EBV-positive Burkitt's Lymphoma Cell Research Articles

Activation of Epstein–Barr virus latent genes protects human B cells from death by apoptosis

Epstein-Barr virus (EBV), a human herpesvirus, establishes a persistent asymptomatic infection of the circulating B-lymphocyte pool. The mechanism of virus persistence is not understood but, given the limited lifespan of most B cells in vivo, it seems most likely that EBV-infected cells must gain access to the long-lived memory B-cell pool. Here we show in an in vitro system that EBV, through expression of the full set of eight virus-coded 'latent' proteins, can protect human B cells from programmed cell death (apoptosis), the deletion mechanism which normally restricts entry into memory. We have found that EBV-positive Burkitt's lymphoma (BL) cell clones retaining the original tumour cell phenotype and expressing only one of the virus latent proteins, the nuclear antigen EBNA 1, are extremely sensitive to apoptosis; in this respect they resemble the tumour's normal cell of origin found in the germinal centres of lymphoid tissue. By contrast, isogenic BL cell clones which have activated expression of all eight EBV latent proteins are resistant to the induction of apoptosis. The EBV latent proteins should therefore be seen not just as activators of B-cell proliferation but, perhaps more importantly, as mediators of enhanced B-cell survival.

Epstein-Barr virus latent membrane protein (LMP1) and nuclear proteins 2 and 3C are effectors of phenotypic changes in B lymphocytes: EBNA-2 and LMP1 cooperatively induce CD23

Latent Epstein-Barr virus (EBV) infection and growth transformation of B lymphocytes is characterized by EBV nuclear and membrane protein expression (EBV nuclear antigen [EBNA] and latent membrane protein [LMP], respectively). LMP1 is known to be an oncogene in rodent fibroblasts and to induce B-lymphocyte activation and cellular adhesion molecules in the EBV-negative Burkitt's lymphoma cell line Louckes. EBNA-2 is required for EBV-induced growth transformation; it lowers rodent fibroblast serum dependence and specifically induces the B-lymphocyte activation antigen CD23 in Louckes cells. These initial observations are now extended through an expanded study of EBNA- and LMP1-induced phenotypic effects in a different EBV-negative B-lymphoma cell line, BJAB. LMP1 effects were also evaluated in the EBV-negative B-lymphoma cell line BL41 and the EBV-positive Burkitt's lymphoma cell line, Daudi (Daudi is deleted for EBNA-2 and does not express LMP). Previously described EBNA-2- and LMP1-transfected Louckes cells were studied in parallel. EBNA-2, from EBV-1 strains but not EBV-2, induced CD23 and CD21 expression in transfected BJAB cells. In contrast, EBNA-3C induced CD21 but not CD23, while no changes were evident in vector control-, EBNA-1-, or EBNA-LP-transfected clones. EBNAs did not affect CD10, CD30, CD39, CD40, CD44, or cellular adhesion molecules. LMP1 expression in all cell lines induced growth in large clumps and expression of the cellular adhesion molecules ICAM-1, LFA-1, and LFA-3 in those cell lines which constitutively express low levels. LMP1 expression induced marked homotypic adhesion in the BJAB cell line, despite the fact that there was no significant increase in the high constitutive BJAB LFA-1 and ICAM-1 levels, suggesting that LMP1 also induces an associated functional change in these molecules. LMP1 induction of these cellular adhesion molecules was also associated with increased heterotypic adhesion to T lymphocytes. The Burkitt's lymphoma marker, CALLA (CD10), was uniformly down regulated by LMP1 in all cell lines. In contrast, LMP1 induced unique profiles of B-lymphocyte activation antigens in the various cell lines. LMP1 induced CD23 and CD39 in BJAB; CD23 in Louckes; CD39 and CD40 in BL41; and CD21, CD40, and CD44 in Daudi. In BJAB, CD23 surface and mRNA expression were markedly increased by EBNA-2 and LMP1 coexpression, compared with EBNA-2 or LMP1 alone. This cooperative effect was CD23 specific, since no such effect was observed on another marker, CD21.(ABSTRACT TRUNCATED AT 400 WORDS)

Epstein-Barr virus latent membrane protein: induction of B-cell activation antigens and membrane patch formation does not require vimentin.

The latent membrane protein (LMP) of Epstein-Barr virus (EBV) forms patches associated with the vimentin intermediate filament system in EBV-transformed lymphoblastoid cell lines, EBV-infected Burkitt's lymphoma cells, and LMP-transfected, EBV-negative Burkitt's lymphoma cells. By gene transfer, LMP induces the expression of vimentin and B-cell activation antigens in EBV-negative Burkitt's lymphoma cells. We have now expressed LMP in an EBV-positive Burkitt's lymphoma cell line, Daudi, which does not express any LMP or vimentin. In these Daudi transfectants, LMP still formed plasma membrane patches in the absence of vimentin. LMP did not resist nonionic detergent extraction in Daudi cells as it does in vimentin-expressing cells. LMP still retained functional activity as judged by induction of B-cell activation antigens. These data indicate that LMP can form plasma membrane patches and induce B-lymphocyte activation independent of vimentin association.

