EBV-negative Burkitt's Lymphoma Cell Research Articles

Intracellular localization of the Epstein-Barr virus BFRF1 gene product in lymphoid cell lines and oral hairy leukoplakia lesions.

A novel protein encoded by the BFRF1 gene of the Epstein-Barr virus was identified recently [Farina et al. (2000) J Virol 74:3235-3244], which is antigenic "in vivo" and expressed early in the viral replicative cycle. In the present study, its subcellular localization was examined in greater detail comparing Epstein-Barr virus (EBV) induced producing and nonproducing cell lines by immunofluorescence: in 12-0-tetradecanoyl phorbol-13-acetate (TPA)-induced Raji and B95-8 cells, as well as in anti-IgG-stimulated Akata cells, the protein appeared to be localized over the cell nuclear membrane. A similar nuclear membrane localization was observed in epithelial cells of oral hairy leukoplakia, a pathological manifestation of permissive EBV infection. In contrast, upon transfection of BFRF1 in the EBV-negative Burkitt's lymphoma cell line DG75, the protein was localized predominantly over the plasma membrane. The membrane localization was abolished when DG75 cells were transfected with a C-terminal deletion mutant of BFRF1 lacking the transmembrane domain. Because induced Raji cells do not produce virus, the above observations indicate that the nuclear membrane localization is not associated with viral production, but requires the expression of EBV genes, and suggest that additional proteins, expressed early during viral lytic infection, might be necessary to target the protein to the nuclear membrane. Immunogold electron microscopy on ultrathin cryosections of induced B95-8 cells showed that BFRF1 on the nuclear membranes was concentrated over multilayered domains representing areas of active viral replication or at the sites of viral budding, suggesting that BFRF1 is involved in the process of viral assembly.

Gene Array Identification of Epstein Barr Virus-Regulated Cellular Genes in EBV-Converted Burkitt Lymphoma Cell Lines

Epstein Barr virus (EBV) is associated with various B-cell neoplasms such as post-transplant lymphoproliferative disease or Burkitt lymphoma. B-lymphocyte reprogramming by EBV involves the control of numerous cellular genes. To identify such EBV-deregulated genes, we have compared the gene expression profile of EBV-negative Burkitt lymphoma cell lines (BL) (BL2, BL30, BL70) with their EBV-converted counterpart (BL2-B95, BL30-B95, BL70-B95) by cDNA array. Statistical analysis of the results was made using Ward's cluster analysis method. Results showed that the expression of up to 26% of the 1176 cellular genes analyzed may be modified in EBV-converted BL cells. Within this set of genes, a subset of genes markedly regulated in EBV-converted BL cells was defined as those for which expression in EBV+ cells was increased or decreased more than 2-fold. Expression of various genes was modulated in agreement with their previously reported regulation by EBV or by transcription factors activated by EBV. Numerous genes were newly identified as modulated in EBV-converted BL cells. Some of these results were verified by both semiquantitative RT-PCR and Western blotting, and were consistent with functional studies. Functional classification of EBV-regulated genes gave a comprehensive picture of cellular reprogramming by EBV in BL, by pointing out cellular modules such as cell cycle, apoptosis, and signal transduction pathways, including BCR and TNF receptor family and interferon pathways. Furthermore, and perhaps most importantly, cDNA array results point to three families of transcription factors, Rel/NF-κΒ, STAT1, and Ets-related proteins Spi-B, Elf-1, and Ets-1 as putative cellular targets of EBV.

Epstein-Barr virus latent membrane protein-1 activates CD25 expression in lymphoma cells involving the NFkappaB pathway.

