tetracycline and Burkitt-Lymphoma

Topics: Burkitt Lymphoma; Epstein-Barr Virus Nuclear Antigens; Gene Expression Regulation, Viral; Genetic Vectors; Herpesvirus 4, Human; Humans; Phenotype; Protein Binding; Tetracycline; Transcription Factors; Tumor Cells, Cultured; Viral Proteins; Virus Replication

As a new model to elucidate molecular mechanisms in Epstein-Barr virus (EBV) activation, we tested the tetracycline-inducible (Tet-On)/BZLF1-oriP plasmid system in Raji cells. Cells transfected with this Tet-On plasmid did not activate EBV by doxycycline and surprisingly EBV latency was disrupted with large amounts of BMRF1 protein (EA-D) being accumulated in the cells. Brilliant EA-D fluorescence was markedly condensed in small sized cells, intra-cellular vesicles, and extra-cellular particles. Scanning electron microscopy demonstrated the extra-cellular particles to be covered with a membrane. EA-D molecules of 58, 50, 48, and 44kDa were expressed in the cells. The high (58 and 50kDa) and low (48 and 44kDa) EA-D molecules appeared in the early and late stages, respectively. Low EA-D molecules were detected mostly in EA-D positive cells separated into the heaviest density layer of a discontinuous Percoll gradient. Such molecules could be created from high EA-D molecules by protein phosphatase treatment. The EA-D molecules that appeared similar were detected in EBV-activated P3HR-1 and Akata cells. Several hypotheses concerning the accumulation of EA-D molecules of various polymorphic forms and their phosphorylation/dephosphorylation in this model system are presented, with possible biological and clinical relevance.

Topics: Antigens, Viral; Blotting, Western; Burkitt Lymphoma; Cell Line; Cell Membrane; Cytoplasm; Cytoplasmic Vesicles; DNA-Binding Proteins; Gene Expression; Herpesvirus 4, Human; Humans; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Molecular Weight; Phosphoprotein Phosphatases; Plasmids; Tetracycline; Trans-Activators; Viral Proteins; Virus Activation

The product of the proto-oncogene c-myc (myc) is a potent activator of cell proliferation. In Burkitt lymphoma (BL), a human B-cell tumor, myc is consistently found to be transcriptionally activated by chromosomal translocation. The mechanisms by which myc promotes cell cycle progression in B-cells is not known. As a model for myc activation in BL cells, we have established a human EBV-EBNA1 positive B-cell line, P493-6, in which myc is expressed under the control of a tetracycline regulated promoter. If the expression of myc is switched off, P493-6 cells arrest in G0/G1 in the presence of serum. Re-expression of myc activates the cell cycle without inducing apoptosis. myc triggers the expression of cyclin D2, cyclin E and Cdk4, followed by the activation of cyclin E-associated kinase and hyper-phosphorylation of Rb. The transcription factor E2F-1 is expressed in proliferating and arrested cells at constant levels. The Cdk inhibitors p16, p21, p27 and p57 are expressed at low or not detectable levels in proliferating cells and are not induced after repression of myc. Ectopic expression of p16 inhibits cell cycle progression. These data suggest that myc triggers proliferation of P493-6 cells by promoting the expression of a set of cell cycle activators but not by inactivating cell cycle inhibitors.

Topics: Burkitt Lymphoma; Cell Cycle; Cell Cycle Proteins; Cyclin-Dependent Kinase Inhibitor p16; Humans; Neoplasm Proteins; Nuclear Proteins; Phosphorylation; Protein Synthesis Inhibitors; Proto-Oncogene Mas; Proto-Oncogene Proteins c-myc; Retinoblastoma Protein; Retinoblastoma-Like Protein p107; Tetracycline; Tumor Cells, Cultured

The Burkitt's lymphoma (BL) cell line Akata retains the latency I program of Epstein-Barr virus (EBV) gene expression and cross-linking of its surface immunoglobulin G (IgG) by antibodies results in activation of viral replication. When EBV nuclear antigen 2 (EBNA2) was artificially expressed by a constitutive expression vector, the Cp EBNA promoter remained inactive and accordingly the latency III program was not induced. In contrast, expression of LMP2A and activity of the Fp lytic promoter were activated. Consistent with this Fp activity, the rate of spontaneous activation of the EBV replicative cycle was increased significantly, suggesting the possibility that EBNA2 can induce EBV replication. The efficiency of anti-IgG-induced activation of the viral replication was reduced in Akata cells expressing EBNA2. To obtain more direct evidence for EBNA2-induced activation of the EBV replicative cycle, this protein was next expressed by a tetracycline-regulated expression system. EBNA2 was undetectable with low doses (<0.5 microgram/ml) of tetracycline, while its expression was rapidly induced after removal of the antibiotic. This induced expression of EBNA2 was immediately followed by expression of EBV replicative cycle proteins in up to 50% of the cells, as shown by indirect immunofluorescence and immunoblot analysis. These results suggest an unexpected potential of EBNA2 to disrupt EBV latency and to activate viral replication.

Topics: Burkitt Lymphoma; Epstein-Barr Virus Nuclear Antigens; Gene Expression Regulation; Humans; Tetracycline; Tumor Cells, Cultured; Virus Latency

We wish to identify developmental changes in germinal center B cells that may contribute to their rapid growth. SHP-1 is an SH2 domain-containing phosphotyrosine phosphatase that negatively regulates activation of B cells and other cells of hematopoietic lineages. We have found that in all 13 EBV-negative and 11 EBV-positive Burkitt lymphomas with a nonlymphoblastoid phenotype, the mean concentration of SHP-1 was reduced to 5% of that of normal B and T cells. The possibility that this diminished expression of SHP-1 was related to the germinal center phenotype of Burkitt lymphomas was supported by the low to absent immunofluorescent staining for SHP-1 in germinal centers, and by the inverse relationship between the concentration of SHP-1 and the expression of the germinal center marker CD38 on purified tonsillar B cells. In CD38-high B cells, SHP-1 concentration was 20% of that of mantle zone B cells from the same donor. This reduction in SHP-1 is comparable to that of cells from motheaten viable mev/mev mice in which there is dysregulated, spontaneous signaling by cytokine and antigen receptors. Therefore, germinal center B cells may have a developmentally regulated, low threshold for cellular activation.

Topics: B-Lymphocytes; Burkitt Lymphoma; Cell Differentiation; Down-Regulation; Gene Expression Regulation; Germinal Center; Humans; Intracellular Signaling Peptides and Proteins; Plasmids; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Protein Tyrosine Phosphatases; SH2 Domain-Containing Protein Tyrosine Phosphatases; Tetracycline; Transfection; Tumor Cells, Cultured