Short, discontinuous exposure to butyrate effectively sensitizes latently EBV-infected lymphoma cells to nucleoside analogue antiviral agents

Abstract

Antiviral drugs alone have been unsuccessful in the treatment of Epstein Barr virus (EBV)-associated malignancies because the virus maintains a latent state of replication in these tumors. In recent years, novel therapeutic approaches are being investigated wherein lytic replication of the virus is induced prior to the use of cytotoxic antiviral drugs. The choice of suitable agents to induce lytic replication has been a critical step in this novel approach. We have previously demonstrated that butyrate derivatives induce a lytic pattern of EBV gene expression in patient-derived EBV-positive lymphoblastoid cell lines and, together with nucleoside analog ganciclovir, effectively reduce or eliminate tumor growth in humans. Butyrate has drawbacks as a therapeutic agent, however, as constant intravenous infusion is required to achieve detectable plasma levels of this drug. In this study, we investigated whether discontinuous exposure to butyrate is capable of initiating lytic-phase gene expression and thymidine kinase induction, and sensitizing EBV-positive lymphoma cells to ganciclovir-mediated cell growth arrest and apoptosis. We demonstrate that multiple daily 6 hr exposures of the EBV-positive Burkitt's lymphoma cell line P3HR1 to butyrate induced sustained expression of the EBV lytic-phase protein BMRF. Viral thymidine kinase was also induced by intermittent exposure, although to a lower level than with continuous exposure treatment. However, discontinuous exposure to butyrate in combination with ganciclovir induced a similar level of tumor cell growth inhibition as did continuous treatment, as measured by serial enumeration of viable cells, MTT cell proliferation assays, and measurement of cellular DNA content. We further demonstrated that those cells which survived initial exposure to butyrate plus ganciclovir remained susceptible to further cycles of combination treatment. These findings suggests that continuous infusion of butyrate may not be necessary for maintaining viral thymidine kinase gene expression and sensitization to anti-viral agents in EBV-associated tumors, and that therapeutic regimens which employ more convenient, discontinuous exposure to butyrate may also be effective clinically.

Histone deacetylase inhibitors are potent inducers of gene expression in latent EBV and sensitize lymphoma cells to nucleoside antiviral agents

“…Indeed, we have reported previously that shorter durations of exposure to butyrate also efficiently killed EBV ϩ lymphoma cells in the presence of GCV. 33 Based on these data, 1 patient with refractory EBV ϩ lymphoma was treated in a protocol using butyrate for 5 days and GCV or valganciclovir for 21 days, and the lymphoma burden was dramatically reduced within 1 cycle. Furthermore, previously high EBV viral loads, as well as the viral loads of 2 other herpesviruses (CMV and HHV6), became undetectable.…”

“…We have reported previously that exposure of P3HR1 cells to butyrate induces EBV early antigen-diffuse protein (EA-D or BMRF1), a major protein of the EBV lytic replication cycle. 33 We evaluated EA-D protein expression in P3HR1 cells that were exposed to some of the individual HDAC inhibitors used in this study. As shown in Figure 4B, LBH589 strongly induced the EA-D protein, whereas PXD101 did so very weakly and the other HDAC inhibitors did not.…”

“…Relative quantification of gene expression was determined by the comparative threshold method (⌬CT), as described previously. 33 Expression of the ␤-actin mRNA in each individual sample was used to normalize the dataset.…”

Using Epstein-Barr Viral Load Assays To Diagnose, Monitor, and Prevent Posttransplant Lymphoproliferative Disorder

“…First, PTLD often includes scattered cells expressing markers of lytic viral replication among the more predominant latently infected tumor cell population, and animal studies show that such replicative capability enhances tumor growth (87). Second, intriguing data suggest that ganciclovir is rendered a more potent killer of infected cells when combined with replication inducers (58,65,97,147). This is because the EBV lytic enzyme thymidine kinase phosphorylates ganciclovir to launch the process by which activated ganciclovir carries out its cytotoxic effect (57).…”

Epstein-Barr Virus-Encoded miRNAs in Epstein-Barr Virus-Related Malignancy

“…9 During latency, only a few viral genes are transcribed, no viral progeny are produced, and infected cells are protected from apoptotic stimuli and, in some circumstances, driven to proliferate. Based on serology, about 95% of the world's adult population is infected with EBV, and following primary infection, hosts remain lifelong carriers of the virus.…”