Epstein-Barr virus (EBV) is associated with several human malignancies including Burkitt's lymphoma (BL), Hodgkin's disease (HD) and nasopharyngeal carcinoma. A variety of cytokines and receptors have been described to be activated by EBV. Here we show that the IL-2 receptor (IL-2R) alpha-chain, which is weakly expressed on normal resting lymphoid cells, is activated by EBV. Comparison of EBV-negative BL cell lines and their EBV convertants showed an enhanced CD25 expression in EBV-positive BL cells. Transient expression of the oncogenic virus protein latent membrane protein-1 (LMP1) in L428 Hodgkin's lymphoma cells and in Burkitt's lymphoma cells (BL2, BL41, BL30) cells leads to enhanced CD25 expression. Both C-terminal activating regions (CTARs) of LMP1 are involved in CD25 activation. Inhibition of LMP1-mediated NFkappaB enhancement by a constitutive repressive form of IkappaB-alpha resulted in decreased CD25 surface expression, indicating that NFkappaB is involved in CD25 gene regulation. Furthermore, LMP1-mediated CD25 activation was associated with enhanced levels of the soluble form of CD25 (sCD25) in L428 Hodgkin's lymphoma cells but not in BL cells. LMP1 associated enhanced expression of membrane CD25 and soluble CD25 may have immunomodulatory functions and could be involved in biology of EBV-associated diseases.

Tissue Inhibitor of Metalloproteinase-1 Alters the Tumorigenicity of Burkitt’s Lymphoma via Divergent Effects on Tumor Growth and Angiogenesis

Epstein-Barr virus (EBV)-positive Burkitt's lymphoma cells and EBV-infected B cells elicit humoral factors that inhibit tumor-induced angiogenesis, resulting in tumor necrosis and regression. Of the chemokine factors identified in association with this growth behavior, none have induced complete tumor regression. We have previously identified tissue inhibitors of metalloproteinase (TIMP)-1 in various B cell lymphoma cell lines. Here we show that induction of TIMP-1 expression in an EBV-negative Burkitt's lymphoma cell line results in a biphasic, in vivo tumor growth pattern in the nude mouse that is essentially identical to EBV-positive Burkitt's lymphoma cell lines. The initial effect of TIMP-1 is to enhance tumor growth, consistent with the reported anti-apoptotic effect of TIMP-1 on B cell growth. Tumor necrosis and regression then follow the initial period of rapid, increased tumor growth. Only microscopic foci of residual, proliferating tumor cells are observed on biopsy of the tumor site. This latter effect is mediated by TIMP-1 inhibition of an angiogenic response within the developing tumor mass, as demonstrated by immunostaining and microvessel counts. These findings suggest that TIMP-1 is an important mediator of the in vivo growth properties of EBV-positive Burkitt's lymphoma.

EBNA promoter usage in EBV-negative Burkitt lymphoma cell lines converted with a neomycin-resistant EBV strain.

Latent Epstein-Barr virus (EBV) uses two alternative strategies to express the Epstein-Barr nuclear antigens (EBNAs). Resting normal B cells harboring latent virus and Burkitt's lymphoma (BL) cells use monocistronic messages generated from the Q promoter (restricted strategy). EBV-transformed immunoblasts express all EBNAs by using giant messages generated from the W/C promoter (full program). Whether the virus establishes the restricted program on primary infection of a BL cell (or its progenitor) or, alternatively, whether such cells are generated by phenotypic down-regulation from the immunoblast is unclear. We found previously that conversion of EBV-negative BL lines to EBV-positive sublines required repeated exposure to large virus doses. The converted sublines used the full program. However, the possibility that cells with a full program had a selective advantage during the long period of in vitro passage could not be excluded. We therefore infected EBV-negative BL lines with recombinant EBV carrying a neomycin resistance marker. Most convertants of the 12 lines tested were positive for YUK splicing, indicative of the full program, but some were also positive for the restricted QUK splice program. One convertant DG75 line showing both YUK and QUK was cloned and gave rise to stable QUK users. We conclude that EBV infection of established BL lines can give rise to subclones with either the full or the restricted program. The fact that all EBVs carrying BL lines use the restricted program in vitro may be a consequence of immunoselection.

Epstein-Barr virus small RNAs potentiate tumorigenicity of Burkitt lymphoma cells independently of an effect on apoptosis.

