Induction Of Lytic Cycle Research Articles

P0127 Curcuminoids as putative ebv lytic induction agents for adjuvant treatment in EBV-positive carcinoma

Background Epstein-Barr virus (EBV) is causally involved in various lymphoid and non-lymphoid cancers, including nasopharyngeal (NPC) and stomach carcinomas (EBVaGC). NPC is endemic in south east Asia, including Indonesia. EBVaGC accounts for about 10% of GC worldwide. We recently initiated efforts to target EBV inside tumour cells by cytolytic virus activation therapy (CLVA), currently being validated in a phase 1/2 trial. We aim to develop oral drugs to improve CLVA treatment availability for low-income countries. Curcumin (diferuloylmethane) from Curcuma longa is used as spice in Asian countries without any toxic effects and has anti-cancer activity. Several curcumin analogues (curcuminoids), developed to increase efficacy and bioavailability, are currently being tested in clinical studies. Curcuminoids affect multiple pathways, including inhibition of COX2 and NF-kB and have potential as oral CLVA regimens for EBV-positive carcinoma. Methods We investigated EBV-reactivating effects of curcuminoids on EBV-positive NPC (C666.1) and EBVaGC cell lines (SNU179 and AGS-BX1). EBV-negative GC (AGS) line was used as a negative control. Standard CLVA, SAHA, and bortezomib were used as positive controls. EBV lytic (Zebra, EA-D) activity was measured by semi-quantitative immunoblot, FACS and indirect immunofluorescence. Effects on cell viability were measured by MTT assay. Findings Curcumin induced EBV lytic activity in AGS-BX1 cells but had marginal effect on natural EBV lines (C666.1,SNU179). However, several curcumin analogues induced EBV reactivation in all three EBV-positive cell lines in a dose- and time-dependent manner. MTT assay showed curcuminoids to be more toxic to EBVaGC cells than to C666.1 cells. Noteworthy curcuminoids showed synergistic effects with current lytic induction agents. Interpretation Curcumin analogues serve as non-toxic drugs to reactivate EBV from latently infected NPC and GC cells, but seem to reactivate EBV differently in NPC versus GC. Curcuminoids at pharmacologically relevant concentrations may promote cytolysis of EBV-positive carcinoma when combined with CLVA drugs. The molecular mechanisms of EBV lytic cycle induction by curcuminoids will be studied to gain further insights in EBV reactivation from latency in different carcinoma models.

HTERT Inhibition Triggers Epstein–Barr Virus Lytic Cycle and Apoptosis in Immortalized and Transformed B Cells: A Basis for New Therapies

Induction of viral lytic cycle, which induces death of host cells, may constitute a useful adjunct to current therapeutic regimens for Epstein-Barr virus (EBV)-driven malignancies. Human telomerase reverse transcriptase (hTERT), essential for the oncogenic process, may modulate the switch from latent to lytic infection. The possible therapeutic role of hTERT inhibition combined with antiviral drugs was investigated. EBV-negative BL41 and convertant EBV-positive BL41/B95.8 Burkitt's lymphoma cell lines and lymphoblastoid cell lines (LCL) were infected with retroviral vector encoding short hairpin RNA (shRNA) anti-hTERT and cultured with or without the prodrug ganciclovir. The effects on EBV lytic replication, cell proliferation, and apoptosis were characterized. hTERT silencing by shRNA induced the expression of BZLF1, EA-D, and gp350 EBV lytic proteins and triggered a complete lytic cycle. This effect was associated with downregulation of BATF, a negative regulator of BZLF1 transcription. hTERT silencing also resulted in antiproliferative and proapoptotic effects. In particular, hTERT inhibition induced an accumulation of cells in the S-phase, an effect likely due to the dephosphorylation of 4E-BP1, an AKT1-dependent substrate, which results in a decreased availability of proteins needed for cell-cycle progression. Besides inducing cell death through activation of complete EBV lytic replication, hTERT inhibition triggered AKT1/FOXO3/NOXA-dependent apoptosis in EBV-positive and -negative Burkitt's lymphoma cells. Finally, ganciclovir enhanced the apoptotic effect induced by hTERT inhibition in EBV-positive Burkitt's lymphomas and LCLs. These results suggest that combination of antiviral drugs with strategies able to inhibit hTERT expression may result in therapeutically relevant effects in patients with EBV-related malignancies.

