The Experts below are selected from a list of 9636 Experts worldwide ranked by ideXlab platform
George Miller - One of the best experts on this subject based on the ideXlab platform.
-
Valpromide Inhibits Lytic Cycle Reactivation of Epstein-Barr Virus
mBio, 2016Co-Authors: Kelly L. Gorres, Derek Daigle, Sudharshan Mohanram, Grace E. Mcinerney, Danielle E. Lyons, George MillerAbstract:ABSTRACT Reactivation of Epstein-Barr virus (EBV) from latency into the Lytic phase of its life Cycle allows the virus to spread among cells and between hosts. Valproic acid (VPA) inhibits initiation of the Lytic Cycle in EBV-infected B lymphoma cells. While VPA blocks viral Lytic gene expression, it induces expression of many cellular genes, because it is a histone deacetylase (HDAC) inhibitor. Here we show, using derivatives of VPA, that blockade of EBV reactivation is separable from HDAC inhibition. Valpromide (VPM), an amide derivative of valproic acid that is not an HDAC inhibitor, prevented expression of two EBV genes, BZLF1 and BRLF1, that mediate Lytic reactivation. VPM also inhibited expression of a viral late gene, but not early genes, when BZLF1 was exogenously expressed. Unlike VPA, VPM did not activate Lytic expression of Kaposi’s sarcoma-associated herpesvirus. Expression of cellular immediate-early genes, such as FOS and EGR1, is kinetically upstream of the EBV Lytic Cycle. VPM did not activate expression of these cellular immediate-early genes but decreased their level of expression when induced by butyrate, an HDAC inhibitor. VPM did not alter expression of several other cellular immediate-early genes, including STAT3, which were induced by the HDAC inhibitors in cells refractory to Lytic induction. Therefore, VPM selectively inhibits both viral and cellular gene expression. VPA and VPM represent a new class of antiviral agents. The mechanism by which VPA and VPM block EBV reactivation may be related to their anticonvulsant activity. IMPORTANCE Epstein-Barr virus, (EBV), a human tumor virus, establishes a life-long latent infection. Reactivation of EBV into the Lytic phase of its life Cycle allows the virus to spread. Previously, we showed that EBV reactivation was blocked by valproic acid (VPA), an inhibitor of cellular histone deacetylases (HDACs). VPA alters the expression of thousands of cellular genes. In this study, we demonstrate that valpromide (VPM), an amide derivative of valproic acid that is not an HDAC inhibitor, prevented initiation of the EBV Lytic Cycle. VPA induced Lytic reactivation of Kaposi’s sarcoma-associated herpesvirus (KSHV), but VPM did not. Unlike VPA, VPM did not activate cellular immediate-early gene expression. VPM is a new type of antiviral agent. VPM will be useful in probing the mechanism of EBV Lytic reactivation and may have therapeutic application.
-
Valproic Acid Antagonizes the Capacity of Other Histone Deacetylase Inhibitors To Activate the Epstein-Barr Virus Lytic Cycle
Journal of virology, 2011Co-Authors: Derek Daigle, Lyn Gradoville, Kelly L. Gorres, David Tuck, Vince Schulz, Ruth Wang'ondu, George MillerAbstract:Diverse stimuli reactivate the Epstein-Barr virus (EBV) Lytic Cycle in Burkitt lymphoma (BL) cells. In HH514-16 BL cells, two histone deacetylase (HDAC) inhibitors, sodium butyrate (NaB) and trichostatin A (TSA), and the DNA methyltransferase inhibitor azacytidine (AzaCdR) promote Lytic reactivation. Valproic acid (VPA), which, like NaB, belongs to the short-chain fatty acid class of HDAC inhibitors, fails to induce the EBV Lytic Cycle in these cells. Nonetheless, VPA behaves as an HDAC inhibitor; it causes hyperacetylation of histone H3 (J. K. Countryman, L. Gradoville, and G. Miller, J. Virol. 82:4706-4719, 2008). Here we show that VPA blocked the induction of EBV early Lytic proteins ZEBRA and EA-D in response to NaB, TSA, or AzaCdR. The block in Lytic activation occurred prior to the accumulation of BZLF1 transcripts. Reactivation of EBV in Akata cells, in response to anti-IgG, and in Raji cells, in response to tetradecanoyl phorbol acetate (TPA), was also inhibited by VPA. MS-275 and apicidin, representing two additional classes of HDAC inhibitors, and suberoylanilide hydroxamic acid (SAHA) reactivated EBV in HH514-16 cells; this activity was also inhibited by VPA. Although VPA potently blocked the expression of viral Lytic-Cycle transcripts, it did not generally block the transcription of cellular genes and was not toxic. The levels and kinetics of specific cellular transcripts, such as Stat3, Frmd6, Mad1, Sepp1, c-fos, c-jun, and egr1, which were activated by NaB and TSA, were similar in HH514-16 cells treated with VPA. When combined with NaB or TSA, VPA did not inhibit the activation of these cellular genes. Changes in cellular gene expression in response to VPA, NaB, or TSA were globally similar as assessed by human genome arrays; however, VPA selectively stimulated the expression of some cellular genes, such as MEF2D, YY1, and ZEB1, that could repress the EBV Lytic Cycle. We describe a novel example of functional antagonism between HDAC inhibitors.