The tumorigenic potential of the Burkitt lymphoma (BL) cell line Akata is dependent on the restricted latency program of Epstein-Barr virus (EBV) that is characteristically maintained in BL tumors. Within these cells, EBV-mediated inhibition of apoptosis correlates with an up-regulation of BCL-2 levels in concert with a down-regulation in c-MYC expression that occurs under growth-limiting conditions. Here we addressed whether EBV's effects on apoptosis and tumorigenicity are mediated by the EBV small RNAs EBER-1 and EBER-2. Stable expression of the EBERs in EBV-negative Akata BL cells, at levels comparable to those in EBV-positive cells, significantly enhanced the tumorigenic potential of EBV-negative BL cells in SCID mice, but did not fully restore tumorigenicity relative to EBV-positive Akata cells. Furthermore, wild-type or greater levels of EBER expression in EBV-negative Akata cells did not promote BL cell survival. These data therefore suggest that EBV can contribute to BL through at least two avenues: an EBER-dependent mechanism that enhances tumorigenic potential independent of a direct effect on apoptosis, and a second mechanism, mediated by an as-yet-unidentified EBV gene(s), that offsets the proapoptotic consequences of deregulated c-MYC in BL.

The bfl-1 gene is transcriptionally upregulated by the Epstein-Barr virus LMP1, and its expression promotes the survival of a Burkitt's lymphoma cell line.

The recently identified bfl-1 gene (also known as A1 or GRS), a homologue of bcl-2, encodes an antiapoptotic protein that suppresses apoptosis induced by the p53 tumor suppressor protein and exhibits proliferative and potent cooperative transforming activities. We show that elevated levels of bfl-1 mRNA are a feature of Epstein-Barr virus (EBV)-immortalized B-cell lines and Burkitt's lymphoma cell lines expressing the full spectrum of EBV latent proteins. Using an EBV-negative Burkitt's lymphoma cell line in which the expression of EBV latent membrane protein 1 (LMP1) is inducibly regulated by tetracycline, we demonstrate that LMP1 expression coincides with a dramatic increase in the level of bfl-1 mRNA. Also in this system, an increase in the level of Bcl-2 protein was seen to occur earlier than that of bcl-2 mRNA, suggesting that both transcriptional and translational mechanisms are involved in the control of Bcl-2 expression by LMP-1. We show that elevated bfl-1 mRNA stability can contribute to this effect of LMP-1, thus providing evidence of a novel mechanism of gene regulation by this EBV protein. Upregulation of bfl-1 by LMP1 was not observed in the T-cell line Jurkat or the epithelial cell line C33A. Ectopic expression of Bfl-1 in an EBV-positive cell line exhibiting a latency type I infection protects against apoptosis induced by growth factor deprivation, thereby providing a functional role for Bfl-1 in this cellular context and adding Bfl-1 to the list of antiapoptotic proteins whose expression is modulated by EBV. This is the first report of the regulation of bfl-1 expression by a viral protein, and this novel finding may thus represent an important link between the EBV oncoprotein LMP1 and its cellular growth-transforming properties.

The Epstein-Barr virus nuclear antigen 2 (EBNA2), a protein required for B lymphocyte immortalization, induces the synthesis of type I interferon in Burkitt's lymphoma cell lines.

Epstein-Barr virus nuclear antigen 2 (EBNA2), a protein involved in cell transformation, interferes with the cellular response to type I interferons (IFN-alpha/beta). We investigated the function of conditionally expressed EBNA2 in the context of the IFN response in Burkitt's lymphoma cell lines. Expression of EBNA2 led to the transcriptional activation of both endogenous or transfected IFN-stimulated genes (ISGs), genes which contain within their promoters either the interferon-stimulated response element (ISRE) or the gamma interferon activation site (GAS). In search of a molecular mechanism for the transcriptional induction of ISGs, we observed an EBNA2-dependent synthesis of IFN-beta mRNA at low levels and the secretion of low amounts of IFN. A transfected IFN-beta promoter responded to EBNA2 activation, and a sequence closely resembling a RBP-Jkappa binding site was pinpointed as a potential target of EBNA2 activity. EBNA2-dependent transcriptional induction of the IFN-beta promoter occurred in EBV-negative Burkitt's lymphoma cells, indicating that other EBV genes were not required for the induction of IFN-beta synthesis.

