Burkitt's Lymphoma Line Daudi Research Articles

Autocrine lymphotoxin production in Epstein-Barr virus-immortalized B cells: induction via NF-kappaB activation mediated by EBV-derived latent membrane protein 1.

Epstein-Barr virus (EBV)-immortalized lymphoblastoid cells express high levels of lymphotoxin and use this molecule as an autocrine growth factor. We hypothesized that the EBV-derived latent membrane protein 1 (LMP1) mediates lymphotoxin production by inducing NF-kappaB binding to the lymphotoxin promoter. We assessed lymphotoxin production, LMP1 expression, and NF-kappaB activation in Z-43 (EBV-positive lymphoblastoid cells), Daudi (EBV-positive Burkitt's cells), and 3A4 (EBV-negative Burkitt's cells containing a stably transfected tetracycline-inducible LMP1 construct). Z-43 cells expressed high levels of LMP1 (immunoblot) and lymphotoxin (ELISA); the EBV-positive Burkitt's lymphoma line Daudi expressed neither LMP1 nor lymphotoxin. Similarly, induction of LMP1 in the 3A4 cells (exposed to tetracycline) was accompanied by a 13-fold increase in lymphotoxin levels (ELISA) as compared to uninduced (LMP1-negative) cells. EMSAs demonstrated high levels of NF-kappaB activation in Z-43 and tetracycline-induced 3A4 cells, but much lower levels in the uninduced 3A4 cells. Exposure of these cells to Bay 11-7082 (an inhibitor of IkappaB phosphorylation and, therefore, NF-kappaB activation) abrogated NF-kappaB binding and lymphotoxin production in a dose-dependent manner in both Z-43 and 3A4 cells. Therefore, in our model system, autocrine lymphotoxin production is largely driven by NF-kappaB activation, which is in turn mediated by EBV-derived LMP1 signaling.

A novel putative G-protein-coupled receptor expressed in lung, heart and lymphoid tissue

cDNA encoding a novel putative G-protein-coupled receptor, named LyGPR ( ly mphocyte derived G - p rotein-coupled r eceptor) was cloned using a reverse transcription-PCR approach. The LyGPR amino acid sequence is 375 residues long and shows similarity (about 30–35% identity) both to the angiotensin receptors and members of the chemokine receptor family. Northern blot analysis revealed a 3.1-kb LyGPR transcript expressed predominantly in lung, heart and lymphoid tissues. LyGPR expression was also detected in the pre-B acute lymphoblastoid leukemia cell lines Reh and Nalm-6, in the Burkitt's lymphoma line Daudi, and in hematopoietic progenitor cells from bone marrow, as well as in B cells, T cells and monocytes from peripheral blood.

Induction of an exceptionally high-level, nontranslated, Epstein-Barr virus-encoded polyadenylated transcript in the Burkitt's lymphoma line Daudi.

An Epstein-Barr virus transcript (designated D-HIT [Daudi high-level-inducible transcript]), constitutively expressed at low levels in the Burkitt's lymphoma (BL)-derived cell line Daudi, can be induced with tetradecanoylphorbol acetate or n-butyrate or, in combination, to about 1% of the levels of high-molecular-weight RNAs in cells. The transcript can also be induced in some other EBV-positive BL-derived cells but to a much lesser extent, particularly in lines that can give rise to productive infection. D-HIT is viral in origin and is composed largely of repetitive sequence. It is polyadenylated but mainly nuclear in location and is highly structured, sensitive only to double-strand-specific RNase. It is endogenously expressed in interferon-sensitive Daudi strains but not in an insensitive strain, Daudi 100K. D-HIT contains a part of a viral open reading frame (designated LF3, and deleted in the prototype B95-8 strain), using an internal polyadenylation (AAUAAA) sequence as a signal to specify processing of its 3' end. In Daudi cells, the promoter contains a putative hinge structure, as found in some interferon-inducible genes and c-myc. Since D-HIT lies adjacent to, probably even encompassing, one of the two viral lytic origins (D(R)) of replication, it may have a role in the regulation of DNA replication. Alternatively, or in addition via its double-stranded structure, D-HIT may play a regulatory role in interferon pathways. Its promoter could be of value for studying expression in constructions containing heterologous genes.