Highlights of This Issue

Using a chemosensitivity RNA interference screen against 133 genes with known somatic mutations in breast cancer, Singel and colleagues identified the kinesin family member 14 (KIF14) and talin 1 as modulators of response to docetaxel in 4 triple-negative breast cancer cell lines but not in 3 hormonereceptor positive cell lines. Knockdown of these genes did not affect chemosensitivity to normal mammary epithelial cells. Their results show that high KIF14 expression is prognostic of poor relapse-free survival (946 patients) and poor overall survival (652 patients). Therefore, decreasing expression of these genes is an attractive target to sensitize triple negative human breast cancer cells to taxane treatment.Induction of the viral lytic cycle is a promising strategy for treatment of Epstein–Barr virus (EBV)–driven malignancies because viral lytic replication is associated with death of EBV-carrying tumor cells. Giunco and colleagues show that inhibition of human telomerase reverse transcriptase (hTERT), the catalytic component of telomerase, promotes induction of the viral lytic cycle and triggers apoptosis of EBV-immortalized and fully transformed B cells. These results provide the rationale to activate clinical studies based on the combination of antiviral drugs with inhibitors of hTERT expression/activity for the treatment of EBV-driven malignancies.The multikinase inhibitor sorafenib successfully displayed antitumor activity in preclinical models of metastatic osteosarcoma. Unfortunately, in the clinical setting tumor shrinkage and disease stabilization with sorafenib were short-lived, owing to drug resistance. Pignochino and colleagues showed mTOR complex 2 (mTORC2) upregulation in sorafenib-treated osteosarcoma models, thereby suggesting a potential escape mechanism. The combination of sorafenib with the mTOR inhibitor everolimus achieved total blockage of both mTORC1 and mTORC2 through an increase of reactive oxygen species and the activation of AMP-dependent kinase. This complete inhibition eventually enhances the antitumor, antimetastatic, and antiangiogenic effects in osteosarcoma xenograft models.In a phase II clinical study in metastatic colorectal cancer, Amatu and colleagues show for the first time that dacarbazine activity is confined to patients with tumors defective in O6-methylguanine-DNA-methyltransferase function due to promoter CpG hypermethylation. In the context of cancer science, these data therefore highlight a previously unidentified subgroup of patients with colorectal cancer who benefit from treatment with methylating agents based on a specific epigenetic alteration in individual tumors. This is a step in the direction of personalized cancer medicine.

Epstein-Barr Virus Transcription Activator R Upregulates BARF1 Expression by Direct Binding to Its Promoter, Independent of Methylation

Epstein-Barr virus (EBV) BamHI-A rightward frame 1 (BARF1) is considered a major viral oncogene in epithelial cells and has immune-modulating properties. However, in B cells and lymphomas, BARF1 expression is restricted to the viral lytic replication cycle. In this report, the transcriptional regulation of BARF1 during lytic replication is unraveled. Bisulfite sequencing of various cell lines indicated a high level of methylation of the BARF1 gene control region. A BARF1 promoter luciferase reporter construct was created using a CpG-free vector, enabling true assessment of promoter methylation. Induction of the EBV lytic cycle is mediated by the immediate-early proteins BZLF1 (Z) and BRLF1 (R). R was found to activate expression of the BARF1 promoter up to 250-fold independently of Z and unaffected by BARF1 promoter methylation. Chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA), and specific mutagenesis of the R-responsive elements (RREs) demonstrated direct binding of R to RREs between nucleotides -554 and -327 relative to the BARF1 transcriptional ATG start site. The kinetics of BARF1 expression upon transactivation by R showed that BARF1 mRNA was expressed within 6 h in the context of the viral genome. In conclusion, expression of the BARF1 protein during lytic replication is regulated by direct binding of R to multiple RREs in the gene control region and is independent of the promoter methylation status. The early kinetics of BARF1 upon transactivation by R confirm its status as an early gene and emphasize the necessity of early immune modulation during lytic reactivation.

Roles of Cell Signaling Pathways in Cell-to-Cell Contact-Mediated Epstein-Barr Virus Transmission