-
Upregulation of STAT3 Marks Burkitt Lymphoma Cells Refractory to Epstein-Barr Virus Lytic Cycle Induction by HDAC Inhibitors
Journal of virology, 2009Co-Authors: Derek Daigle, Lyn Gradoville, George Miller, Ayman El-guindy, Cynthia M. Megyola, David Tuck, Sumita Bhaduri-mcintoshAbstract: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.
-
histone hyperacetylation occurs on promoters of Lytic Cycle regulatory genes in epstein barr virus infected cell lines which are refractory to disruption of latency by histone deacetylase inhibitors
Journal of Virology, 2008Co-Authors: J Countryman, Lyn Gradoville, George MillerAbstract:Activation of the Epstein-Barr virus (EBV) Lytic Cycle is mediated through the combined actions of ZEBRA and Rta, the products of the viral BZLF1 and BRLF1 genes. During latency, these two genes are tightly repressed. Histone deacetylase inhibitors (HDACi) can activate viral Lytic gene expression. Therefore, a widely held hypothesis is that Zp and Rp, the promoters for BZLF1 and BRLF1, are repressed by chromatin and that hyperacetylation of histone tails, by allowing the access of positively acting factors, leads to transcription of BZLF1 and BRLF1. To investigate this hypothesis, we used chromatin immunoprecipitation (ChIP) to examine the acetylation and phosphorylation states of histones H3 and H4 on Zp and Rp in three cell lines, Raji, B95-8, and HH514-16, which differ in their response to EBV Lytic induction by HDACi. We studied the effects of three HDACi, sodium butyrate (NaB), trichostatin A (TSA), and valproic acid (VPA). We also examined the effects of tetradecanoyl phorbol acetate (TPA) and 5-aza-2'-deoxycytidine, a DNA methyltransferase inhibitor, on histone modification. In Raji cells, TPA and NaB act synergistically to activate the EBV Lytic Cycle and promote an increase in histone H3 and H4 acetylation and phosphorylation at Zp and Rp. Surprisingly, however, when Raji cells were treated with NaB or TSA, neither of which is sufficient to activate the Lytic Cycle, an increase of comparable magnitude of hyperacetylated and phosphorylated histone H3 at Zp and Rp was observed. In B95-8 cells, NaB inhibited Lytic induction by TPA, yet NaB promoted hyperacetylation of H3 and H4. In HH514-16 cells, NaB and TSA strongly activated the EBV Lytic Cycle and caused hyperacetylation of histone H3 on Zp and Rp. However, when HH514-16 cells were treated with VPA, Lytic Cycle mRNAs or proteins were not induced, although histone H3 was hyperacetylated as measured by immunoblotting or by ChIP on Zp and Rp. Taken together, our data suggest that open chromatin at EBV BZLF1 and BRLF1 promoters is not sufficient to activate EBV Lytic Cycle gene expression.