Up regulation of the Epstein-Barr virus (EBV)-encoded membrane protein LMP in the Burkitt's lymphoma line Daudi after exposure to n-butyrate and after EBV superinfection

The Burkitt's lymphoma line Daudi carries a nontransforming Epstein-Barr virus (EBV) strain that has a deletion in the BamHI WYH region of the genome coding for the EBV nuclear antigen 2 (EBNA-2). Daudi cells fail to express the EBV-encoded latent membrane protein (LMP) (D. Ghosh and E. Kieff, J. Virol. 64:1855-1858, 1990). We show that LMP expression can be up regulated by exposure to n-butyrate and by superinfection with the B95-8 (B virus)- and P3HR1 (P virus)-derived EBV strains. Two LMP polypeptides of 60 and 48 kilodaltons (kDa) were detected in immunoblots of Daudi cells that had been exposed to 3 mM n-butyrate for 24 h. The intensity of the 48-kDa LMP increased during 72 h, in parallel with the appearance of early antigen-positive cells. The 60-kDa LMP was expressed at a low level and remained constant. Superinfection of Daudi cells with B and P virus induced the 60-kDa LMP within 3 h. In addition, P virus induced the 48-kDa LMP at a low level. The B virus-encoded EBNA-2 and EBNA-5 were detected 12 h after superinfection. The B virus-encoded 63-kDa LMP was coexpressed with the endogenous LMP after 48 h. Inactivation of the virus by UV illumination abolished the expression of the B virus-encoded antigens but did not affect the induction of the endogenous LMP. The B-cell activation marker CD23 was up regulated by B virus superinfection but not by n-butyrate exposure. CD23 was also expressed at a higher level in a stable B virus-converted subline, E95A-Daudi, that was EBNA-2 positive and coexpressed the Daudi virus- and B virus-encoded LMP. The results suggest that LMP expression is regulated by the interaction of cellular and viral factors. Binding of the virus to its membrane receptor might be involved in the triggering of cellular control mechanisms. Viral gene products are not directly involved in this function but may contribute to create a permissive cellular environment for LMP expression.

Sublines of the Burkitt line Daudi, selected on the basis of reactivity with soybean agglutinin, differ in tumorigenicity in athymic mice

Two subpopulations were isolated on the basis of soybean agglutinin (SBA) binding, from the human Burkitt lymphoma line Daudi. The low- and high-binder sublines maintained this characteristic in continuous passages. Their surface marker profiles, antibodies, scanning electron microscope (SEM), cytochemical reactions and binding of other lectins (concanavalin A and wheatgerm agglutinin) were not different. They differed, however, in growth potential in athymic mice. The low-binder subline had lower frequency of takes, tumor weight and volume, and did not metastasize as compared to the high-binder subline. However, the reaction with F-SBA of all the tumor cells examined was strong (> 70%), indicating in vivo selection and tumor development of high binder cells.

The t(8;14) chromosome translocation of the Burkitt lymphoma cell line Daudi occurred during immunoglobulin gene rearrangement and involved the heavy chain diversity region.

Recent molecular analyses of Burkitt lymphomas carrying the t(8;14) chromosome translocation have indicated that a dichotomy exists regarding the molecular mechanisms by which the translocations occur. Most sporadic Burkitt tumors carry translocations that apparently arise due to mistakes in the immunoglobulin isotype-switching process. In contrast, there is evidence that the translocations of most endemic Burkitt lymphomas occur as a consequence of aberrant V-D-J recombination of variable, diversity, and joining gene segments, catalyzed by the recombinase enzymes. This phenomenon was first noted in follicular lymphomas and chronic lymphocytic leukemias of the B-cell lineage and has been described in T-cell malignancies as well. In each of these cases, analysis of the nucleotide sequence at chromosome breakpoints demonstrated the involvement of immunoglobulin heavy chain JH or T-cell-receptor alpha-chain J alpha gene segments in the translocation. We now have cloned and sequenced both the 8q- and 14q+ translocation breakpoints deriving from the t(8;14) translocation of the endemic Burkitt lymphoma line Daudi. Our data show that the translocation resulted from a reciprocal exchange between the DH region on chromosome 14 and sequences far 5' of the MYC protooncogene on chromosome 8. Features of the nucleotide sequences surrounding the breakpoint further implicate the V-D-J joining machinery in the genesis of chromosome translocations in endemic Burkitt lymphomas and, more generally, in other lymphoid malignancies as well.

Induction of Epstein-Barr virus early antigens by corticosteroids: inhibition by TPA and retinoic acid.

Corticosteroids can induce the synthesis of EBV antigens in the Burkitt lymphoma line Daudi. As early as 12 h after application of the drug, an increase of EA-positive cells can be seen, the maximum induction being reached after 2 days. Nanogram amounts per ml of hormone are sufficient for measurable effects. Early antigen induction by corticosteroids does not require replication of viral DNA. Induction by corticosteroid differs from induction by other systems in two major respects: (1) it does not cooperate with other inducers, and (2) it is specifically inhibited by 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Induction by corticosteroids, however, shares at least one retinoic acid-sensitive step with induction by chemicals such as TPA, 5-iodo-2-deoxyuridine (IdUrd), n-butyric acid (n-BA) or inducing serum factor. This study defines three qualitatively different effects of TPA in Daudi cells: an inhibitory effect on EBV induction by corticosteroids and two differential types of synergistic effects with serum factor or n-BA, respectively. In this particular cell line, TPA exhibits no inducing capacity when applied alone.