Epstein-Barr virus (EBV), a human gamma herpesvirus, establishes a life-long latent infection in B lymphocytes and epithelial cells following primary infection. Several lines of evidence indicate that the efficiency of EBV infection in epithelial cells is accelerated up to 10(4)-fold by coculturing with EBV-infected Burkitt's lymphoma (BL) cells compared to infection with cell-free virions, indicating that EBV infection into epithelial cells is mainly mediated via cell-to-cell contact. However, the molecular mechanisms involved in this pathway are poorly understood. Here, we establish a novel assay to assess cell-to-cell contact-mediated EBV transmission by coculturing an EBV-infected BL cell line with an EBV-negative epithelial cell line under stimulation for lytic cycle induction. By using this assay, we confirmed that EBV was transmitted from BL cells to epithelial cells via cell-to-cell contact but not via cell-to-cell fusion. The inhibitor treatments of extracellular signal-regulated kinase (ERK) and nuclear factor (NF)-κB pathways blocked EBV transmission in addition to lytic induction. The blockage of the phosphoinositide 3-kinase (PI3K) pathway impaired EBV transmission coupled with the inhibition of lytic induction. Knockdown of the RelA/p65 subunit of NF-κB reduced viral transmission. Moreover, these signaling pathways were activated in cocultured BL cells and in epithelial cells. Finally, we observed that viral replication was induced in cocultured BL cells. Taken together, our data suggest that cell-to-cell contact induces multiple cell signaling pathways in BL cells and epithelial cells, contributing to the induction of the viral lytic cycle in BL cells and the enhancement of viral transmission to epithelial cells.

Signal transducer and activator of transcription 3 (STAT3) controls susceptibility to Epstein-Barr virus reactivation in B cells

Epstein-Barr Virus (EBV) is known to cause malignancies in immunocompromised individuals, including lymphomas and lymphoproliferative disorders. Reactivation of EBV from latency is important in the pathogenesis of such malignancies. Therapeutically, oncolysis of tumors harboring lytic EBV holds promise. However, exposure of latently infected cultured B cells to lytic cycle inducing stimuli results in virus reactivation within only 20% to 50% of cells. Host cell determinants, that govern this susceptibility to lytic reactivat ion within latently infected B cells, are not well understood. Our research has previously identified that higher levels of signal transducer and activator of transcription 3 (STAT3) within cells correlate with resistance to EBV lytic reactivation. We now investigate whether inhibiting function of STAT3 promotes susceptibility of latently infected B cells to known lytic cycle inducing stimuli or results in induction of EBV lytic cycle. We found that pharmacological inhibition of STAT3 phosphorylation by Janus Kinase 2 (JAK2) inhibitors (AG490 or WP1066) in latently infected cells (HH514-16 Burkitt lymphoma and B95-8 lymphoblastoid cells) increased lytic reactivation by chemical stimuli that function by distinct mechanisms. Moreover, functional inhibition of STAT3 alone resulted in lytic reactivation in these cells. EBV-Lymphoblastoid cell lines (LCL) newly generated from healthy individuals also demonstrated increased susceptibility to lytic cycle inducing stimuli in the presence of AG490 but variable susceptibility to lytic reactivation when exposed to AG490 alone. Since this lack of uniform response to AG490 alone could be due to inadequate functional suppression of STAT3, we examined EBV-LCLs derived from patients with Autosomal Dominant Hyperimmunoglobulin-E syndrome (AD-HIES or Job syndrome). Patients with AD-HIES carry a dominant negative mutation in their Stat3 gene resulting in lower basal levels of functional STAT3. When LCLs from AD-HIES patients were exposed to a JAK2 inhibitor alone, we observed a strong increase in lytic reactivation by expression of early and late lytic antigens over LCLs derived from healthy individuals. Lytic reactivation in the presence of AG490 occurred in these cells despite lack of discernable increase from basal levels of expression of ZEBRA, the viral lytic switch protein, when compared to cells not exposed to AG490. Thus, STAT3 is important in determining susceptibility to EBV reactivation. Fully understanding how STAT3 governs such susceptibility can lead to novel therapeutic strategies for EBV-related diseases.

Abstract 4715: Activation of lytic cycle of Epstein-Barr virus by suberoylanilide hydroxamic acid affects apoptosis and tumor growth suppression of nasopharyngeal carcinoma