Identification of a cytotoxic T-lymphocyte response to the novel BARF0 protein of Epstein-Barr virus: a critical role for antigen expression.

The Epstein-Barr virus (EBV)-encoded BARF0 open reading frame gene products are consistently expressed in EBV-positive Burkitt's lymphoma (BL) cell lines, nasopharyngeal carcinoma cell lines, and lymphoblastoid cell lines (LCLs). Here we show that the BARF0 sequence includes an HLA A*0201-restricted cytotoxic T-lymphocyte (CTL) epitope. By using theoretically predicted HLA A2 binding motifs and peptide-loaded antigen presentation-deficient T2 cells, polyclonal BARF0-specific CD8(+) CTLs were isolated from four different healthy EBV-seropositive donors but not from two seronegative donors. These CTL lines recognized the peptide epitope LLWAARPRL, which was found to be conserved in 33 of 34 virus strains originating from Caucasian, African, and Asian individuals. The BARF0-specific CTL lines could lyse EBV-negative BL cells stably transfected with the BARF0 gene but did not kill HLA A2-matched EBV-positive BL cells and LCLs in a standard 51Cr release assay. Reverse transcriptase PCR analysis demonstrated that these EBV-positive cell lines expressed significantly lower levels of BARF0 mRNA than transfected cells. This data indicated that the BARF0 epitope could be endogenously processed; however, antigen levels in the target cell were a limiting factor for the effective interaction between BARF0-expressing cells and CTLs. The limited expression of BARF0 antigen in EBV-infected BL cells and LCLs might contribute to the escape of immune recognition from virus-specific CTLs present in the host.

Absence of IL-6 receptor expression in fresh childhood Burkitt's lymphoma cells and induction of IL-6 receptors by Epstein-Barr virus in vitro.

Interleukin-6 (IL-6) is a cytokine which is necessary for the differentiation of activated B cells and growth of early haemopoietic progenitors. It is used for ex-vivo expansion of myeloid progenitors in the setting of high-dose chemotherapy with autologous bone marrow transplantation (BMT). Expression of the IL-6 receptor (IL-6R) was examined in six fresh Burkitt's lymphoma (BL) cell preparations and 12 BL cell lines by reverse transcriptase polymerase chain reaction (RT-PCR) and flow cytometry (FCM). Inducibility of IL-6R mRNA expression by Epstein-Barr virus (EBV) was studied by comparing two uninfected cell lines with the same cell lines infected by EBV The phenotype of the BL cells lines was analysed by FCM and by proliferation assays in the presence of anti-IgM antibodies. None of the fresh BL cells expressed the IL-6 receptor. The BL cell lines expressed varying degrees of IL-6R mRNA and protein. In vitro infection of EBV-negative BL cell lines resulted in up-regulation of IL-6R mRNA. There was no proliferative response of the BL cell lines to IL-6, including the cells that expressed the receptor. Compared to uninfected BL cell lines, EBV-infected cell lines and lymphoblastoid cell lines (LCLs) showed a weaker or no response to anti-IgM antibodies, indicating a more mature phenotype of these cells. We conclude that the IL-6 receptor is not expressed in fresh childhood BLs, but only in BL cell lines. EBV infection in vitro leads to an up-regulation of IL-6R mRNA but not to increased proliferation. This makes growth stimulation of contaminating BL cells in the setting of autologous BMT unlikely.