-
De Novo Protein Synthesis Is Required for Lytic Cycle Reactivation of Epstein-Barr Virus, but Not Kaposi's Sarcoma-Associated Herpesvirus, in Response to Histone Deacetylase Inhibitors and Protein Kinase C Agonists
Journal of virology, 2007Co-Authors: Lyn Gradoville, Derek Daigle, George MillerAbstract:The oncogenic human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), are latent in cultured lymphoma cells. We asked whether reactivation from latency of either virus requires de novo protein synthesis. Using Northern blotting and quantitative reverse transcriptase PCR, we measured the kinetics of expression of the Lytic Cycle activator genes and determined whether abundance of mRNAs encoding these genes from either virus was reduced by treatment with cycloheximide (CHX), an inhibitor of protein synthesis. CHX blocked expression of mRNAs of EBV BZLF1 and BRLF1, the two EBV Lytic Cycle activator genes, when HH514-16 Burkitt lymphoma cells were treated with histone deacetylase (HDAC) inhibitors, sodium butyrate or trichostatin A, or a DNA methyltransferase inhibitor, 5-Aza-2′-deoxycytidine. CHX also inhibited EBV Lytic Cycle activation in B95-8 marmoset lymphoblastoid cells by phorbol ester phorbol-12-myristate-13-acetate (TPA). EBV Lytic Cycle induction became resistant to CHX between 4 and 6 h after application of the inducing stimulus. KSHV Lytic Cycle activation, as assessed by ORF50 mRNA expression, was rapidly induced by the HDAC inhibitors, sodium butyrate and trichostatin A, in HH-B2 primary effusion lymphoma cells. In HH-B2 cells, CHX did not inhibit, but enhanced, expression of the KSHV Lytic Cycle activator gene, ORF50. In BC-1, a primary effusion lymphoma cell line that is dually infected with EBV and KSHV, CHX blocked EBV BRLF1 Lytic gene expression induced by TPA and sodium butyrate; KSHV ORF50 mRNA induced simultaneously in the same cells by the same inducing stimuli was resistant to CHX. The experiments show, for the cell lines and inducing agents studied, that the EBV BZLF1 and BRLF1 genes do not behave with “immediate-early” kinetics upon reactivation from latency. KSHV ORF50 is a true “immediate-early” gene. Our results indicate that the mechanism by which HDAC inhibitors and TPA induce Lytic Cycle gene expression of the two viruses differs and suggest that EBV but not KSHV requires one or more proteins to be newly synthesized between 4 and 6 h after application of an inducing stimulus.
Lyn Gradoville - One of the best experts on this subject based on the ideXlab platform.
-
Valproic Acid Antagonizes the Capacity of Other Histone Deacetylase Inhibitors To Activate the Epstein-Barr Virus Lytic Cycle
Journal of virology, 2011Co-Authors: Derek Daigle, Lyn Gradoville, Kelly L. Gorres, David Tuck, Vince Schulz, Ruth Wang'ondu, George MillerAbstract:Diverse stimuli reactivate the Epstein-Barr virus (EBV) Lytic Cycle in Burkitt lymphoma (BL) cells. In HH514-16 BL cells, two histone deacetylase (HDAC) inhibitors, sodium butyrate (NaB) and trichostatin A (TSA), and the DNA methyltransferase inhibitor azacytidine (AzaCdR) promote Lytic reactivation. Valproic acid (VPA), which, like NaB, belongs to the short-chain fatty acid class of HDAC inhibitors, fails to induce the EBV Lytic Cycle in these cells. Nonetheless, VPA behaves as an HDAC inhibitor; it causes hyperacetylation of histone H3 (J. K. Countryman, L. Gradoville, and G. Miller, J. Virol. 82:4706-4719, 2008). Here we show that VPA blocked the induction of EBV early Lytic proteins ZEBRA and EA-D in response to NaB, TSA, or AzaCdR. The block in Lytic activation occurred prior to the accumulation of BZLF1 transcripts. Reactivation of EBV in Akata cells, in response to anti-IgG, and in Raji cells, in response to tetradecanoyl phorbol acetate (TPA), was also inhibited by VPA. MS-275 and apicidin, representing two additional classes of HDAC inhibitors, and suberoylanilide hydroxamic acid (SAHA) reactivated EBV in HH514-16 cells; this activity was also inhibited by VPA. Although VPA potently blocked the expression of viral Lytic-Cycle transcripts, it did not generally block the transcription of cellular genes and was not toxic. The levels and kinetics of specific cellular transcripts, such as Stat3, Frmd6, Mad1, Sepp1, c-fos, c-jun, and egr1, which were activated by NaB and TSA, were similar in HH514-16 cells treated with VPA. When combined with NaB or TSA, VPA did not inhibit the activation of these cellular genes. Changes in cellular gene expression in response to VPA, NaB, or TSA were globally similar as assessed by human genome arrays; however, VPA selectively stimulated the expression of some cellular genes, such as MEF2D, YY1, and ZEB1, that could repress the EBV Lytic Cycle. We describe a novel example of functional antagonism between HDAC inhibitors.
-
Upregulation of STAT3 Marks Burkitt Lymphoma Cells Refractory to Epstein-Barr Virus Lytic Cycle Induction by HDAC Inhibitors
Journal of virology, 2009Co-Authors: Derek Daigle, Lyn Gradoville, George Miller, Ayman El-guindy, Cynthia M. Megyola, David Tuck, Sumita Bhaduri-mcintoshAbstract: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.