Abstract Nasopharyngeal carcinoma (NPC) is strongly associated with Epstein-Barr virus (EBV). We examined the in vitro and in vivo effects of suberoylanilide hydroxamic acid (SAHA), a FDA-approved histone deacetylase inhibitor, on EBV lytic cycle induction in NPC and investigated the cellular consequences. Micromolar concentrations of SAHA potently induced EBV lytic cycle with replication of EBV DNA, expression of immediate early (Zta and Rta), early (BMRF1) and late (gp350) lytic proteins and production of infectious viral particles in culture supernatants in three of four EBV-positive NPC cell lines. One NPC cell line had abortive EBV lytic cycle (no EBV DNA replication or late lytic protein expression) induction. Same concentrations of SAHA effected enhanced killing of EBV-positive NPC cells in comparison with EBV-negative counterparts, as measured by MTT assay. Increased percentages of annexin V-positive and TUNEL-positive cells and proteolytic cleavage of poly (ADP-ribose) polymerase (PARP), caspase-3, -7 and -9 in EBV-positive versus EBV-negative NPC were also observed. Immunofluorescence staining and flow cytometry showed that the majority of NPC cells (>85%) expressing Zta, BMRF1 or gp350 lytic protein co-expressed cleaved caspase-3. Tracking of expression of EBV lytic protein and cleaved caspase-3 showed that proportion of NPC cells co-expressing EBV lytic protein and cleaved caspase-3 increased over time. Furthermore, knockdown of Zta expression by short-hairpin RNA significantly decreased proteolytic cleavage of caspase-3 in SAHA-treated EBV-positive NPC cells whilst overexpression of Zta induced apoptosis of EBV-positive NPC cells, confirming that lytic cycle induction programmed NPC cells to apoptosis. Interestingly, inhibition of EBV DNA replication and late lytic protein expression by phosphonoformic acid did not impact on SAHA's induced cell death in NPC, suggesting that early rather than late phase of EBV lytic cycle contributed to the apoptotic effect. In vivo effects of SAHA on EBV lytic cycle induction and tumor growth suppression were observed in NPC xenografts established in nude mice. Taken together, our data indicated that activation of lytic cycle from latent cycle of EBV by SAHA effects apoptosis and tumor growth suppression of NPC thereby providing experimental evidence for virus-targeted therapy against EBV-positive cancer. This project is funded by NPC Area of Excellence (AoE/M 06/08 Center for Nasopharyngeal Carcinoma Research) and Epstein-Barr virus research (# 20004525) grants of AKSC. 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 4715. doi:1538-7445.AM2012-4715

Activation of lytic cycle of Epstein‐Barr virus by suberoylanilide hydroxamic acid leads to apoptosis and tumor growth suppression of nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is strongly associated with Epstein-Barr virus (EBV). We reported that suberoylanilide hydroxamic acid (SAHA) induced EBV lytic cycle in EBV-positive gastric carcinoma cells and mediated enhanced cell death. However, expression of EBV lytic proteins was thought to exert antiapoptotic effect in EBV-infected cells. Here, we examined the in vitro and in vivo effects of SAHA on EBV lytic cycle induction in NPC cells and investigated the cellular consequences. Micromolar concentrations of SAHA significantly induced EBV lytic cycle in EBV-positive NPC cells. Increased apoptosis and proteolytic cleavage of poly(ADP-ribose) polymerase and caspase-3, -7 and -9 in EBV-positive versus EBV-negative NPC cells were observed. More than 85% of NPC cells expressing immediate-early (Zta), early (BMRF1) or late (gp350/220) lytic proteins coexpressed cleaved caspase-3. Tracking of expression of EBV lytic proteins and cleaved caspase-3 over time demonstrated that NPC cells proceeded to apoptosis following EBV lytic cycle induction. Inhibition of EBV DNA replication and late lytic protein expression by phosphonoformic acid did not impact on SAHA's induced cell death in NPC, indicating that early rather than late phase of EBV lytic cycle contributed to the apoptotic effect. In vivo effects of SAHA on EBV lytic cycle induction and tumor growth suppression were also observed in NPC xenografts in nude mice. Taken together, our data indicated that activation of lytic cycle from latent cycle of EBV by SAHA leads to apoptosis and tumor growth suppression of NPC thereby providing experimental evidence for virus-targeted therapy against EBV-positive cancer.

Differences in Shiga toxin and phage production among stx2g-positive STEC strains

Shiga toxin-producing Escherichia coli (STEC) are characterized by the production of Shiga toxins (Stx) encoded by temperate bacteriophages. Stx production is linked to the induction of the phage lytic cycle. Several stx variants have been described and differentially associated with the risk of developing severe illness. The variant named stx2g was first identified in a STEC strain isolated from the faeces of healthy cattle. Analysis of stx2g-positive strains isolated from humans, animals, and environmental sources have shown that they have a close relationship. In this study, stx2g-positive STEC isolated from cattle were analyzed for phage and Stx production, with the aim to relate the results to differences observed in cytotoxicity. The presence of inducible phages was assessed by analyzing the bacterial growth/lysis curves and also by plaque assay. Bacterial growth curves in the absence of induction were similar for all isolates, however, notably differed among induced cultures. The two strains that clearly evidenced bacteriolysis under this condition also showed higher phage titers in plaque assays. However, only the phage plaques produced by one of these strains (FB 62) hybridized with a stx2-probe. Furthermore, the production of Stx was evaluated by enzyme immunoassay (EIA) and Western immunoblotting in overnight supernatants. By EIA, we detected Stx only in supernatants of FB 62, with a higher signal for induced than uninduced cultures. By immunoblotting, Stx2 could be detected after induction in all stx2g-positive isolates, but with lower amounts of Stx2B subunit in those supernatants where phages could not be detected. Taking into account all the results, several differences could be found among stx2g-positive strains. The strain with the highest cytotoxic titer showed higher levels of stx2-phages and toxin production by EIA, and the opposite was observed for strains that previously showed low cytotoxic titers, confirming that in stx2g-positive strains Stx production is phage-regulated.