Induction of Pleckstrin by the Epstein–Barr Virus Nuclear Antigen 3 Family

The Epstein–Barr virus (EBV) encoded nuclear antigens, EBNA-3, -4, and -6 (EBNA 3 family) are expressed in latently infected human B-cells and are involved in the transformation of lymphocytes by EBV. Human Burkitt's lymphoma (BL) dG75 cells which stably expressed either the complete EBNA 3 gene family or the vector alone were generated and changes in gene activity in these transfectants were assayed using the differential display of mRNA technique. For the first time, the human gene pleckstrin, which is thought to be involved in signalling and differentiation of hemopoietic cells, was found to be upregulated in the presence of the EBNA 3 protein family, but not in cells expressing the individual EBNA-3, -4, or -6 gene. Pleckstrin was increased up to sevenfold in different cell clones and the bulk culture of EBNA 3 gene family expressing cells as demonstrated by Northern blot, RT-PCR, and immunoblot. In contrast to EBV-negative BL cells, pleckstrin RNA and protein were highly expressed in EBV growth transformed lymphoblastoid cell lines which are thought to play an important role in EBV persistencein vivo.These data suggest that induction of pleckstrin might be important for the EBV-controlled activation of cells and offers a unique biological system for analyzing pleckstrin function.

Transcription of the Epstein-Barr virus nuclear antigen 1 (EBNA1) gene occurs before induction of the BCR2 (Cp) EBNA gene promoter during the initial stages of infection in B cells

The purpose of this study was to gain insights into the regulation of Epstein-Barr virus (EBV) gene transcription during the establishment of viral latency in B cells. During the early stages of EBV infection in B lymphocytes, transcription of six viral nuclear antigens (EBNAs) is initiated from an early promoter (Wp). This is followed by a switch of promoter usage to an upstream promoter, Cp, whose activity is autoregulated by both EBNA1 and EBNA2. Previously it was demonstrated that infection of primary B cells with EBNA2-negative (EBNA2-) EBNA4-mutant (EBNA4mut) virus resulted only in the expression of mutant EBNA4 protein and failure to express the other EBNA gene products (C. Rooney H. G. Howe, S. H. Speck, and G. Miller, J. Virol. 63:1531-1539, 1989). We extended this research to demonstrate that Wp-to-Cp switching did not occur upon infection of primary B cells with an EBNA2- EBNA4mut virus (M. Woisetschlaeger, X. W. Jin, C. N. Yandara, L. A. Furmanski, J. L. Strominger, and S. H. Speck, Proc. Natl. Acad. Sci. USA 88:3942-3946, 1991). Further characterization of this phenomenon led to the identification of an EBNA2-dependent enhancer upstream of Cp. On the basis of these data, a model was proposed in which initial transcription from Wp gives rise to the expression of EBNA2 and EBNA4, and then transcription is upregulated from Cp via the EBNA2- dependent enhancer (Woisetschlaeger et al., as noted above). Implicit in this model is that transcription of the EBNA1 and EBNA3a to -3c genes is dependent on the switch from Wp to Cp, since primary cells infected with EBNA2- EBNA4mut virus fail to switch and also fail to express these viral antigens. Here we critically evaluate this model and demonstrate, in contrast to the predictions of the model, that transcription of both the EBNA1 and EBNA2 genes precedes activation of Cp. Furthermore, the level of EBNA1 gene transcription was strongly reduced in primary B cells infected with EBNA2- EBNA4mut virus compared with that of cells infected with wild-type virus. Switching to Cp, as well as EBNA1 gene transcription, was observed upon infection of EBV-negative Burkitt's lymphoma (BL) cell lines with EBNA2- EBNA4mut virus, thus establishing a correlation between early EBNA1 gene transcription and upregulation of transcription initiation from Cp. However, in EBV-negative BL cell lines infected with EBNA2- EBNA4mut virus, transcription of the EBNA1 gene at early time points postinfection initiated from Qp, the EBNA1 gene promoter active in group I BL cells (B. C. Schaefer, J. L. Strominger, and S. H. Speck, Proc. Natl. Acad. Sci. USA 92:10565-10569, 1995), rather than from Wp. The data support a model in which EBNA1 plays an important role in the cascade of events leading to successful switching from Wp to Cp and subsequent immortalization of the infected B cell.

Redefining the Epstein-Barr virus-encoded nuclear antigen EBNA-1 gene promoter and transcription initiation site in group I Burkitt lymphoma cell lines.

