We have demonstrated that EBV can directly contribute to the tumorigenic potential of BL in the context of a type I latency program. With the possible exception of EBNA-1 (see below), EBV genes expressed during type I latency have not been directly linked to either B-cell immortalization or oncogenic transformation associated with EBV infection (24
). In fact, the EBER and RK-BARF0 genes are dispensable for EBV-induced immortalization of B lymphocytes in vitro (44
). B-cell immortalization by EBV, however, occurs in the context of a type III latency (expression of all 12 EBV latency-associated genes), and thus one cannot exclude a possible role for EBNA-1, RK-BARF0, or the EBERs in growth-related functions of the type I latency program. The reservoir of EBV in healthy infected individuals consists of B lymphocytes that, like BL cells, preclude the expression of known growth-promoting EBV proteins (7
). Thus, one or more of the EBV genes expressed during the type I latency program may perform a critical role in modulating B-cell growth or survival in vivo.
The concept that the type I latency program of EBV promotes BL cell growth and survival is strongly supported by data presented here demonstrating that (i) EBV-positive Akata BL cells are more resistant to apoptosis than are EBV-negative Akata cells under growth-limiting conditions; (ii) under conditions that favor increased sensitivity to apoptotic stimuli, there is an EBV-dependent reduction in c-MYC, an oncoprotein known to augment the apoptotic program in BL (35
) and other cell lineages (3
); (iii) EBV-positive Akata BL cells generally, but not always, express higher levels of the antiapoptotic protein Bcl-2 than do their EBV-negative counterparts throughout all stages of the cell growth cycle; and (iv) tumorigenicity in Akata BL cells is strictly dependent on a latent EBV infection characteristic of BL tumors. Based on these observations, we propose that EBV contributes to tumorigenicity in BL by inhibiting c-MYC-induced apoptosis through at least two mechanisms: a modest up-regulation of Bcl-2 expression and, most importantly, a concomitant decrease in c-MYC expression, apparently at the level of translation, under growth-limiting conditions.
Although earlier studies failed to detect Bcl-2 expression in BL cells that maintain type I latency (21
), our results clearly indicate that Bcl-2 is expressed in Akata and other group I BL cell lines (Fig. and data not shown). However, the level of Bcl-2 expression needed to effectively inhibit apoptosis in group I BL cells equals or exceeds the much higher levels of Bcl-2 observed in LCLs and BL cells that maintain type III latency (34
). Because EBV-negative Akata cells express only slightly lower levels of Bcl-2 than their EBV-positive counterparts, Bcl-2 levels characteristic of EBV-positive BL cells that maintain type I latency seem unlikely to be sufficient alone to fully restore tumorigenicity to EBV-negative Akata BL cells.
The striking finding that EBV targets the down-regulation of c-MYC protein under growth-limiting conditions in BL suggests that this is the principal mechanism by which EBV promotes cell survival, particularly given that c-MYC is the primary mediator of apoptosis in BL (35
) and that Akata BL cells are null for p53 (15
). To determine if down-regulation of c-MYC is indeed responsible for the increased survival of BL cells, we have attempted to repress expression of c-MYC in EBV-negative Akata cells during stationary phase, using antisense c-myc
oligonucleotides that have been successfully used in BL (35
). Unfortunately, we were unable to demonstrate a repression of c-MYC by such means and therefore have been unable to directly test whether repression of c-MYC is sufficient to promote group I BL cell survival. Interestingly, our observations regarding BL cells contrast those for EBV-transformed lymphoblastoid cells, in which expression of c-MYC protein is sustained under growth-restrictive conditions (8
), most likely through an EBV-dependent stabilization of c-myc
mRNA that occurs during type III latency (26
). Although these opposing effects of EBV on c-MYC
expression likely result from the dominant influence of the respective EBV latency program maintained by a cell, we cannot exclude the possibility that the EBV-dependent decrease in c-MYC protein observed here is unique to BL. Presumably, the documented ability of cells that maintain type III latency to substantially up-regulate the antiapoptotic proteins Bcl-2 and A20 would protect such cells having sustained c-MYC
Although expression of the EBV EBNA-1 protein may be responsible for the protracted development of B-cell lymphoma in some lines of transgenic mice (59
), our observations indicate that neither EBV-dependent tumorigenicity nor regulation of c-MYC and Bcl-2 expression in BL can be attributed to EBNA-1 alone. Other EBV gene products that are consistently expressed in BL are the small RNAs EBER-1 and EBER-2 and the more recently identified RK-BARF0 protein (2
). Whereas the function of RK-BARF0 is unknown, at least two functions have been ascribed to the EBER RNAs: first, EBERs bind to the double-stranded RNA-dependent protein kinase PKR and disrupt the ability of PKR, a protein with potential tumor suppressor activity, to inhibit translation (10
); second, EBER RNAs bind to the ribosomal protein L22 and sequester L22 to the nucleus (58
). Given that the EBERs are capable of targeting two proteins associated with the translational process and our data suggesting that down-regulation of c-MYC expression is most likely mediated at the level of translation, the possibility that the EBERs contribute to cell survival as defined here is particularly attractive.
Although our observations provide important insights into the long-standing debate over the role of EBV in BL, equally important are the implications of these findings with respect to the maintenance of EBV latency in the healthy EBV-immune host. Because the EBV-dependent down-regulation of c-MYC in BL appears to occur through a posttranscriptional mechanism, this type of regulation may also be operational within normal latently infected B cells in vivo. Thus, EBV may contribute to its persistence by promoting the survival of proliferating latently infected B cells. Although a major reservoir of EBV in the peripheral blood appears to be resting B cells that lack EBNA-1 mRNA but which express LMP-2A transcripts (37
), cells that express EBNA-1 mRNA in the absence of transcripts that encode the other EBNA proteins and LMP-1 are detectable as well (7
). This finding suggests that there is a subpopulation of infected B cells in vivo that maintains a pattern of EBV gene expression similar or identical to that observed in BL. Such B cells may be actively dividing in response to physiological signals and essential to maintain a critical pool of latently infected B cells that is necessary to sustain long-term infection (reviewed in reference 56
). The potential of EBV to usurp physiological pathways of cell proliferation, coupled with its ability to limit cell death under growth-restrictive conditions as shown here, would enable EBV to circumvent the need for virus-induced proliferation and the associated expression of the LMP-1 oncoprotein and other EBV proteins, such as the EBNA-3 family, that are capable of evoking a strong cellular immune response.