)-associated tumors involve upregulation of viral lytic gene expression. Evidence has been presented that the unfolded protein response (UPR) leads to EBV lytic gene expression. Clofoctol, an antibacterial antibiotic, has been reported to upregulate the UPR in prostate cancer cell lines and to slow their growth. We investigated the effects of clofoctol on an EBV-positive Burkitt lymphoma cell line and confirmed the upregulation of all three branches of the UPR and activation of EBV lytic gene expression. While immediate early, early, and late EBV RNAs were all upregulated, immediate early and early viral proteins but not late viral proteins were expressed. Furthermore, infectious virions were not produced. The use of clofoctol in combination with a protein kinase R-like endoplasmic reticulum kinase inhibitor led to expression of late viral proteins. The effects of clofoctol on EBV lytic protein upregulation were not limited to lymphoid tumor cell lines but also occurred in naturally infected epithelial gastric cancer and nasopharyngeal cancer cell lines. An agent that upregulates lytic viral protein expression but that does not lead to the production of infectious virions may have particular value for lytic induction strategies in the clinical setting.IMPORTANCE Epstein-Barr virus is associated with many different cancers. In these cancers the viral genome is predominantly latent; i.e., most viral genes are not expressed, most viral proteins are not synthesized, and new virions are not produced. Some strategies for treating these cancers involve activation of lytic viral gene expression. We identify an antibacterial antibiotic, clofoctol, that is an activator of EBV lytic RNA and protein expression but that does not lead to virion production.

Upregulation of the Chemokine Receptor CCR2B in Epstein‒Barr Virus-Positive Burkitt Lymphoma Cell Lines with the Latency III Program.

CCR2 is the cognate receptor to the chemokine CCL2. CCR2–CCL2 signaling mediates cancer progression and metastasis dissemination. However, the role of CCR2–CCL2 signaling in pathogenesis of B-cell malignancies is not clear. Previously, we showed that CCR2B was upregulated in ex vivo peripheral blood B cells upon Epstein‒Barr virus (EBV) infection and in established lymphoblastoid cell lines with the EBV latency III program. EBV latency III is associated with B-cell lymphomas in immunosuppressed patients. The majority of EBV-positive Burkitt lymphoma (BL) tumors are characterized by latency I, but the BL cell lines drift towards latency III during in vitro culture. In this study, the CCR2A and CCR2B expression was assessed in the isogenic EBV-positive BL cell lines with latency I and III using RT-PCR, immunoblotting, and immunostaining analyses. We found that CCR2B is upregulated in the EBV-positive BL cells with latency III. Consequently, we detected the migration of latency III cells toward CCL2. Notably, the G190A mutation, corresponding to SNP CCR2-V64I, was found in one latency III cell line with a reduced migratory response to CCL2. The upregulation of CCR2B may contribute to the enhanced migration of malignant B cells into CCL2-rich compartments.

CD21-independent Epstein-Barr virus entry into NK cells

Extranodal natural killer (NK)/T-cell lymphoma is an aggressive malignant disease that is associated with Epstein-Barr viral (EBV) infection. To date, the mechanism of viral entry into NK cells remains uncertain. Here, we investigated this mechanism using human NK cells in vitro. CD21 mRNA expression, an EBV-entry receptor, was transiently detected in NK cells after exosome treatment, and levels decreased after further culture. CD21 protein expression was also transiently transferred to NK cells after co-culture with an EBV-positive Burkitt lymphoma cell line (Raji) via trogocytosis. However, EBV did not infect NK cells through CD21-mediated trogocytosis. Unexpectedly, when NK cell leukemia cells, as well as primary NK cells, were treated with viral supernatant, EBV genes, but not RNA, were detected in the NK cells, at latency stage 0. Therefore, these results suggest that EBV-NK cell infection results from the direct transfer of viral episomes, independent of EBV-positive B cells.

Expression of the chemokine receptors CCR1 and CCR2B is up-regulated in peripheral blood B cells upon EBV infection and in established lymphoblastoid cell lines

In immunocompetent individuals, EBV establishes in B cells an asymptomatic lifelong latent infection controlled by the immune system. Chemokine receptors regulate immune system function. CCR1 and CCR2 share protein sequence similarity and exert responses to multiple chemokines. The role of these receptors in B cells is largely unknown. We show that the mRNA and functional protein expression of CCR1 and CCR2 is induced in ex vivo B cells upon EBV infection and in established lymphoblastoid cell lines (LCLs). The CCR1 and CCR2B ORF transcripts were determined in LCLs. In contrast, in both the EBV-negative and EBV-positive Burkitt lymphoma cell lines, neither the CCR1, CCR2A, and CCR2B ORF transcripts nor their corresponding proteins were detected. Our data suggest that CCR1/CCR2B could be involved in clearing EBV-infected latency III B cells in immunocompetent individuals via directing the migration of these cells and attracting the chemokines-expressing immune cells.