The Epstein-Barr virus-encoded nuclear antigen EBNA-1 gene promoter for the restricted Epstein-Barr virus (EBV) latency program operating in group I Burkitt lymphoma (BL) cell lines was previously identified incorrectly. Here we present evidence from RACE (rapid amplification of cDNA ends) cloning, reverse transcription-PCR, and S1 nuclease analyses, which demonstrates that the EBNA-1 gene promoter in group I BL cell lines is located in the viral BamHI Q fragment, immediately upstream of two low-affinity EBNA-1 binding sites. Transcripts initiated from this promoter, referred to as Qp, have the previously reported Q/U/K exon splicing pattern. Qp is active in group I BL cell lines but not in group III BL cell lines or in EBV immortalized B-lymphoblastoid cell lines. In addition, transient transfection of Qp-driven reporter constructs into both an EBV-negative BL cell line and a group I BL cell line gave rise to correctly initiated transcripts. Inspection of Qp revealed that it is a TATA-less promoter whose architecture is similar to the promoters of housekeeping genes, suggesting that Qp may be a default promoter which ensures EBNA-1 expression in cells that cannot run the full viral latency program. Elucidation of the genetic mechanism responsible for the EBNA-1-restricted program of EBV latency is an essential step in understanding control of viral latency in EBV-associated tumors.

Latent membrane protein-1 induces cyclin D2 expression, pRb hyperphosphorylation, and loss of TGF-beta 1-mediated growth inhibition in EBV-positive B cells.

The normal cell cycle is regulated by several molecules, such as the tumor-suppressor protein pRb, the G1 cyclins, the cyclin-dependent kinases, and their inhibitors. These regulators are targeted by negative growth regulatory signals, such as that provided by TGF-beta. Here, we show that the presence of either wild-type EBV or its transforming latent membrane protein-1 (LMP-1) results in the loss of TGF-beta 1-mediated growth inhibition in human B cells. Chemical cross-linking with 125I-labeled TGF-beta 1 showed an essentially normal TGF-beta receptor profile in EBV-positive and EBV-negative Burkitt's lymphoma cell lines, and these receptors were shown to be functional in transducing signals, as evidenced by the TGF-beta 1-mediated modulation of junB gene expression. However, TGF-beta 1 did not induce dephosphorylation of pRb in EBV (or LMP-1)-positive cells as opposed to EBV-negative cells, suggesting a dichotomy in the TGF-beta 1 signaling pathway leading to separable gene regulatory and growth inhibitory responses. Furthermore, LMP-1 was found to induce the expression of cyclin D2; normal B cells or EBV-negative Burkitt's lymphoma cells do not express D-type cyclins. Taken together, these data point to a potential mechanism underlying EBV-mediated B cell transformation whereby constitutive induction of key cell cycle regulators by LMP-1 can lead to pRb hyperphosphorylation and uncontrolled cell proliferation.

Expression of a B‐cell marker, CD24, on nasopharyngeal carcinoma cells

Random sequencing of clones from a lambda gt10 cDNA library, made from mRNA expressed in an Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) has revealed the gene transcript of human CD24. The CD24 antigen, a glycosylphosphatidylinositol-anchored cell surface molecule, has been identified as a B-cell marker that is lost during cell maturation. We show here that it is expressed on 3 NPC xenografts, previously defined as consisting of poorly differentiated epithelial cells, and on an NPC biopsy. In the case of the former, the level of expression of CD24 corresponds to the EBV load. A B-lymphoblastoid cell line carrying the same EBV genome as one of the tumours, C15, and an EBV-negative Burkitt's lymphoma cell line do not display the antigen, but epithelial-like cells of a laryngeal tumour cell line (Hep2) do express it. Our data suggest that CD24 may be a marker of cell differentiation not only for B cells but also for epithelial cells and may have an indirect association with EBV gene expression.

