Epstein-Barr Virus-Induced Resistance to Drugs That Activate the Mitotic Spindle Assembly Checkpoint in Burkitt's Lymphoma Cells

Abstract

Epstein-Barr virus (EBV)is associated with a number of human cancers, and latent EBV gene expression has been reported to interfere with cell cycle checkpoints and cell death pathways. Here we show that latent EBV can compromise the mitotic spindle assembly checkpoint and rescue Burkitt's lymphoma (BL)-derived cells from caspase-dependent cell death initiated in aberrant mitosis. This leads to unscheduled mitotic progression, resulting in polyploidy and multi-and/or micronucleation. The EBV latent genes responsible for this phenotype are expressed from the P3HR1 strain of virus and several viruses with similar genomic deletions that remove the EBNA2 gene. Although EBNA2 and the latent membrane proteins are not expressed, the EBNA3 proteins are present in these BL cells. Survival of the EBV-positive cells is not consistently associated with EBV lytic gene expression or with the genes that are expressed in EBV latency I BL cells (i.e., EBNA1, EBERs, and BARTs) but correlates with reduced expression of the cellular proapoptotic BH3-only protein Bim. These data suggest that a subset of latent EBV gene products may increase the likelihood of damaged DNA being inherited because of the impaired checkpoint and enhanced survival capacity. This could lead to greater genetic diversity in progeny cells and contribute to tumorigenesis. Furthermore, since it appears that this restricted latent EBV expression interferes with the responses of Burkitt's lymphoma-derived cells to cytotoxic drugs, the results of this study may have important therapeutic implications in the treatment of some BL. Epstein-Barr virus (EBV) is a gammaherpesvirus that pro-duces an asymptomatic infection in the majority of the human population. However, EBV is also associated with a number of human tumors of B-cell, T-cell, and epithelial origin. These include Burkitt's lymphoma (BL), some types of Hodgkin's lymphoma, immunoblastic B lymphoma in the immunosuppressed, nasopharyngeal carcinoma, and gastric carcinoma. Although the precise contribution EBV makes to the development of these diseases is not yet known, it has been suggested that interference with cell cycle checkpoints and cell death pathways by EBV may play an important role in B lymphomagenesis (reviewed in reference 20).In vitro, EBV has the ability to infect and transform primary B cells into continuously proliferating lymphoblastoid cell lines (LCLs) that have a pattern of viral gene expression known as latency III. This is characterized by the expression of nine viral proteins: six Epstein-Barr nuclear antigens (EBNAs) (EBNA1, -2, -3A, -3B, -3C, and -LP) and three latent membrane proteins (LMPs) (LMP1, -2A, and -2B). Additional RNA species (the EBV-encoded RNAs [EBERs] and the BamHIA rightward transcripts [BARTs]) are also expressed during latency III, but their significance is not fully understood (3). It has been reported from studies with recombinant EBV that only six of the latency III proteins (EBNA1, -2, -3A, -3C, -LP, and -LMP1) are essential for efficient transformation of B cells...

Two Epstein–Barr virus (EBV) oncoproteins cooperate to repress expression of the proapoptotic tumour-suppressor Bim: clues to the pathogenesis of Burkitt's lymphoma

“…(d) Protein extracts from BL31 cells and BL31 cells infected with KO and revertant viruses were also subjected to western-blot analysis probing for expression of the anti-apoptotic factors (and targets of LMP-1), A20 and Bcl-2; g-tubulin was used as a loading control. EBNA3A and EBNA3C are both required for repression of Bim expression It was shown previously that latent infection of BL cells with EBV results in a significant reduction in the expression of the proapoptotic Bcl-2-family member, Bim (Clybouw et al, 2005;Leao et al, 2007). Moreover, this downregulation appears not to require expression of EBNA2 or the LMP proteins (Leao et al, 2007).…”

“…Analysis of EBV latent gene expression patterns in various EBV-positive BL-derived cell lines suggested that the EBNA3 family might play an important role in this survival phenotype. Correlation between survival and EBV-mediated repression of the BH3-only, Bcl-2-family member Bim (Bcl-2-interacting mediator of cell death) was also observed (Leao et al, 2007).…”

“…EBNA3A and EBNA3C cooperate to rescue BL cells from nocodazole-induced death When multiple cell lines established with each of the recombinant viruses had been validated by western blotting, cell samples were treated with nocodazole for 72 h. We showed previously that after this length of time all BL31 cells were dead as a result of caspase-dependent programmed cell death (Leao et al, 2007). Here, counting viable cells confirmed this and also showed that the B95.8 wild-type virus protects up to 70% of the BL31 cells from death (Figures 2a and b).…”

“…Recently, we and others discovered that in BL cells latent EBV provides significant protection from programmed cell death induced by a variety of cytotoxic agents (Kelly et al, 2005(Kelly et al, , 2006Leao et al, 2007). Analysis of EBV latent gene expression patterns in various EBV-positive BL-derived cell lines suggested that the EBNA3 family might play an important role in this survival phenotype.…”

Three Epstein–Barr virus latency proteins independently promote genomic instability by inducing DNA damage, inhibiting DNA repair and inactivating cell cycle checkpoints

“…The checkpoint-mediating proteins, Bub1, Cenp-F, BubR1, Bub3 and Mad2, are recruited to the kinetochore and stabilize the microtubule-kinetochore complex. It was shown earlier that EBV compromises the spindle assembly checkpoint and rescues BL cells from caspase-dependent cell death initiated by aberrant mitosis (Leao et al, 2007) but the mechanism was not explored. We have found that disruption of the mitotic checkpoint in EBNA-3C-expressing cells (Figure 5a) is associated with a dramatic decrease in BubR1 protein levels ( Figure 5b) and its transcriptional downregulation (Figure 5c).…”