Abstract 4568: Inhibition of autophagy in EBV-positive Burkitt's lymphoma cells enhances EBV lytic genes expression and replication

Abstract Autophagy, an important degradation system involved in maintaining cellular homeostasis, serves also to eliminate pathogens and process their fragments for presentation to the immune system. Several viruses have been shown to interact with the host autophagic machinery to suppress or make use of this cellular catabolic pathway to enhance their survival and replication. Epstein Barr virus (EBV) is a γ-herpes virus associated with a number of malignancies of epithelial and lymphoid origin in which establishes a predominantly latent infection. EBV lytic cycle characterized by the sequential expression of immediate early (IE), early and late antigens results in the production of infectious particles which allow the virus to spread. In this study we analyzed the relationship between EBV and autophagy after inducing the virus productive cycle in Burkitt's lymphoma (BL) cells. By monitoring autophagy markers and EBV lytic genes expression, we demonstrate that autophagy is enhanced in the early phases of EBV lytic activation but decreases thereafter concomitantly with increased levels of EBV lytic proteins. In a cell line defective for late antigens expression, we found an inverse correlation between EBV early antigens expression and autophagosomes formation indicating that early after activation, the virus is able to suppress autophagy. We report that inhibition of autophagy by Bafilomycin A1 or shRNA knockdown of beclin1 gene, enhance EBV lytic genes expression as well as intracellular viral DNA and viral progeny yield. Taken together, these findings indicate that viral replication induces an autophagic response which can inhibit the further expression of EBV early lytic products. Moreover, our findings open the possibility to utilize pharmacological modulators of autophagy to control EBV infection and treat EBV-related lymphomas. Citation Format: Alessandra De Leo, Francesca Colavita, Fabiola Ciccosanti, Gian Maria Fimia, Paul Lieberman, Elena Mattia. Inhibition of autophagy in EBV-positive Burkitt's lymphoma cells enhances EBV lytic genes expression and replication. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4568. doi:10.1158/1538-7445.AM2015-4568

Delivery of miR-155 to retinal pigment epithelial cells mediated by Burkitt's lymphoma exosomes.

Exosomes are extracellularly secreted vesicles ranging from 40 to 100nm in diameter that are thought to play important roles in intercellular communication. Exosomes contain numerous proteins, RNA, and lipids that can affect the status of recipient cells under various pathological conditions. MicroRNAs (miRNAs) are small non-coding RNAs that play a major role in post-transcriptional gene silencing by interacting with the 3'-untranslated regions of target genes. Epstein-Barr virus (EBV) has been reported to induce sustained elevation of cellular miRNAs such as miR-155. We hypothesized that miRNAs delivered by exosomes might affect the angiogenesis of retinal pigment epithelial (RPE) cells. Here, we demonstrated that co-culture of EBV-positive Burkitt's lymphoma (BL) cells (Raji) with retinal pigment epithelial (ARPE-19) cells increased the level of miR-155 in recipient cells whereas no major difference was detected for co-culture with EBV-negative BL cells (Ramos). Isolated Raji exosomes increased transcriptional and translational levels of VEGF-A in ARPE-19 cells, which was reversely correlated with von Hippel-Lindau expression. A human umbilical vein endothelial cell tube formation assay showed that delivery of ectopic miR-155 rendered ARPE-19 cells proangiogenic. Our results demonstrate that sustained accumulation of miR-155 mediated by exosomes might affect remote recipient cells such as retinal pigment epithelial cells.