-
histone hyperacetylation occurs on promoters of Lytic Cycle regulatory genes in epstein barr virus infected cell lines which are refractory to disruption of latency by histone deacetylase inhibitors
Journal of Virology, 2008Co-Authors: J Countryman, Lyn Gradoville, George MillerAbstract:Activation of the Epstein-Barr virus (EBV) Lytic Cycle is mediated through the combined actions of ZEBRA and Rta, the products of the viral BZLF1 and BRLF1 genes. During latency, these two genes are tightly repressed. Histone deacetylase inhibitors (HDACi) can activate viral Lytic gene expression. Therefore, a widely held hypothesis is that Zp and Rp, the promoters for BZLF1 and BRLF1, are repressed by chromatin and that hyperacetylation of histone tails, by allowing the access of positively acting factors, leads to transcription of BZLF1 and BRLF1. To investigate this hypothesis, we used chromatin immunoprecipitation (ChIP) to examine the acetylation and phosphorylation states of histones H3 and H4 on Zp and Rp in three cell lines, Raji, B95-8, and HH514-16, which differ in their response to EBV Lytic induction by HDACi. We studied the effects of three HDACi, sodium butyrate (NaB), trichostatin A (TSA), and valproic acid (VPA). We also examined the effects of tetradecanoyl phorbol acetate (TPA) and 5-aza-2'-deoxycytidine, a DNA methyltransferase inhibitor, on histone modification. In Raji cells, TPA and NaB act synergistically to activate the EBV Lytic Cycle and promote an increase in histone H3 and H4 acetylation and phosphorylation at Zp and Rp. Surprisingly, however, when Raji cells were treated with NaB or TSA, neither of which is sufficient to activate the Lytic Cycle, an increase of comparable magnitude of hyperacetylated and phosphorylated histone H3 at Zp and Rp was observed. In B95-8 cells, NaB inhibited Lytic induction by TPA, yet NaB promoted hyperacetylation of H3 and H4. In HH514-16 cells, NaB and TSA strongly activated the EBV Lytic Cycle and caused hyperacetylation of histone H3 on Zp and Rp. However, when HH514-16 cells were treated with VPA, Lytic Cycle mRNAs or proteins were not induced, although histone H3 was hyperacetylated as measured by immunoblotting or by ChIP on Zp and Rp. Taken together, our data suggest that open chromatin at EBV BZLF1 and BRLF1 promoters is not sufficient to activate EBV Lytic Cycle gene expression.
-
De Novo Protein Synthesis Is Required for Lytic Cycle Reactivation of Epstein-Barr Virus, but Not Kaposi's Sarcoma-Associated Herpesvirus, in Response to Histone Deacetylase Inhibitors and Protein Kinase C Agonists
Journal of virology, 2007Co-Authors: Lyn Gradoville, Derek Daigle, George MillerAbstract:The oncogenic human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), are latent in cultured lymphoma cells. We asked whether reactivation from latency of either virus requires de novo protein synthesis. Using Northern blotting and quantitative reverse transcriptase PCR, we measured the kinetics of expression of the Lytic Cycle activator genes and determined whether abundance of mRNAs encoding these genes from either virus was reduced by treatment with cycloheximide (CHX), an inhibitor of protein synthesis. CHX blocked expression of mRNAs of EBV BZLF1 and BRLF1, the two EBV Lytic Cycle activator genes, when HH514-16 Burkitt lymphoma cells were treated with histone deacetylase (HDAC) inhibitors, sodium butyrate or trichostatin A, or a DNA methyltransferase inhibitor, 5-Aza-2′-deoxycytidine. CHX also inhibited EBV Lytic Cycle activation in B95-8 marmoset lymphoblastoid cells by phorbol ester phorbol-12-myristate-13-acetate (TPA). EBV Lytic Cycle induction became resistant to CHX between 4 and 6 h after application of the inducing stimulus. KSHV Lytic Cycle activation, as assessed by ORF50 mRNA expression, was rapidly induced by the HDAC inhibitors, sodium butyrate and trichostatin A, in HH-B2 primary effusion lymphoma cells. In HH-B2 cells, CHX did not inhibit, but enhanced, expression of the KSHV Lytic Cycle activator gene, ORF50. In BC-1, a primary effusion lymphoma cell line that is dually infected with EBV and KSHV, CHX blocked EBV BRLF1 Lytic gene expression induced by TPA and sodium butyrate; KSHV ORF50 mRNA induced simultaneously in the same cells by the same inducing stimuli was resistant to CHX. The experiments show, for the cell lines and inducing agents studied, that the EBV BZLF1 and BRLF1 genes do not behave with “immediate-early” kinetics upon reactivation from latency. KSHV ORF50 is a true “immediate-early” gene. Our results indicate that the mechanism by which HDAC inhibitors and TPA induce Lytic Cycle gene expression of the two viruses differs and suggest that EBV but not KSHV requires one or more proteins to be newly synthesized between 4 and 6 h after application of an inducing stimulus.