Reactive Oxygen Species Production and Antioxidant Enzyme Expression after Epstein–Barr Virus Lytic Cycle Induction in Raji Cell Line

In a previous study, we have described oxidative stress during Epstein-Barr virus lytic cycle induction. Oxidative stress was evidenced by the observed high MDA levels and the decreased activities of antioxidant enzymes. We hypothesised that the lower activities of the antioxidant enzymes decrease were the result of either the excessive production of reactive oxygen radical species (ROS) or a negative regulation of the antioxidant enzyme gene expressions. In an attempt to clarify this situation, EBV lytic cycle was induced in Raji cell line by a non-stressing dose of 12-0-tetradecanoylphorbol-13-acetate. BZLF-1, superoxide dismutase, and catalase gene expressions were then analysed using semi-quantitative RT-PCR, simultaneously at a kinetic of 6, 12, 24, 36, and 48h. ROS production was evaluated by chemiluminescence. A study was conducted to establish whether ROS production, BZLF-1, and the expression of antioxidant genes were inter-correlated. Induction of the lytic cycle resulted in increased expressions of the genes of superoxide dismutase and catalase, which began at 24h (p < 0.05) and reached a peak at 48h (p < 0.05). Significant increases of the ROS levels were observed in TPA-treated Raji cell line at 12h, as compared with untreated cells, reaching a peak at 48h after EBV lytic cycle induction. ROS production correlates positively with BZLF-1, SOD, and CAT gene expressions (p < 0.05; r = 0.913, r = 0.978, and r = 0.955, respectively). A positive correlation was also observed between BZLF-1 and antioxidant gene expressions (p < 0.05; r = 0.961 and r = 0.987, respectively). In conclusion, the observed increases of the SOD and CAT gene expressions eliminate the hypothesis of a repression of the respective genes during the induction of the lytic cycle. On the other hand, the observed direct correlation between the BZLF-1 gene expression and the ROS production is indicative of a role of this gene in oxidative stress.

Down-regulation of BLIMP1α by the EBV oncogene, LMP-1, disrupts the plasma cell differentiation program and prevents viral replication in B cells: implications for the pathogenesis of EBV-associated B-cell lymphomas

AbstractAn important pathogenic event in Epstein-Barr virus (EBV)-associated lymphomas is the suppression of virus replication, which would otherwise lead to cell death. Because virus replication in B cells is intimately linked to their differentiation toward plasma cells, we asked whether the physiologic signals that drive normal B-cell differentiation are absent in EBV-transformed cells. We focused on BLIMP1α, a transcription factor that is required for plasma cell differentiation and that is inactivated in diffuse large B-cell lymphomas. We show that BLIMP1α expression is down-regulated after EBV infection of primary germinal center B cells and that the EBV oncogene, latent membrane protein-1 (LMP-1), is alone capable of inducing this down-regulation in these cells. Furthermore, the down-regulation of BLIMP1α by LMP-1 was accompanied by a partial disruption of the BLIMP1α transcriptional program, including the aberrant induction of MYC, the repression of which is required for terminal differentiation. Finally, we show that the ectopic expression of BLIMP1α in EBV-transformed cells can induce the viral lytic cycle. Our results suggest that LMP-1 expression in progenitor germinal center B cells could contribute to the pathogenesis of EBV-associated lymphomas by down-regulating BLIMP1α, in turn preventing plasma cell differentiation and induction of the viral lytic cycle.