EBV Microrna Mir-BHRF1-2 Targets PRDM1/Blimp1: Potential Role in EBV Lymphomagenesis

PRDM1/Blimp1, a master regulator of B-cell terminal differentiation, has been identified as a tumor suppressor gene in the pathogenesis of diffuse large B-cell lymphoma (DLBCL). In DLBCL, PRDM1 is inactivated by mutations and deletions; however, there is also evidence that PRDM1 is down-regulated by microRNAs (miRNAs) in DLBCL and Hodgkin/Reed-Sternberg cells of classical Hodgkin lymphoma (cHL). A decrease in PRDM1 activity contributes to the pathogenesis of DLBCL and cHL by inhibiting plasma cell differentiation triggered by signal transduction pathways such as the NF-kB pathway. Since malignant EBV-positive B-cell lymphoproliferations are often associated with increased NF-kB activity, it is conceivable that abnormal PRDM1 down-regulation may play a role in their pathogenesis.EBV-positive B-cell lymphomas are postulated to originate from EBV-infected B-cells with latency III growth program of EBV gene expression. Thus, EBV-immmortalized lymphoblastoid cell lines (LCLs), which are of latency III type, serve as a good model to study EBV lymphomagenesis. We observed discordance in PRDM1 mRNA and protein levels in LCLs. By quantitative real-time reverse transcriptase PCR, PRDM1 mRNA levels in LCLs varied from 14.6% to 1259.7% relative to the multiple myeloma cell line U266, which expresses high levels of PRDM1. However, PRDM1 protein was discordantly low in LCLs compared to U266 based on immunohistochemistry and Western blotting assays, consistent with post-transcriptional regulation. EBV encodes 25 viral miRNAs, and we postulate that one of more of them may function to dampen PRDM1 expression. Indeed, a miRNA binding site containing seed match to bases 2-7 of EBV miR-BHRF1-2 was identified in positions 1565 to 1589 of PRDM1 3’ untranslated region. MiR-BHRF1-2 functionally targeted this specific binding site and repressed luciferase reporter activity. Mutation in the seed region of this site relieved the repression in comparison to the wild type control.MiR-BHRF1-2 was highly expressed in LCLs, while it was barely detectable in the EBV-positive Burkitt lymphoma cell line MUTU I, which has latency type I. Importantly, immunoblotting assay demonstrated an up-regulation of PRDM1 protein level in CCL156 and CCL159 LCL cells transfected with miR-BHRF1-2 inhibitor relative to those transfected with miRNA Inhibitor negative control, supporting a role of miR-BHRF1-2 in PRDM1 down-regulation in vivo. To examine the biological consequences of increased PRDM1 expression in LCL cells, PRDM1 was over-expressed in JY25 and CCL159 LCL cell lines. Enforced expression of PRDM1 induced apoptosis in both cell lines. Furthermore, bromodeoxyuridine (Brdu) incorporation study demonstrated that overexpression of PRDM1 reduced the percentage of S phase from 43.4% to 27.6% in CCL159 cells, and 39.5% to 27.9% in JY25 cells, respectively. Whole transcriptome sequencing (RNA-seq) identified a set of potential PRDM1 direct target genes whose expressions decreased in both LCL cell lines upon PRDM1 over-expression. These genes have broad functions including cell proliferation and survival, transcription and translation, mitochondrial functions, and cytoskeleton. Although no significant changes in cell cycle and apoptosis were observed upon transfection of miR-BHRF1-2 inhibitor, RNA-seq analysis of CLL159 cells transfected with miR-BHRF1-2 inhibitor revealed a small subset of repressed genes which overlapped with those identified by PRDM1 over-expression. This finding suggests that the increase in PRDM1 expression upon miR-BHRF1-2 inhibition, albeit small, is capable of repressing a subset of PRDM1 target genes with potential biological effects.In summary, our findings demonstrate that PRDM1 is a target of EBV miR-BHRF1-2. MiR-BHRF1-2 mediated PRDM1 down-regulation may contribute to the pathogenesis of EBV-associated B-cell lymphomas by inhibiting the transcription repression program of PRDM1 and limiting PRDM1-mediated cellular changes detrimental to tumor growth, including cell cycle arrest and apoptosis. Further characterization of the target genes whose expression is up-regulated by miR-BHRF1-2-mediated PRDM1 down-regulation may provide important clues to the pathogenetic function of miR-BHRF1-2 and EBV oncogenesis in general. DisclosuresNo relevant conflicts of interest to declare.

Hypermethylation of the tumor suppressor gene PRDM1/Blimp-1 supports a pathogenetic role in EBV-positive Burkitt lymphoma.