-
Lytic Cycle switches of oncogenic human gammaherpesviruses.
Advances in cancer research, 2007Co-Authors: George Miller, Ayman El-guindy, J Countryman, Lyn GradovilleAbstract:The seminal experiments of George and Eva Klein helped to define the two life Cycles of Epstein-Barr Virus (EBV), namely latency and Lytic or productive infection. Their laboratories described latent nuclear antigens expressed during latency and discovered several chemicals that activated the viral Lytic Cycle. The mechanism of the switch between latency and the Lytic Cycle of EBV and Kaposi's sarcoma-associated herpesvirus (KSHV) can be studied in cultured B cell lines. Lytic Cycle activation of EBV is controlled by two viral transcription factors, ZEBRA and Rta. The homologue of Rta encoded in ORF50 is the Lytic Cycle activator of KSHV. Control of the Lytic Cycle can be divided into two distinct phases. Upstream events control expression of the virally encoded Lytic Cycle activator genes. Downstream events represent tasks carried out by the viral proteins in driving expression of Lytic Cycle genes and Lytic viral DNA replication. In this chapter, we report three recent groups of experiments relating to upstream and downstream events. Azacytidine (AzaC) is a DNA methyltransferase inhibitor whose Lytic Cycle activation capacity was discovered by G. Klein and coworkers. We find that AzaC rapidly activates the EBV Lytic Cycle but does not detectably alter DNA methylation or histone acetylation on the promoters of the EBV Lytic Cycle activator genes. AzaC probably acts via a novel, yet to be elucidated, mechanism. The Lytic Cycle of both EBV and KSHV can be activated by sodium butyrate (NaB), a histone deacetylase inhibitor whose activity in disrupting latency was also discovered by G. Klein and coworkers. Activation of EBV by NaB requires protein synthesis; activation of KSHV is independent of protein synthesis. Thus, NaB works by a different pathway on the two closely related viruses. ZEBRA, the major downstream mediator of EBV Lytic Cycle activation is both a transcription activator and an essential replication protein. We show that phosphorylation of ZEBRA at its casein kinase 2 (CK2) site separates these two functions. Phosphorylation by CK2 is required for ZEBRA to activate Lytic replication but not to induce expression of early Lytic Cycle genes. We discuss a number of unsolved mysteries about Lytic Cycle activation which should provide fertile territory for future research.
Alan K. S. Chiang - One of the best experts on this subject based on the ideXlab platform.
-
intracellular iron chelation by a novel compound c7 reactivates epstein barr virus ebv Lytic Cycle via the erk autophagy axis in ebv positive epithelial cancers
Cancers, 2018Co-Authors: Stephanie Pei Tung Yiu, Kwai Fung Hui, Chung King Choi, Richard Y. T. Kao, Chi Wang, Dan Yang, Alan K. S. ChiangAbstract:Pharmaceutical reactivation of Lytic Cycle of Epstein⁻Barr virus (EBV) represents a potential therapeutic strategy against EBV-associated epithelial malignancies, e.g., gastric carcinoma (GC) and nasopharyngeal carcinoma (NPC). A novel Lytic-inducing compound, C7, which exhibits structural similarity to Di-2-Pyridyl Ketone 4, 4-Dimethyl-3-Thiosemicarbazone (Dp44mT), a known chelator of intracellular iron, is found to reactivate EBV Lytic Cycle in GC and NPC. This study aims to investigate the role of intracellular iron chelation by C7 and other iron chelators in Lytic reactivation of EBV in GC and NPC. Testing of six structural analogs of C7 revealed only those which have high affinity towards transition metals could induce EBV Lytic Cycle. Precomplexing C7 and iron chelators to iron prior to treatment of the cells abolished EBV Lytic reactivation. Though hypoxia signaling pathway was activated, it was not the only pathway associated with EBV reactivation. Specifically, C7 and iron chelators initiated autophagy by activating extracellular signal-regulated kinase (ERK1/2) to reactivate EBV Lytic Cycle since autophagy and EBV Lytic reactivation were abolished in cells treated with ERK1/2 blockers whilst inhibition of autophagy by 3-Methyladenine (3-MA) and atg5 knockdown significantly abolished EBV Lytic reactivation. In summary, we discovered a novel mechanism of reactivation of the EBV Lytic Cycle through intracellular iron chelation and induction of ERK-autophagy axis in EBV-positive epithelial malignancies, raising the question whether clinically available iron chelators can be incorporated into existing therapeutic regimens to treat these cancers.