NF-κB-Mediated Modulation of Inducible Nitric Oxide Synthase Activity Controls Induction of the Epstein-Barr Virus Productive Cycle by Transforming Growth Factor Beta 1

Transforming growth factor beta 1 (TGF-β1) signal transduction has been implicated in many second-messenger pathways, including the NF-κB pathway. We provide evidence of a novel TGF-β1-mediated pathway that leads to extracellular signal-regulated kinase (ERK) 1/2 phosphorylation, which in turn induces expression of an Epstein-Barr virus (EBV) protein, ZEBRA, that is responsible for the induction of the viral lytic cycle. This pathway includes two unexpected steps, both of which are required to control ERK 1/2 phosphorylation: first, a quick and transient activation of NF-κB, and second, downregulation of inducible nitric oxide synthase (iNOS) activity that requires the participation of NF-κB activity. Although necessary, NF-κB alone is not sufficient to produce downregulation of iNOS, suggesting that another uncharacterized event(s) is involved in this pathway. Dissection of the steps involved in the switch from the EBV latent cycle to the lytic cycle will be important to understand how virus-host relationships modulate the innate immune system.

Induction of epstein-barr virus (EBV) lytic cycle in vitro causes lipid peroxidation, protein oxidation and DNA damage in lymphoblastoid B cell lines

BackgroundWe investigated the oxidative modifications of lipids, proteins and DNA, potential molecular targets of oxidative stress, in two lymphoblastoid cell lines: B95-8 and Raji, after EBV lytic cycle induction. Conjugated dienes level was measured as biomarker of lipid peroxidation. Malondialdehyde adduct and protein carbonyl levels, as well as protein thiol levels were measured as biomarkers of protein oxidation. DNA fragmentation was evaluated as biomarker of DNA oxidation.ResultsAfter 48 h (peak of lytic cycle), a significant increase in conjugated dienes level was observed in B95-8 and Raji cell lines (p = 0.0001 and p = 0.019 respectively). Malondialdehyde adduct, protein carbonyl levels were increased in B95-8 and Raji cell lines after EBV lytic cycle induction as compared to controls (MDA-adduct: p = 0.008 and p = 0.006 respectively; Carbonyl: p = 0.003 and p = 0.0039 respectively). Proteins thiol levels were decreased by induction in B95-8 and Raji cell lines (p = 0.046; p = 0.002 respectively). DNA fragmentation was also detected in B95-8 and Raji cell lines after EBV lytic cycle induction as compared to controls.ConclusionThe results of this study demonstrate the presence of increased combined oxidative modifications in lipids, proteins in B95-8 and Raji cells lines after EBV lytic cycle induction. These results suggest that lipid peroxidation, protein oxidation and DNA fragmentation are generally induced during EBV lytic cycle induction and probably contribute to the cytopathic effect of EBV.

Epstein-Barr Virus and Kaposi's Sarcoma Herpesvirus Lytic Cycle Induction with Bortezomib Is a Response to ER Stress

AbstractAbstract 1736EBV and KSHV are associated with a variety of lymphoid malignancies. Bortezomib, a proteasome inhibitor, is among the most potent activators of EBV and KSHV lytic infection and is active at nanomolar concentrations. Studying EBV Burkitt's cell lines, KSHV primary effusion lymphoma cell lines and EBV myeloma cell lines, we found that lytic gene expression is a class effect shared by other proteasome inhibitors, such as MG-132 and epoxomicin. Proteasome inhibition results in IKB stabilization and inhibition of NFKB activity and this effect has been implicated in many of the effects of bortezomib. We studied the promoters of EBV and KSHV immediate early viral protein reporters (EBV Zta and KSHV Rta) and found that IKB super-repressor did not activate promoter reporters suggesting that other pathways must be important in activation. Therefore, we sought to better understand the drug's impact on viral gene expression in virus-associated tumor cells. ER stress has been implicated in bortezomib's antitumor effects. Thapsigargin and tunicamycin are classic activators of ER stress that do not act through proteasomal inhibition. These agents were found to be potent activators of the EBV and KSHV lytic cycle. Previous studies have identified C/EBP family members (C/EBP alpha and beta) as activators of EBV and KSHV immediate early gene expression. In other studies, C/EBP family members (C/EBP beta, CHOP10) have been implicated in regulating the ER stress response. We found that bortezomib, thapsigargin and tunicamycin increased levels of the activating C/EBP beta LAP isoform as assessed by immunoblot and by real-time RT-PCR. Treatment also led to increase in ATF4, XBP1(s), CHOP10, and ATF6 cleavage, all consistent with induction of the ER stress response. Additionally, we showed that treatment with bortezomib increased C/EBP beta binding to previously characterized binding sites in the Zta promoter and expression of C/EBP beta LAP isoform was sufficient to activate EBV immediate early lytic promoters. Finally, we demonstrated that in tumor cell lines with C/EBP beta silenced by doxycycline regulated siRNA, induction of EBV lytic induction by bortezomib and thapsigargin was blocked. These results demonstrate that both human lymphoma associated herpesviruses (EBV and KSHV) are activated into lytic cycle by bortezomib and that these effects are mediated through ER stress pathways and specifically involve C/EBP beta. Disclosures:No relevant conflicts of interest to declare.