Induction of anti-EBNA-1 protein by 12-O-tetradecanoylphorbol-13-acetate treatment of human lymphoblastoid cells

Binding of the Epstein-Barr virus (EBV) nuclear antigen (EBNA-1) to BamHI-C DNA was studied by affinity column chromatography followed by immunoblotting with human serum specific for EBNA-1. Two species of EBNA-1 (68 and 70 kilodaltons) were identified in nuclear extracts of the EBV-positive Burkitt's lymphoma cell line Raji and not in nuclear extracts of the EBV-negative Burkitt's lymphoma cell line BJAB. Both EBNA-1s bound specifically to the region required for EBV plasmid DNA maintenance (oriP) located in the BamHI-C fragment. Upon treatment with 12-O-tetradecanoylphorbol-13-acetate, which activates latent EBV genome in Raji cells, the 68-kilodalton EBNA-1 was uncoupled from binding to EBV oriP. Nuclear extracts from 12-O-tetradecanoylphorbol-13-acetate-treated BJAB cells also uncoupled the binding of both EBNA-1s to oriP. DNA-cellulose column chromatography identified two protein species which competed for and uncoupled the binding of EBNA-1 to oriP. The two cellular competitors we called anti-EBNA-1 proteins had molecular masses of 60 and 40 kilodaltons, respectively. They were not found in nuclear extracts of BJAB cells not activated by 12-O-tetradecanoylphorbol-13-acetate.

Expression of CRl and CR2 Complement Receptors following Epstein‐Barr Virus Infection of Burkitt's Lymphoma Cell Lines

Epstein-Barr virus (EBV) infection of human B lymphocytes involves a specific receptor closely associated with, or identical to, the C3d complement receptor, CR2. Thus, 25 out of 29 EBV-positive Burkitt's lymphoma (BL) cell lines but none of 15 EBV-negative BL lines were found to express C3 receptors. Furthermore, in vitro infection with EBV of six EBV-negative cell lines resulted in the expression of C3 receptors in association with that of EBV-determined nuclear antigen (EBNA). Rosette assays using erythrocytes coated with human C3b, C3bi, and C3d, inhibition of rosette formation with anti-receptor antibodies, and flow cytometry analysis of stained cells demonstrated that EBV-converted lines expressed C3b and C3d receptors, CR1 and CR2. Anti-receptor antibodies recognized an average of 40,700 anti-CR1 and 140,000 anti-CR2 binding sites on an EBV-converted line (BL41/B95), whereas no specific binding occurred on the corresponding EBV-negative (BL41) cells. Because CR1 and CR2 are involved in B-cell proliferation and/or differentiation, enhanced expression of C3 receptors following the interaction between EBV and B cells and/or subsequent infection of the cells by EBV may provide a basis for positive control of B lymphocyte proliferation by EBV.

Epstein-Barr virus mediates a switch in responsiveness to transforming growth factor, type beta, in cells of the B cell lineage.

The functional performance of the transforming growth factor beta (TGF beta), appears to depend on the target cell phenotype as well as in vitro culture conditions. We show here that Epstein-Barr virus (EBV)-infection may induce a change in responsiveness to TGF beta, since TGF beta inhibits traverse of the cell cycle of activated normal human B cells, but promotes cell proliferation of EBV-positive Burkitt's lymphoma cell lines as well as EBV-infected B cells. We present evidence that the switch in the responsiveness to TGF beta is mediated by EBV infection, irrespective of the proliferative status of target cells, and thus may contribute to the initiation as well as the maintenance of certain B cell neoplasias.

Diploid human lymphoblastoid and Burkitt lymphoma cell lines: susceptibility to murine NK cells and heterotransplantation to nude mice.

Human lymphoid cell lines which had been classified on the basis of studies on clonality and morphological, on the basis of studies on clonality and morphological, chromosomal and functional parameters as lymphoblastoid cell line (LCL) of presumed non-neoplastic origin and Burkitt lymphoma (BL) lines of proven malignant origin, were tested for susceptibility to natural killer (NK) cells obtained from the spleens of athymic nude mice. The 20 lines included normal diploid LCL and aneuploid BL lines. All cells carried the Epstein-Barr virus (EBV) genome. In addition, two EBV-negative BL lines were tested. The pronase-induced release of 14C-DNA from 14C-thymidine-labelled target cells was used to assess the sensitivity of the cell lines to NK activity. When attempts were made to correlate the growth of the EBV-positive LCL and the EBV-positive BL cell lines in the subcutaneous space of adult nude mice with their susceptibility to NK cells, no significant correlation was observed. The EBV-negative BL cell line, Ramos, however, could be transplanted subcutaneously in nude mice and was more resistant to NK activity than was the EBV-negative BL cell line, BJAB, which cannot be transplanted subcutaneously. Growth of heterotransplanted EBV-converted cell lines in the subcutaneous space of adult nude mice may be influenced by immune effectors other than NK cells.