-
Identification of Novel Small Organic Compounds with Diverse Structures for the Induction of Epstein-Barr Virus (EBV) Lytic Cycle in EBV-Positive Epithelial Malignancies
PloS one, 2015Co-Authors: Chung King Choi, Kwai Fung Hui, Richard Y. T. Kao, Alan K. S. ChiangAbstract:Phorbol esters, which are protein kinase C (PKC) activators, and histone deacetylase (HDAC) inhibitors, which cause enhanced acetylation of cellular proteins, are the main classes of chemical inducers of Epstein-Barr virus (EBV) Lytic Cycle in latently EBV-infected cells acting through the PKC pathway. Chemical inducers which induce EBV Lytic Cycle through alternative cellular pathways may aid in defining the mechanisms leading to Lytic Cycle reactivation and improve cells’ responsiveness towards Lytic induction. We performed a phenotypic screening on a chemical library of 50,240 novel small organic compounds to identify novel class(es) of strong inducer(s) of EBV Lytic Cycle in gastric carcinoma (GC) and nasopharyngeal carcinoma (NPC) cells. Five hit compounds were selected after three successive rounds of increasingly stringent screening. All five compounds are structurally diverse from each other and distinct from phorbol esters or HDAC inhibitors. They neither cause hyperacetylation of histone proteins nor significant PKC activation at their working concentrations, suggesting that their biological mode of action are distinct from that of the known chemical inducers. Two of the five compounds with rapid Lytic-inducing action were further studied for their mechanisms of induction of EBV Lytic Cycle. Unlike HDAC inhibitors, Lytic induction by both compounds was not inhibited by rottlerin, a specific inhibitor of PKCδ. Interestingly, both compounds could cooperate with HDAC inhibitors to enhance EBV Lytic Cycle induction in EBV-positive epithelial cancer cells, paving way for the development of strategies to increase cells’ responsiveness towards Lytic reactivation. One of the two compounds bears structural resemblance to iron chelators and the other strongly activates the MAPK pathways. These structurally diverse novel organic compounds may represent potential new classes of chemicals that can be used to investigate any alternative mechanism(s) leading to EBV Lytic Cycle reactivation from latency.
-
Abstract 4715: Activation of Lytic Cycle of Epstein-Barr virus by suberoylanilide hydroxamic acid affects apoptosis and tumor growth suppression of nasopharyngeal carcinoma
Experimental and Molecular Therapeutics, 2012Co-Authors: Alan K. S. Chiang, Kwai Fung HuiAbstract: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 SAHA9s 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.
International journal of cancer, 2012Co-Authors: Kwai Fung Hui, Jaap M Middeldorp, Chi Man Tsang, George S.w. Tsao, Alan K. S. ChiangAbstract: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.
-
Suberoylanilide hydroxamic acid induces viral Lytic Cycle in Epstein-Barr virus-positive epithelial malignancies and mediates enhanced cell death†
International journal of cancer, 2010Co-Authors: Kwai Fung Hui, Alan K. S. ChiangAbstract: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.
Mara Cirone - One of the best experts on this subject based on the ideXlab platform.
-
stat3 phosphorylation affects p53 p21 axis and kshv Lytic Cycle activation
Virology, 2019Co-Authors: Roberta Santarelli, Valentina Carillo, Maria Anele Romeo, Aurelia Gaeta, Cristina Nazzari, Roberta Gonnella, Marisa Granato, Alberto Faggioni, Gabriella Dorazi, Mara CironeAbstract:Abstract The Tyr705 STAT3 constitutive activation, besides promoting PEL cell survival, contributes to the maintenance of viral latency. We found indeed that its de-phosphorylation by AG490 induced KSHV Lytic Cycle. Moreover, Tyr705 STAT3 de-phosphorylation, mediated by the activation of tyrosine phosphatases, together with the increase of Ser727 STAT3 phosphorylation contributed to KSHV Lytic Cycle induction by TPA. We then observed that p53-p21 axis, essential for the induction of KSHV replication, was activated by the inhibition of Tyr705 and by the increase of Ser727 STAT3 phosphorylation. As a possible link between STAT3, p53-p21 and KSHV Lytic Cycle, we found that TPA and AG490 reduced the expression of KAP-1, promoting p53 stability, p21 transcription and KSHV Lytic Cycle activation in PEL cells.