Epstein-Barr virus lytic cycle activation alters proteasome subunit expression in Burkitt's lymphoma cells

We have shown that Epstein-Barr virus (EBV) lytic cycle activation in Burkitt's lymphoma (BL) cells down-regulates chymotrypsin- and caspase-like activities of the proteasome. The aim of the present study was to evaluate whether EBV activation might also affect proteasome subunit composition. Our results indicate that, independently of the latency program established in the host cells, induction of the EBV lytic cycle reduces the expression of the proteasomal components β5, β1 and β2i, whereas it increases that of β2, β1i, PA28α and PA28β. The modulation of the composition and enzymatic activities of the proteolytic complex are indicative of a less efficient generation of viral immunoepitopes.

Abstract 5445: Suberoylanilide hydroxamic acid induces Epstein-Barr virus lytic cycle and enhanced apoptosis in nasopharyngeal carcinoma

Abstract Nasopharyngeal carcinoma (NPC) has uniquely high prevalence in Southern Chinese populations and a predilection to affect young adult males. Epstein-Barr virus (EBV) is harbored in every tumour cell of NPC but it persists in tightly latent form expressing few viral proteins and evading the immune system. Induction of EBV lytic cycle will lead to expression of a much larger number of viral proteins which may serve as potential therapeutic targets against the cancer. We reported that suberoylanilide hydroxamic acid (SAHA), a FDA-approved histone deacetylase inhibitor, could strongly induce viral lytic cycle in EBV-positive gastric carcinoma cells. Here, we demonstrated that SAHA (at 5-10 μM) could potently induce EBV lytic cycle in a panel of EBV-positive NPC cell lines, as evidenced by increased viral DNA replication, expression of immediate early (Zta and Rta), early (BMRF1) and late (VCA-p40 and gp350/220) viral lytic proteins and production of infectious viral particles. Immunofluorescent staining demonstrated expression of viral lytic proteins in more than 40% of the NPC cells. Enhanced killing of EBV-positive NPC cells when compared with that of EBV-negative counterparts was observed and cleaved form of PARP was predominantly expressed in EBV-positive than EBV-negative NPC cells, following treatment with SAHA for 24, 48 and 72 hours. The kinetics of expression of cleaved PARP correlated with that of Zta viral protein in EBV-positive NPC cells. In conclusion, SAHA is found to be a potent EBV lytic cycle inducing agent and mediates enhanced apoptosis of NPC cells. Further investigation into SAHA's development as a novel virus-targeted therapeutic drug in NPC is warranted. This work is funded by EBV research and NPC AoE grants of AKSC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5445.

Suberoylanilide hydroxamic acid induces viral lytic cycle in Epstein‐Barr virus‐positive epithelial malignancies and mediates enhanced cell death

In Epstein-Barr virus (EBV)-associated malignancies, the virus is harbored in every tumor cell and persists in tightly latent forms expressing a very limited number of viral latent proteins. Induction of EBV lytic cycle leads to expression of a much larger number of viral proteins, which may serve as potential therapeutic targets. We found that 4 histone deacetylase inhibitors, trichostatin A (TSA), sodium butyrate (SB), valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA), all significantly induced EBV lytic cycle in EBV-positive gastric carcinoma cells (AGS/BX1, latency II) but only weakly induced in Burkitt lymphoma cells (AK2003, latency I) and did not induce in lymphoblastoid cells (LCLs, latency III). Interestingly, SAHA potently induced viral lytic cycle in AGS/BX1 cells at micromolar concentrations (evidenced by 8-fold increase in viral DNA replication, strong expression of viral lytic proteins and production of infectious virus particles) and mediated enhanced cell death of EBV-positive AGS/BX1 cells when compared with that of EBV-negative AGS cells, possibly related to cell cycle arrest at G2/M phase. Furthermore, SAHA effected strong induction of EBV lytic cycle in nasopharyngeal carcinoma but not in NK lymphoma cells (both expressing EBV latency II pattern), indicating preferential viral lytic induction in epithelial rather than lymphoid malignancies. In conclusion, SAHA is found to be a potent EBV lytic cycle inducing agent, which warrants further investigation into its potential application as a novel virus-targeted drug for treatment of EBV-associated epithelial malignancies.