The properties of C4 nephritic factor

cDNA encoding a novel putative G-protein-coupled receptor, named LyGPR ( ly mphocyte derived G - p rotein-coupled r eceptor) was cloned using a reverse transcription-PCR approach. The LyGPR amino acid sequence is 375 residues long and shows similarity (about 30–35% identity) both to the angiotensin receptors and members of the chemokine receptor family. Northern blot analysis revealed a 3.1-kb LyGPR transcript expressed predominantly in lung, heart and lymphoid tissues. LyGPR expression was also detected in the pre-B acute lymphoblastoid leukemia cell lines Reh and Nalm-6, in the Burkitt's lymphoma line Daudi, and in hematopoietic progenitor cells from bone marrow, as well as in B cells, T cells and monocytes from peripheral blood.

Purification of human interleukin 2 to apparent homogeneity and its molecular heterogeneity.

Interleukin 2 (IL-2), produced with and without co-stimulation by the Burkitt's lymphoma line Daudi, was purified 37,000-fold to apparent homogeneity from lymphocyte conditioned medium by (NH4)2SO4 precipitation, DEAE-cellulose ion-exchange chromatography, gel filtration, and chromatography on blue agarose and on Procion-red agarose. The purified IL-2 showed a 10(6) U/mg protein sp act. IL-2 produced in the absence of Daudi cells had a mol wt of 26,000 as measured by gel filtration and an isoelectric point of 6.7. This IL-2 showed a 16,000 and 17,000 mol wt in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). IL-2, produced in the presence of Daudi cells (10(6)/ml), showed a mol wt of approximately 14,000, as measured by both gel filtration and SDS-PAGE, and an isoelectric point of 8.1. The purified IL-2 lacked detectable interferon (alpha and gamma), granulocyte-macrophage colony-stimulating factor, B cell growth factor, T cell-replacing factor, and thymocyte-differentiating activity and was free of any contaminating proteins as judged by silver staining in SDS-PAGE. All three molecular forms of IL-2 were biologically active at concentrations of 10(-11) - 10(-10) M, supporting the growth of human and murine cytotoxic T cell lines.

Serological distinction between DR antigens and lymphocyte activating determinants.

The Burkitt lymphoma (BL)-derived, HLA-DR antigen positive B cell line, EB1, is a consistently low stimulator in MLC. A rabbit antiserum raised against the strongly stimulating BL line DAUDI, after appropriate absorption with EB1, inhibits MLC stimulation by both B cell lines and allogeneic lymphocytes, whilst lectin-induced proliferation is not significantly affected. Indirect immunofluorescence and 125I-staphylococcal protein A binding to cells pre-incubated with this antiserum suggest that the antigen is present on both peripheral B and T cells, as well as on B lymphoblastoid and myeloma lines. We suggest that this antiserum is directed against lymphocyte activating determinant(s) (LADs) and that these are distinct from the serologically defined DR antigens.

Biosynthesis of HLA-A and HLA-B antigens in vivo.

The biosynthesis of the HLA-A and -B antigens was studied in the B lymphoblastoid cell line BRI 8 and the Burkitt lymphoma line DAudi. The heavy chains of the HLA-A and -B antigens were inserted asymmetrically into the rough endoplasmic reticulum of BRI 8 cells as transmembrane polypeptides. During the first 5 min after synthesis, the majority of the heavy chains became associated with beta 2-microglobulin. At this stage, the heavy chains carried a high mannose (core) oligosaccharide. Subsequent processing of the oligosaccharide unit occurred during the next 20 to 25 min, resulting in the conversion of the oligosaccharide from the high mannose to the complex form. About 30 to 40 min after synthesis, the mature antigens were expressed at the cell surface. Glycosylation was not required for asymmetric insertion, intracellular transport, or surface expression of the HLA-A and -B antigens. However, studies with Daudi cells indicated that combination with beta 2-microglobulin was necessary for subsequent processing and intracellular transport of the heavy chains after their synthesis in the rough endoplasmic reticulum.

The EBV-carrying, β2M/HLA deficient Burkitt lymphoma line Daudi is sensitive to EBV-specific killer T-cells of mononucleosis patients

The beta2-microglobulin/HLA deficient Burkitt lymphoma line Daudi was tested for sensitivity to EBV-specific cytotoxicity mediated by natural killer (NK)-depleted T-cells from acute mononucleosis patients. While the Daudi line was not as sensitive as the reference EBV-genome-positive target line, it was clearly sensitive in the majority of cases. This would speak against a major role of syngeneic restriction in this system.

Chromosome banding, isoenzyme studies and determination of epstein‐barr virus DNA content on human burkitt lymphoma/mouse hybrids

Four independently fused hybrid clones derived from a cross between the mouse mammary carcinoma TA3Ha and the human Burkitt lymphoma line Daudi were tested for the EBV-determined nuclear antigen (EBNA), EBV-DNA and the presence of human chromosomes, in the course of serial propagation in vitro. EBNA and EBV-DNA were lost in parallel with the loss of human chromosomes. It seems that the persistence of EB-viral genetic information does not require the presence of a specific human chromosome(s) in this particular hybrid combination.