-
Cytotoxic Drugs Activate KSHV Lytic Cycle in Latently Infected PEL Cells by Inducing a Moderate ROS Increase Controlled by HSF1, NRF2 and p62/SQSTM1
MDPI AG, 2018Co-Authors: Marisa Granato, Alberto Faggioni, Maria Saveria Gilardini Montani, Camilla Angiolillo, Gabriella D’orazi, Mara CironeAbstract:Previous studies have indicated that cytotoxic treatments may induce or not activate viral Lytic Cycle activation in cancer cells latently infected by Kaposi’s sarcoma-associated herpesvirus (KSHV). To investigate the molecular mechanisms responsible for such an effect, we compared two cytotoxic treatments able to induce the viral Lytic Cycle, named 12-O-tetradecanoylphorbol 13-acetate (TPA) (T) in combination with sodium butyrate (B) and bortezomib (BZ), with two cytotoxic treatments that did not activate this process, named metformin (MET) and quercetin (Q). Our results indicated that TB and bortezomib increased levels of oxygen reactive species (ROS) while metformin and quercetin reduced them. The finding that N-acetylcysteine (NAC), a reactive oxigen species (ROS) scavenger, counteracted K-bZIP expression induced by TB or bortezomib, confirmed that an ROS increase played a role in KSHV Lytic Cycle activation. Moreover, we found that TB and bortezomib up-regulated p62/Sequestosome1(p62/SQSTM1) protein, while metformin and quercetin down-regulated it. p62/SQSTM1 silencing or the inhibition of NF-E2-related factor 2 (NRF2) or Heat Shock Factor 1 (HSF1), that mediate p62/SQSTM1 transcription, also reduced KSHV Lytic antigen expression induced by TB or bortezomib. Interestingly, such combination treatments further increased intracellular ROS and cytotoxicity induced by the single TB or bortezomib treatment, suggesting that NRF2, HSF1 and p62/SQSTM1 keep the ROS level under control, allowing primary effusion lymphoma (PEL) cells to continue to survive and KSHV to replicate
-
oxidant species are involved in t b mediated erk1 2 phosphorylation that activates p53 p21 axis to promote kshv Lytic Cycle in pel cells
Free Radical Biology and Medicine, 2017Co-Authors: Roberta Gonnella, Roberta Santarelli, Marisa Granato, Alberto Faggioni, Gabriella Dorazi, Maria Saveria Gilardini Montani, Shivangi Yadav, Alessia Garufi, Mara CironeAbstract:Abstract KSHV is a gammaherpesvirus strongly associated to human cancers such as Primary Effusion Lymphoma (PEL) and Kaposi's Sarcoma. The naturally virus-infected tumor cells usually display latent infection since a minority of cells undergoes spontaneous viral replication. The Lytic Cycle can be induced in vitro upon appropriate stimuli such as TPA (T), alone or in combination with butyrate (B), (T/B). In previous studies, Protein Kinase C (PKC) δ, Extracellular Signal-regulated Kinase1/2 (ERK1/2) and p53-p21 axis have been separately reported to play a role in KSHV reactivation from latency. Here, we found that these pathways were interconnected to induce KSHV Lytic Cycle in PEL cells treated with T/B. T/B also increased H2O2 that played an important role in the activation of these pathways. Oxidant specie production correlated with PKC δ activation, as the PKC δ inhibitor rottlerin reduced both H2O2 and KSHV Lytic antigen expression. H2O2 contributed to T/B-mediated ERK1/2 activation that mediated p53 phosphorylation at serine 15 (Ser15) and increased p21 expression. Oxidant specie inhibition by quercetin indeed strongly reduced the activation of these pathways, Lytic antigen expression and interestingly it also increased T/B-induced cell death. The use of ERK inhibitor PD98059 or p53 silencing demonstrated the importance of p53Ser15 phosphorylation and of p53-p21 axis in KSHV Lytic Cycle activation. Understanding the role of oxidant species and the molecular mechanisms involved in KSHV Lytic Cycle induction is particularly important since oxidant species represent the most physiological stimulus for viral reactivation in vivo and it is known that viral production contributes to the maintenance/progression of KSHV associated malignancies.
-
Bortezomib promotes KHSV and EBV Lytic Cycle by activating JNK and autophagy.