PRDM1/Blimp-1 is a tumor suppressor gene in the activated B-cell subtype of diffuse large B-cell lymphomas. Its inactivation contributes to pathogenesis in this setting by impairing terminal B-cell differentiation induced by constitutive nuclear factor-κB activation. The role of PRDM1 in Burkitt lymphoma (BL) lymphomagenesis is not known. Here we identified hypermethylation of the promoter region and exon 1 of PRDM1 in all six Epstein–Barr virus (EBV)-positive BL cell lines and 12 of 23 (52%) primary EBV-positive BL or BL-related cases examined, but in none of the EBV-negative BL cell lines or primary tumors that we assessed, implying a tumor suppressor role for PRDM1 specifically in EBV-associated BL. A direct induction of PRDM1 hypermethylation by EBV is unlikely, as PRDM1 hypermethylation was not observed in EBV-immortalized B lymphoblastoid cell lines. Treatment of EBV-positive BL cells with 5′ azacytidine resulted in PRDM1 induction associated with PRDM1 demethylation, consistent with transcriptional silencing of PRDM1 as a result of DNA methylation. Overexpression of PRDM1 in EBV-positive BL cell lines resulted in cell cycle arrest. Our results expand the spectrum of lymphoid malignancies in which PRDM1 may have a tumor suppressor role and identify an epigenetic event that likely contributes to the pathogenesis of BL.

Indole-3-carbinol induces cMYC and IAP-family downmodulation and promotes apoptosis of Epstein–Barr virus (EBV)-positive but not of EBV-negative Burkitt's lymphoma cell lines

Indole-3-carbinol (I3C) is a natural product found in broadly consumed plants of the Brassica genus, such as broccoli, cabbage, and cauliflower, which exhibits anti-tumor effects through poorly defined mechanisms. I3C can be orally administered and clinical trials have demonstrated that I3C and derivatives are safe in humans. In this study we show that I3C efficiently induces apoptosis in cell lines derived from EBV-positive Burkitt's lymphomas (virus latency I/II), while it does not have any cytotoxic activity against EBV-negative Burkitt's lymphomas and immortalized EBV-infected lymphoblastoid cell lines (virus latency III). The effect of I3C in EBV-positive Burkitt's lymphoma is very specific, since only I3C and its C6-methylated derivative, but not other 3-substituted indoles, have an effect on cell viability. I3C treatment caused apoptosis characterized by loss of mitochondria membrane potential and caspase activation. I3C alters the expression of proteins involved in the control of apoptosis and transcription regulation in EBV-positive Burkitt's lymphoma cell lines. Among those, cMYC, cIAP1/2 and XIAP downmodulation at mRNA and protein level precede apoptosis induction, thus suggesting a role in I3C cytotoxicity. We also showed that I3C and, more particularly, its condensation dimer 3,3′-diindolylmethane (DIM) prolonged survival and reduced tumor burden of mice xenotransplanted with EBV-positive Burkitt's lymphoma Daudi cells.In summary these results, together with previous reports from clinical trials indicating the lack of toxicity in humans of I3C and derivatives, support the use of these compounds as a new therapeutic approach for treating patients with endemic (EBV-positive) Burkitt's lymphoma.

Enhanced susceptibility of B lymphoma cells to measles virus by Epstein–Barr virus type III latency that upregulates CD150/signaling lymphocytic activation molecule

Measles virus (MV) is one of the candidates for the application of oncolytic virotherapy (OVT). Although an advanced clinical study has been reported on a T-cell lymphoma, the potential of MV OVT against B-cell lymphomas remains to be clarified. We found that an EBV-transformed B lymphoblastoid cell line, a model for diffuse large B-cell lymphoma, and EBV-positive Burkitt's lymphoma cells bearing type III latency were highly susceptible to the cytolysis induced by an MV vaccine strain CAM-70. As analyzed by EBV-positive and -negative counterparts of the same cytogenetic background, type III EBV latency, not type I, was shown to augment the susceptibility of B lymphoma cells to MV-induced cytolysis. Cell surface levels of CD150/signaling lymphocytic activation molecule, a receptor of MV, were upregulated in B lymphoma cell lines with type III EBV latency by 3.8-fold, on average. The cytolytic activity of CD150-tropic WT MV was akin to that of CD46- and CD150-tropic CAM-70, suggesting that CD150 is critical for the susceptibility to MV-induced cytolysis. Among EBV-encoded genes, latent membrane protein 1 was responsible for the CD150 upregulation. It was notable that the majority of B lymphoma cell lines of type III EBV latency showed higher susceptibility to the non-Edmonston-derived CAM-70 than to the Edmonston-derived Schwarz strain. This is the first report indicating the potential of non-Edmonston MV strain for the application of OVT. Furthermore, a cellular regulator of MV replication was implicated that functions in a vaccine strain-specific fashion. Altogether, the MV OVT should serve as an alternative therapy against EBV-positive diffuse large B-cell lymphoma with type III EBV latency.