Upregulation of STAT3 Marks Burkitt Lymphoma Cells Refractory to Epstein-Barr Virus Lytic Cycle Induction by HDAC Inhibitors

A fundamental problem in studying the latent-to-lytic switch of Epstein-Barr virus (EBV) and the viral lytic cycle itself is the lack of a culture system fully permissive to lytic cycle induction. Strategies to target EBV-positive tumors by inducing the viral lytic cycle with chemical agents are hindered by inefficient responses to stimuli. In vitro, even in the most susceptible cell lines, more than 50% of cells latently infected with EBV are refractory to induction of the lytic cycle. The mechanisms underlying the refractory state are not understood. We separated lytic from refractory Burkitt lymphoma-derived HH514-16 cells after treatment with an HDAC inhibitor, sodium butyrate. Both refractory- and lytic-cell populations responded to the inducing stimulus by hyperacetylation of histone H3. However, analysis of host cell gene expression showed that specific cellular transcripts Stat3, Fos, and interleukin-8 (IL-8) were preferentially upregulated in the refractory-cell population, while IL-6 was upregulated in the lytic population. STAT3 protein levels were also substantially increased in refractory cells relative to untreated or lytic cells. This increase in de novo expression resulted primarily in unphosphorylated STAT3. Examination of single cells revealed that high levels of STAT3 were strongly associated with the refractory state. The refractory state is manifest in a unique subpopulation of cells that exhibits different cellular responses than do lytic cells exposed to the same stimulus. Identifying characteristics of cells refractory to lytic induction relative to cells that undergo lytic activation will be an important step in developing a better understanding of the regulation of the EBV latent to lytic switch.

Cyanobacterial cytoskeleton immunostaining: the detection of cyanobacterial cell lysis induced by planktopeptin BL1125

The aim of this study was to detect cyanobacterial cytoskeletal elements and the cytoskeletal framework using immunostaining with anti-bovine α-tubulin mouse monoclonal antibodies. After strong permeabilization of axenic cyanobacterial cell lines, the cytoskeleton elements become visible in all cells. A mild cell permeabilization procedure allows the discrimination between healthy and senescent or otherwise stressed cyanobacteria whose cell integrity has been jeopardized. This technique can be useful to investigate cyanobacterial bloom lysis provoked by various natural or artificial factors. Viruses, among others, are important mortality agents of cyanobacteria. The presence of non-hepatotoxic cyclic cyanopeptides can provoke lysis of non-axenic Microrystis aeruginosa cell lines. This is presumably due to lytic cycle induction in lysogen cyanobacteria. A susceptible cyano-bacterial cell line exposed to the depsipeptide planktopeptin BL1125 has been analysed with transmission electron microscopy to corroborate the involvement of virus like particles (VLP) in the process of lysis. VLP that correspond in shape and size to tailed cyanophages have been observed only in samples where the process of lysis has been triggered. The immunostaining of cytoskeletal elements by using epifluorescence and confocal microscopy has confirmed that the lysis expands from single infected cells or cell groups, the focal points, to their immediate environment. Our in vitro experiments demonstrate that lysogen focal point formation, which follows induction by endogenous cyanobacterial cyclic peptides, could constitute, also in the natural environment, the basis for an extremely rapid and extensive cyanobacterial bloom collapse.

X-Box Binding Protein 1 Contributes to Induction of the Kaposi's Sarcoma-Associated Herpesvirus Lytic Cycle under Hypoxic Conditions

Kaposi's sarcoma-associated herpesvirus (KSHV), like other herpesviruses, has two stages to its life cycle: latency and lytic replication. KSHV is required for development of Kaposi's sarcoma, a tumor of endothelial origin, and is associated with the B-cell tumor primary effusion lymphoma (PEL) and the plasmablastic variant of multicentric Castleman's disease, all of which are characterized by predominantly latent KSHV infection. Recently, we and others have shown that the activated form of transcription factor X-box binding protein 1 (XBP-1) is a physiological trigger of KSHV lytic reactivation in PEL. Here, we show that XBP-1s transactivates the ORF50/RTA promoter though an ACGT core containing the XBP-1 response element, an element previously identified as a weakly active hypoxia response element (HRE). Hypoxia induces the KSHV lytic cycle, and active HREs that respond to hypoxia-inducible factor 1alpha are present in the ORF50/RTA promoter. Hypoxia also induces active XBP-1s, and here, we show that both transcription factors contribute to the induction of RTA expression, leading to the production of infectious KSHV under hypoxic conditions.