Scientific reports, 2017Co-Authors: Marisa Granato, Roberta Santarelli, Maria Anele Romeo, Roberta Gonnella, Alberto Faggioni, Mariangela Sara Tiano, Maria Saveria Gilardini Montani, Mara CironeAbstract:KSHV and EBV are gammaherpesviruses strictly linked to human cancers. Even if the majority of cancer cells harbor a latent infection, the few cells that undergo viral replication may contribute to the pathogenesis and maintenance of the virus-associated malignancies. Cytotoxic drugs used for the therapies of cancers harboring virus-infection often have, as side effect, the activation of viral Lytic Cycle. Therefore it is important to investigate whether they affect viral reactivation and understand the underlying mechanisms involved. In this study, we found that proteasome inhibitor bortezomib, a cytotoxic drug that efficiently target gammaherpesvirus-associated B cell lymphomas, triggered KSHV or EBV viral Lytic Cycle by activating JNK, in the course of ER stress, and inducing autophagy. These results suggest that the manipulation of these pathways could limit viral spread and improve the outcome of bortezomib treatment in patients affected by gammaherpesvirus-associated lymphomas.
-
The activation of KSHV Lytic Cycle blocks autophagy in PEL cells
Autophagy, 2015Co-Authors: Marisa Granato, Roberta Santarelli, Roberta Gonnella, Alberto Faggioni, Antonella Farina, Mariarosaria Filardi, Maria Rosaria Torrisi, Mara CironeAbstract:This study confirms that autophagy is activated concomitantly with KSHV Lytic Cycle induction, and that autophagy inhibition by BECN1 knockdown reduces viral Lytic gene expression. In addition, we extend previous observations and show that autophagy is blocked at late steps, during viral replication. This is indicated by the lack of colocalization of autophagosomes and lysosomes and by the LC3-II level that does not increase in the presence of bafilomycin A1 in primary effusion lymphoma (PEL) cells induced to enter the Lytic Cycle, either by TPA/sodium butyrate (BC3 and BCBL1) or by doxycycline (TRExBCBL1-Rta). The autophagic block correlates with the downregulation of RAB7, whose silencing with specific siRNA results in an autophagic block in the same cells. Finally, by electron microscopy analysis, we observed viral particles inside autophagic vesicles in the cytoplasm of PEL cells undergoing viral replication, suggesting that they may be involved in viral transport.
Paul Kellam - One of the best experts on this subject based on the ideXlab platform.
-
x box binding protein 1 induces the expression of the Lytic Cycle transactivator of kaposi s sarcoma associated herpesvirus but not epstein barr virus in co infected primary effusion lymphoma
Journal of General Virology, 2011Co-Authors: Paul J Farrell, Paul KellamAbstract:Cells of primary effusion lymphoma (PEL), a B-cell non-Hodgkin's lymphoma, are latently infected by Kaposi's sarcoma-associated herpesvirus (KSHV), with about 80 % of PEL also co-infected with Epstein–Barr virus (EBV). Both viruses can be reactivated into their Lytic replication Cycle in PEL by chemical inducers. However, simultaneous activation of both Lytic cascades leads to mutual Lytic Cycle co-repression. The plasma cell-differentiation factor X-box binding protein 1 (XBP-1) transactivates the KSHV immediate–early promoter leading to the production of the replication and transcription activator protein (RTA), and reactivation of KSHV from latency. XBP-1 has been reported to act similarly on the EBV immediate–early promoter Zp, leading to the production of the Lytic-Cycle transactivator protein BZLF1. Here we show that activated B-cell terminal-differentiation transcription factor X-box binding protein 1 (XBP-1s) does not induce EBV BZLF1 and BRLF1 expression in PEL and BL cell lines, despite inducing Lytic reactivation of KSHV in PEL. We show that XBP-1s transactivates the KSHV RTA promoter but does not transactivate the EBV BZLF1 promoter in non-B-cells by using a luciferase assay. Co-expression of activated protein kinase D, which can phosphorylate and inactivate class II histone deacetylases (HDACs), does not rescue XBP-1 activity on Zp nor does it induce BZLF1 and BRLF1 expression in PEL. Finally, chemical inducers of KSHV and EBV Lytic replication in PEL, including HDAC inhibitors, do not lead to XBP-1 activation. We conclude that XBP-1 specifically reactivates the KSHV Lytic Cycle in dually infected PELs.
-
x box binding protein 1 contributes to induction of the kaposi s sarcoma associated herpesvirus Lytic Cycle under hypoxic conditions
Journal of Virology, 2009Co-Authors: Lucy Daltongriffin, Sam J Wilson, Paul KellamAbstract: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 1α 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.