A Ca2+/calmodulin-dependent protein kinase, CaM kinase-Gr, expressed after transformation of primary human B lymphocytes by Epstein-Barr virus (EBV) is induced by the EBV oncogene LMP1.

CaM kinase-Gr is a multifunctional Ca2+/calmodulin-dependent protein kinase which is enriched in neurons and T lymphocytes. The kinase is absent from primary human B lymphocytes but is expressed in Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines, suggesting that expression of the kinase can be upregulated by an EBV gene product(s). We investigated the basis of CaM kinase-Gr expression in EBV-transformed cells and the mechanisms that regulate its activity therein by using an EBV-negative Burkitt lymphoma cell line, BJAB, and BJAB cells converted to expression of individual EBV proteins by single-gene transfer. CaM kinase-Gr expression was upregulated in BJAB cells by EBV latent-infection membrane protein 1 (LMP1) but not by LMP2A or by nuclear proteins EBNA1, EBNA2, EBNA3A, and EBNA3C. In LMP1-converted BJAB cells, the kinase was functional and was dramatically activated upon cross-linking of surface immunoglobulin M. Overlapping cDNA clones that encode human CaM kinase-Gr were sequenced, revealing 81% amino acid identity between the rat and human proteins. Transfection of BJAB cells with an expression construct for the human enzyme resulted in a functional kinase which was shown by epitope tagging to localize primarily to cytoplasmic and perinuclear structures. Induction of CaM kinase-Gr expression by LMP1 provides the first example of a Ca2+/calmodulin-dependent protein kinase upregulated by a viral protein. In view of the key role played by LMP1 in B-lymphocyte immortalization by EBV, these findings implicate CaM kinase-Gr as a potential mediator of B-lymphocyte growth transformation.

Latent membrane protein of Epstein-Barr virus induces cellular phenotypes independently of expression of Bcl-2.

The stable expression of the Epstein-Barr virus (EBV) latent membrane protein (LMP) in certain EBV-negative Burkitt's lymphoma cell lines correlates with an increased expression of the oncogene Bcl-2 (S. Henderson, M. Rowe, C. Gregory, D. Croom-Carter, F. Wang, R. Longnecker, E. Kieff, and A. Rickinson, Cell 65:1107-1115, 1991). This finding is consistent with a model in which Bcl-2 contributes to the immortalization of B cells mediated by EBV. We therefore asked whether the expression of Bcl-2 protein correlates with the induction of three cellular phenotypes induced by or associated with LMP. The expression of Bcl-2 in primary B cells infected with the B95-8 strain of EBV varied between 1 and 1.8 times that in uninfected cells when 50% of the cells were infected, expressed LMP, and incorporated 20-fold more [3H]thymidine than did uninfected cells. This finding indicates that induced proliferation of these primary cells is not sufficient to induce Bcl-2. We found that BALB/c 3T3 cells and their derivatives transformed by LMP do not express Bcl-2 detectably. The expression of LMP at high levels in lymphoid cells is cytotoxic and correlates with an increased expression of Bcl-2 following stable selection for the introduced LMP gene; 2 days after transfection, control vector- and LMP-transfected populations, however, express equal levels of Bcl-2 protein. We also analyzed transient expression of LMP in an EBV-negative Burkitt's lymphoma cell line. Infection of BJAB cells with the B95-8 strain of EBV results in an increase in Bcl-2 expression with a time course similar to that of LMP expression, and LMP alone transiently induces an increase in Bcl-2 expression in these cells. We interpret these observations to indicate that increased expression of Bcl-2 is unlikely to contribute to the ability of EBV to immortalize primary B cells and that both the transformation of rodent cells and the cytotoxicity mediated by LMP are independent of Bcl-2.

