Epstein-Barr virus (EBV) can immortalize B-cll cells activated by cytokines.

B-type of chronic lymphocytic leukemia (B-CLL) cells are inert to the potent transforming action of Epstein-Barr virus (EBV). The mitogenic action of Staphylococcus aureus Cowan I (SAC), MP6-thioredoxin, and interleukin 2 (IL-2), agents previously shown to induce proliferation in normal as well as in B-CLL cells, lifted this block, and EBV-positive cell lines could be established. It was not possible to establish cell lines of leukemic origin from cultures that were incubated with EBV alone or cytokine mix alone. CLL-cells infected with EBV only, expressed the viral nuclear antigen complex (EBNA), but not the viral latent membrane protein (LMP). They were not activated as measured by cell size and 3H-thymidine incorporation. In contrast, cells incubated with EBV and cytokine mix expressed both EBNA and LMP in parallel with enlargement and increased 3H-thymidine incorporation. These results emphasize that LMP expression is a prerequisite for growth transformation and immortalization and that cytokine activation signals are required for its expression in B-CLLs. Cells incubated with SAC/MP6-thioredoxin/IL-2 did not express any of the viral antigens, but were activated with regard to the mentioned parameters. Nine cell lines were established from six patients. From each of the three patients, we obtained 'twin'-pair lines: one corresponding to the malignant cell and the other to a normal B-lymphoblastoid cell. Thus, malignant and normal B-cell counterparts, from the very same donor, are at hand for comparative studies. The cell lines have been carried out for more than 12 months in culture. We conclude that B-CLL that are refractory to EBV-transformation can be rendered susceptible through in vitro cytokine activation.

Secretion of thioredoxin after in vitro activation of human B cells.

The redox-active enzyme thioredoxin (Trx) is secreted by various virus-transformed cell lines of B- and T-cell origin and has been considered to play an autoregulatory role as a cofactor during cellular growth processes. We show in this paper that exposure of B lymphocytes from normal, healthy donors and B cells from B-type chronic lymphocytic leukemia (B-CLL) to Staphylococcus aureus Cowan I (SAC) induced expression of Trx mRNA. By combining SAC, or the phorbol ester TPA, with IL-2 and the conditioned medium of a T-cell hybridoma (BSF-MP6), we could strongly enhance the Trx expression. After [35S]methionine labeling of stimulated B-CLL cells in vitro, Trx was immunoprecipitated both from cell extracts and from the medium with antibodies against human placenta Trx. Secretion of newly synthesized Trx was also confirmed by a quantitative radioimmunoassay for human Trx. During 24 h cultivation experiments, treatment with SAC induced a 5-fold increase of the Trx content of normal B lymphocytes as well as in B-CLL cells. Approximately two-thirds of the total amount of the enzyme was released into the medium.

Cell phenotype (CD23)-dependent variation in EBV genome copy numbers within lymphoblastoid cell lines (LCL).

Three Epstein-Barr-virus-transformed lymphoblastoid cell lines (LCL) were analysed on the basis of their CD23 expression. Levels of EBV-DNA were compared in the positive and negative subpopulations. Two lines were further analysed with regard to EBNA, cytoplasmic immunoglobulin (cIg) and lytic (EA/VCA) protein expression. Both subpopulations had a similar MHC class-II transcription, but the CD23- subpopulation had a lower plating efficiency and a lower rate of DNA synthesis. In the B6, NAD50 and 0467.3 cell lines, CD23- cells contained 2 +/- 0.2 - 6.4 +/- 3.0 times less EBV DNA than the corresponding CD23+ population. EBNA was expressed in 81 +/- 4.2% - 93 +/- 3.8% of the CD23+ cells and in 0 - 46 +/- 8.0% of the CD23- cells. No CD23+ cells in B6 or NAD50 contained any EA/VCA, while 19 +/- 2.8% - 24 +/- 4.2% of the CD23- cells were positive for the lytic-cycle-associated antigens. Of the CD23- cells, 70 +/- 8.6% - 86 +/- 6.0% were positive for cytoplasmic immunoglobulin compared to 14.7 +/- 2.7% - 14.9 +/- 1.8% in the corresponding CD23+ population. We have previously shown that only 18% of the cIg-positive cells were EBNA-positive in the B6 line compared to 94% in the cIg- population. This was open to 2 alternative interpretations: loss of EBV genomes from a fraction of the cells with subsequent differentiation to secretory immunoglobulin production, or down-regulation of EBNA expression in differentiating, EBV-genome-positive cells. Our present findings speak for the first alternative, indicating that a certain proportion of the cells may lose their EBV genomes in both long-established and freshly transformed LCLs. This is accompanied by a reduced percentage of EBNA-positive cells, the disappearance of at least one activation marker (CD23) associated with the virally induced blast transformation, and an increased synthesis of cIg.

Co-localization of the retinoblastoma protein and the Epstein-Barr virus-encoded nuclear antigen EBNA-5.