Molecular network of chromatin immunoprecipitation followed by deep sequencing-based (ChIP-Seq) Epstein-Barr virus nuclear antigen 1-target cellular genes supports biological implications of Epstein-Barr virus persistence in multiple sclerosis

Objectives Epstein–Barr virus (EBV) infection confers a strong risk factor for development of multiple sclerosis (MS). EBV preferentially infects B lymphocytes to establish a life-long latent infection. EBV nuclear antigen 1 (EBNA1), acting as a transcriptional activator, plays a central role in the replication and maintenance of the latent episomal EBV genome in EBV-infected host cells. However, the comprehensive set of host cellular genes directly regulated by EBNA1 relevant to the immunopathogenesis of MS remains to be elucidated. Methods We investigated the chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) dataset of Raji cells, an EBV-positive Burkitt's lymphoma cell line. We studied the molecular network of target genes by pathway analysis tools of bioinformatics. Results We identified the set of 228 EBNA1-target cellular genes. The EBNA1-binding sites were located mainly in intronic regions of target genes with an existence of the palindromic consensus sequence motif. By pathway analysis using Ingenuity Pathways Analysis and KeyMolnet, the EBNA1-target cellular gene network showed a significant relationship with the networks related to cell death and survival, and transcriptional regulation by interferon-regulatory factor (IRF), and signal transducer and activator of transcription (STAT). Conclusions These results show that the EBNA1-target cellular gene network is closely associated with maintenance of EBV persistence by controlling the fate of EBV-infected host cells, and by aberrantly regulating the production of host-derived antiviral interferons and other cytokines, supporting biological implications of EBV persistence in MS.

Epstein-Barr virus latent membrane protein 1 induces CD69 expression through activation of nuclear factor-κB

Latent membrane protein‑1 (LMP-1) of Epstein‑Barr virus (EBV) promotes tumorigenesis. Here, we report that LMP-1 activates the immunoregulatory molecule CD69 gene transcription through a nuclear factor-κB (NF-κB)‑dependent pathway. CD69 expression was upregulated in LMP-1‑expressing EBV-immortalized human B-cell lines and an EBV-positive Burkitt's lymphoma cell line. LMP-1 expression increased CD69 expression at the transcriptional level. CD69 promoter was regulated by LMP-1 activation of NF-κB via the carboxy-terminal activation region 1 and 2. Promoter deletion analysis indicated that two NF-κB binding sites are necessary for activation of the CD69 promoter. Electrophoretic mobility shift analysis demonstrated that LMP-1 activates both NF-κB binding sites in the CD69 promoter. This is the first report of the regulation of CD69 expression by LMP-1, and this novel finding may, thus, represent an important link between the EBV oncoprotein LMP-1 and its critical role in the development of EBV-associated diseases.

Ascorbic acid kills Epstein–Barr virus positive Burkitt lymphoma cells and Epstein–Barr virus transformed B-cells in vitro, but not in vivo

Ascorbic acid has been shown to kill various cancer cell lines at pharmacologic concentrations. We found that Epstein–Barr virus (EBV)-positive Burkitt lymphoma (BL) cells were more susceptible to ascorbic acid-induced cell killing than EBV-negative BL cells or EBV-transformed lymphoblastoid cells (LCLs). Ascorbic acid did not induce apoptosis in any of the tested cells but did induce the production of reactive oxygen species and cell death. Previously, we showed that bortezomib, a proteasome inhibitor, induces cell death in LCLs and EBV-positive BL cells. We found that ascorbic acid is strongly antagonistic for bortezomib-induced cell death in LCLs and EBV-positive BL cells. Finally, ascorbic acid did not prolong survival of severe combined immunodefiency mice inoculated with LCLs either intraperitoneally or subcutaneously. Thus, while ascorbic acid was highly effective at killing EBV-positive BL cells and LCLs in vitro, it antagonized cell killing by bortezomib and was ineffective in an animal model.