Retinoic Acid Induces Changes in c- fgr Proto-Oncogene mRNA Levels in Burkitt's Lymphoma Cells

The c- fgr proto-oncogene is expressed in Burkitt's lymphoma (BL) cells and cell lines derived from them. When Epstein-Barr virus (EBV)-negative BL cell lines that contain low levels of c- fgr mRNA are infected with EBV, transcription of the c- fgr gene is further induced. In this paper we show that treatment of EBV-negative and EBV-positive BL cell lines with all- trans retinoic acid also stimulates an increase in c- fgr mRNA levels, varying between 2- and 13-fold depending on the cell line. An increase is detectable 12 to 48 h after treatment, depending on the cell line, suggesting that the c- fgr gene is not regulated directly by retinoic acid but responds to other retinoic acid-induced changes in the cell. We also show that treatment of BL cell lines with all- trans retinoic acid either results in a dose-dependent decrease in growth rate, or has no effect on growth, depending on the cell line. It has previously been suggested that the c- fgr gene product might have a role in regulating the growth of BL cells, since treatment of the EBV-positive BL cell line Daudi with α-interferon results in a decrease in c- fgr mRNA levels followed by a decrease in growth rate. Our data indicate that there is no general correlation between c- fgr mRNA levels and growth rate in BL cells and so argue against a role for the c- fgr gene product in growth regulation in these cells.

Expression of bcl-2 in Burkitt’s Lymphoma Cell Lines: Induction by Latent Epstein-Barr Virus Genes

Abstract The bcl-2 oncogene blocks programmed cell death (apoptosis). Epstein- Barr virus (EBV) can immortalize B lymphocytes into continuously growing lymphoblastoid cell lines (LCL) by the coordinate expression of at least 9 latent genes (EBV nuclear antigen [EBNA] 1–6, latent membrane protein [LMP], and terminal proteins [TP] 1 and 2). We analyzed transcription and expression of bcl-2 and latent EBV genes in Burkitt's lymphoma (BL) cell lines with a germinal center phenotype (group I) as well as activated BL cell lines (group III) and LCLs. We found high expression of bcl-2 as well as the full spectrum of latent EBV genes in LCLs and activated group III BL cell lines. Group I BL cells expressed little or no bcl-2, EBNA-2, and LMP. Superinfection with nondefective EBV or an EBNA-2-defective virus as well as transfection with EBNA-2- or LMP-carrying vectors into the EBV-negative cell lines RAMOS, DG75, U698, or BJAB induced upregulation of bcl-2 expression. The strongest effect on bcl-2 was obtained by transfection with LMP, or infection with the nondefective virus. No change of bcl-2 expression was observed with EBNA-1. Our data indicate that the immortalization capacity of EBV and the growth advantage of EBV- positive compared with EBV-negative BL cells in vitro may predominantly be mediated via induction of bcl-2 and the main effectors are EBNA-2 and LMP.

Expression of bcl-2 in Burkitt's lymphoma cell lines: induction by latent Epstein-Barr virus genes

The bcl-2 oncogene blocks programmed cell death (apoptosis). Epstein-Barr virus (EBV) can immortalize B lymphocytes into continuously growing lymphoblastoid cell lines (LCL) by the coordinate expression of at least 9 latent genes (EBV nuclear antigen [EBNA] 1-6, latent membrane protein [LMP], and terminal proteins [TP] 1 and 2). We analyzed transcription and expression of bcl-2 and latent EBV genes in Burkitt's lymphoma (BL) cell lines with a germinal center phenotype (group I) as well as activated BL cell lines (group III) and LCLs. We found high expression of bcl-2 as well as the full spectrum of latent EBV genes in LCLs and activated group III BL cell lines. Group I BL cells expressed little or no bcl-2, EBNA-2, and LMP. Superinfection with nondefective EBV or an EBNA-2-defective virus as well as transfection with EBNA-2- or LMP-carrying vectors into the EBV-negative cell lines RAMOS, DG75, U698, or BJAB induced upregulation of bcl-2 expression. The strongest effect on bcl-2 was obtained by transfection with LMP, or infection with the nondefective virus. No change of bcl-2 expression was observed with EBNA-1. Our data indicate that the immortalization capacity of EBV and the growth advantage of EBV-positive compared with EBV-negative BL cells in vitro may predominantly be mediated via induction of bcl-2 and the main effectors are EBNA-2 and LMP.