A monoclonal antibody (aRB1C1) raised against an Rb fusion protein detects a limited number (4-10) of relatively large intranuclear foci in an EBV-immortalized cord blood cell line (IB4). These domains also bind an anti-EBNA-5 monoclonal antibody. The Rb antibody reactive sites also co-localize with the SV40 T antigen in transformed monkey cells (COS). The nuclear structures stained by aRB1C1 and EBNA-5 antibodies are distinct from the structures detected with antibodies against centromeric proteins and certain snRNP epitopes. EBNA-5/Rb-positive domains do not selectively react with antibodies against the La antigen known to associate with the small EBV-encoded nuclear RNA species designated as the EBERs.

Intranuclear distribution of Epstein-Barr virus-encoded nuclear antigens EBNA-1, -2, -3 and -5.

Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) express at least seven virally encoded proteins. Their functional role, and their relationships to each other and to normal nuclear constituents are virtually unknown. As the first step towards a topographical study, the intranuclear distribution of EBV-encoded nuclear antigens EBNA-1, -2, -3 and -5 (abbreviated E1, E2 etc.) was examined in EBV-transformed LCLs by immunofluorescence and digital image analysis of fluorescence patterns. E1-E3 showed a finely granular distribution. The E2 patterns were virtually identical when comparing indirect staining using an E2-specific mouse monoclonal antibody with anticomplement immunofluorescence using a human antibody, rendered monospecific to E2 by absorption. The E1/E2 patterns showed 32% overlap and the E2/E3 10% overlap in the high overlap category (66.7-100%), while the E2/E2 comparison with two reagents showed 61% overlap in this category. This suggests that E2 and E3 largely appear in different nuclear structures, whereas E1 appears to be randomly distributed with regard to E2. The E5 pattern was radically different from that of E1, E2 and E3. The anti-E5 mouse monoclonal antibody detected 4-10 huge, globular, sharply circumscribed dots, located in dispersed chromatin areas, while the distribution of E1, E2 and E3 showed no obvious relationship to chromatin distribution. The methods described here allow a more refined topographical analysis of the EBNA protein family, mostly in relation to each other, in relation to other nuclear proteins, and with respect to specialized functional domains in interphase chromatin.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of the Epstein-Barr virus nuclear antigen 2 using monoclonal antipeptide antibodies.

The Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) is the only one of the EBNA proteins to have been implicated as an EBV-encoded transforming protein. More detailed studies of this protein have been hampered by the lack of EBNA-2-specific monoclonal antibodies (MAbs) and of purified protein. To overcome these problems, we isolated 5 hybridomas producing MAbs reactive with an 18 residue synthetic peptide corresponding to the carboxyterminus of EBNA-2. Four of the 5 MAbs were specifically reactive with EBNA-2 in its denatured form on immunoblots. The 5th antibody (115E) was reactive with the native form of EBNA-2. By using a one-step immunoaffinity purification method with 115E cross-linked to protein-A-Sepharose, we purified EBNA-2 to homogeneity, i.e., more than 1,200-fold, from Burkitt lymphoma cell extracts. A major 32-kDa associated protein and a less abundant 17-kDa protein were co-purified with EBNA-2. Immunoprecipitation with 115E from 35S-methionine-labelled cell extracts showed that the 32-kDa protein co-precipitated with EBNA-2 from EBV-positive cells, but was not detectable in immunoprecipitates of EBV-negative cells. When the immunoprecipitates or the purified proteins were immunoblotted with EBV-immune sera, only EBNA-2 was reactive, indicating that the associated proteins are of cellular origin. Immunoprecipitation of cells labelled with 32P-orthophosphate showed that EBNA-2, but not the associated proteins, is a phosphoprotein. The expression level of EBNA-2 varied between different EBV-carrying cell lines, as measured by a 2-site ELISA based on antibody 115E. In indirect immunofluorescence, the 115E MAb gave an EBNA-2-specific characteristic granular staining pattern. These characteristics of EBNA-2 resemble those of other viral transforming proteins.

EBV-transformed lymphoblastoid cell lines down-regulate EBNA in parallel with secretory differentiation.

Four monoclonal and one polyclonal lymphoblastoid cell line (LCL) were studied with regard to cytoplasmic immunoglobulin (cIg) expression, presence of the Epstein-Barr virus (EBV)-determined nuclear antigen (EBNA) and DNA synthesis. Each line was found to consist of two subpopulations, with only minimal overlap. Proliferating, EBNA-positive, cIg-negative cells formed the majority. The minority were EBNA-negative, contained abundant cIg and were largely non-proliferating. This suggests the continuous occurrence of a maturation process within each LCL. The concomitant down-regulation of EBNA raises the interesting question whether continued synthesis of the nuclear antigen is incompatible with differentiation for epigenetic reasons, or, alternatively, whether differentiation takes place when the viral genomes are suppressed or lost.