Epstein-Barr nuclear antigen 1 induces expression of the cellular microRNA hsa-miR-127 and impairing B-cell differentiation in EBV-infected memory B cells. New insights into the pathogenesis of Burkitt lymphoma

Epstein-Barr Virus (EBV) is a γ-herpesvirus that infects >90% of the human population. Although EBV persists in its latent form in healthy carriers, the virus is also associated with several human cancers. EBV is strongly associated with Burkitt lymphoma (BL), even though there is still no satisfactory explanation of how EBV participates in BL pathogenesis. However, new insights into the interplay between viruses and microRNAs (miRNAs) have recently been proposed. In particular, it has been shown that B-cell differentiation in EBV-positive BL is impaired at the post-transcriptional level by altered expression of hsa-miR-127. Here, we show that the overexpression of hsa-miR-127 is due to the presence of the EBV-encoded nuclear antigen 1 (EBNA1) and give evidence of a novel mechanism of direct regulation of the human miRNA by this viral product. Finally, we show that the combinatorial expression of EBNA1 and hsa-miR-127 affects the expression of master B-cell regulators in human memory B cells, confirming the scenario previously observed in EBV-positive BL primary tumors and cell lines. A good understanding of these mechanisms will help to clarify the complex regulatory networks between host and pathogen, and favor the design of more specific treatments for EBV-associated malignancies.

Parthenolide induces apoptosis and lytic cytotoxicity in Epstein-Barr virus-positive Burkitt lymphoma

Burkitt lymphoma (BL) has been reported to be strongly associated with Epstein-Barr virus (EBV) infection. The fact that EBV is generally present in cancer cells but rarely found in healthy cells represents an opportunity for targeted cancer therapy. One approach is to activate the lytic replication cycle of the latent EBV. Nuclear factor (NF)-κB is thought to play an essential role in EBV lytic infection. Elevated NF-κB levels inhibit EBV lytic replication. Parthenolide (PN) is a sesquiterpene lactone found in medicinal plants, particularly in feverfew (Tanacetum parthenium). The aim of the present study was to analyze the effect of PN on the survival of Raji EBV-positive lymphoma cells. Raji cells were treated with 0, 4 or 6 μmol/l PN for 48 h. MTT assay and western blot analysis were performed to evaluate the findings. Results showd that PN suppressed the growth of the EBV-positive BL cell line, Raji, and activated the transcription of BZLF1 and BRLF1 by inhibiting NF-κB activity. Most notably, when PN was used in combination with ganciclovir (GCV), the cytotoxic effect of PN was amplified. These data suggest that the induction of lytic EBV infection with PN in combination with GCV may be a viral-targeted therapy for EBV-associated BL.

Abstract LB-181: Piperlongumine inhibits growth and survival of human Burkitt lymphoma by downregulating NF-κB, c-Myc and LMP1

Abstract Piperlongumine (PL), a natural product constituent of the fruit of the Long pepper, Piper longum, has well documented anti-inflammatory and cancer inhibiting properties. The latter include evidence that PL selectively kills solid tumor cells, but spares their normal counterparts. Because it has not been established whether PL can also inhibit hematological malignancies of the B-lymphocyte lineage, we here decided to evaluate the efficacy with which PL suppresses growth and survival of human non-Hodgkin's Burkitt lymphoma (BL) cells in vitro. We found that PL curbed cell growth (IC50 values of PL for 24h treatment: DG-75; 8.5uM, Daudi; 2.84uM, Raji; 7.7uM and Ramos; 4.5uM) and caused Caspase-3-dependent apoptosis of BL cells in a concentration- and time-dependent manner. PL strongly suppressed constitutive NF-κB signaling by inhibition of IκBα degradation and subsequent nuclear translocation of p65 as well as interruption of NF-κB binding to its consensus DNA sequence. PL also disrupted the expression of NF-κB target genes such as E2F1, c-Myb and GADD45B. c-Myc, transcription factor important for BL development and survival, was remarkably reduced after treatment with PL. In EBV-positive BL cell lines that express high level of LMP1 (e.g., Daudi, Raji), PL efficiently repressed LMP1, a known activator of NF-κB. Considering that NF-κB, c-Myc and LMP1 play important roles in the development and in vivo maintenance of B-lineage and other types of blood cells neoplasms, our findings strongly suggest that PL may afford new approaches to the chemoprevention and treatment of blood cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-181. doi:1538-7445.AM2012-LB-181