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

Abstract

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. Epstein-Barr virus (EBV) is a gammaherpesvirus that per-sists as a lifelong infection by remaining in the latent phase of its life cycle within B lymphocytes (17). EBV is associated with human cancers such as Burkitt lymphoma, nasopharyngeal carcinoma, Hodgkin's disease, and EBV-associated lymphoproliferative disease in immunocompromised individuals (11). Efforts to eliminate EBV-positive tumor cells by nucleoside analogue antiviral agents following induction of the viral lytic cycle have shown promising results (15,16,18,38,44,52). These efforts have been preceded by extensive studies on the switch from latency to the EBV lytic cycle in lymphoid cell lines. A fundamental problem in studying the latent to lytic switch and the lytic cycle itself is the lack of a culture system fully permissive to lytic cycle induction (45). In cell culture, EBV can be induced into the lytic cycle by a variety of chemical stimuli, including agents currently being used or investigated as chemotherapeutic drugs, such as the HDAC inhibitors trichostatin A (TSA) and sodium butyrate (NaB) and the DNA methyltransferase inhibitor azacytidine (Aza) (3, 57). However, following treatment of cells latently infected with EBV, only a fraction of cells enter the lytic cycle; the remainder of the population is refractory to lyti...

Valproic Acid Antagonizes the Capacity of Other Histone Deacetylase Inhibitors To Activate the Epstein-Barr Virus Lytic Cycle

“…We have studied extensively the effects of the HDAC inhibitors NaB and TSA and the DNA methyltransferase inhibitor AzaCdR on viral reactivation (22,24,26,41,64,102). We use the Burkitt lymphoma (BL) cell line HH514-16 for these studies, since these cells have a very low level of spontaneous reactivation and are readily induced by NaB, TSA, or AzaCdR.…”

“…Individual samples were assayed in triplicate. Primers either were designed using Primer3 (77) or have been described previously (26,101). The sequences of the primers are listed in Table 1.…”

“…Microarray analysis. Affymetrix U133 2.0 Plus arrays were used for all microarray experiments (26). HH514-16 cells were either left untreated or treated with NaB, TSA, or VPA and were harvested after 2, 3, or 6 h. Two separate gene expression microarray experiments were performed.…”

STAT3 mutations are present in aggressive B-cell lymphomas including a subset of diffuse large B-cell lymphomas with CD30 expression

“…These findings are in agreement with the studies of other groups that have noted increased activation of STAT3 in a subset of B-cell lymphomas. [10][11][12][13] Why some cases of DLBCL show protein overexpression of STAT3 but harbor no STAT3 mutation is not entirely clear; however, it is certainly feasible that there are other mechanisms to increase STAT3 activity via upstream protein regulators.…”

Tracking expression and subcellular localization of RNA and protein species using high-throughput single cell imaging flow cytometry

“…Although all cells in a given culture were treated with valproic acid for the same length of time, individual cells within that culture responded differently to the treatment, with some cells remaining refractory to induction and other cells progressing into various stages of the lytic phase (Daigle et al 2010). Thus, within the same population of cells, different subpopulations could be identified as latent, early, or late stage of lytic infection.…”