demecolcine and Burkitt-Lymphoma

Latent Epstein-Barr virus (EBV) genomes are maintained in human cells as low copy number episomes that are thought to be partitioned by attachment to the cellular mitotic chromosomes through the viral EBNA1 protein. We have identified a human protein, EBP2, which interacts with the EBNA1 sequences that govern EBV partitioning. Here we show that, in mitosis, EBP2 localizes to the condensed cellular chromosomes producing a staining pattern that is indistinguishable from that of EBNA1. The localization of EBNA1 proteins with mutations in the EBP2 binding region was also examined. An EBNA1 mutant (delta325-376) disrupted for EBP2 binding and segregation function was nuclear but failed to attach to the cellular chromosomes in mitosis. Our results indicate that amino acids 325-376 mediate the binding of EBNA1 to mitotic chromosomes and strongly suggest that EBNA1 mediates EBV segregation by attaching to EBP2 on the cellular mitotic chromosomes.

Topics: Antineoplastic Agents, Phytogenic; B-Lymphocytes; Burkitt Lymphoma; Carrier Proteins; Cell Line; Chromosomes; Demecolcine; DNA; Epstein-Barr Virus Nuclear Antigens; Herpesvirus 4, Human; Humans; Microscopy, Fluorescence; Mitosis; Nuclear Proteins; Protein Binding; Protein Structure, Tertiary; RNA-Binding Proteins; Tumor Cells, Cultured

Cells undergoing apoptosis (programmed cell death) display profound morphologic and biochemical changes in the nucleus, cytoplasm, and plasma membrane. We have shown a direct temporal relationship between the onset of apoptosis in Jurkat T-cell lymphoblast cultures and a greater than two-fold increase in the signal intensity of the methylene resonance (at 1.3 ppm) as observed by proton nuclear magnetic resonance spectroscopy (1H NMR). The increase in the methylene resonance intensity was seen when apoptosis was induced by serum deprivation, glucocorticoid, and doxorubicin treatment but not in necrotic (nonapoptotic) cell death. We have found similar changes in a variety of other cell lines undergoing apoptosis including the Hut 78 T-cell leukemia, JY natural killer T-cell leukemia, Daudi B-cell lymphoma, HeLa, and 3T3 fibroblast cell lines. Furthermore, this spectral change was diminished in Bcl-2 overexpressing HL-60 cell cultures treated with doxorubicin, which were relatively resistant to apoptosis, as compared to apoptotic HL-60 cultures. 1H NMR spectroscopy therefore may be useful in detecting apoptotic cell death in vivo.

Topics: 3T3 Cells; Animals; Apoptosis; Burkitt Lymphoma; Cell Line; Culture Media, Serum-Free; Demecolcine; Dexamethasone; DNA Damage; Doxorubicin; HL-60 Cells; Humans; Leukemia; Lymphocyte Subsets; Magnetic Resonance Spectroscopy; Mice; Neoplasm Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Protons; Tumor Cells, Cultured

Human Daudi lymphoblastoid cells, which are highly sensitive to the antiproliferative action of human leukocyte alpha-interferon (IFN-alpha), and IFN-resistant and IFN-sensitive Daudi subclones (Cl2 and Cl1, respectively), contain 2300 (Kd = 20 X 10(-12) M), 3000 (Kd = 45 X 10(-12) M), and 3700 (Kd = 52 X 10(-12) M) IFN-alpha binding sites per cell, respectively. Thus, these IFN-sensitive and IFN-resistant cells have similar numbers of high-affinity IFN-alpha receptors. IFN-receptor complexes that are insoluble in Triton X-100 accumulate in IFN-sensitive but not in IFN-resistant cells. The ligand-induced accumulation of Triton-insoluble complexes in IFN-sensitive cells was inhibited by cytochalasin B. This suggests that the solubility change of IFN-receptor complexes results from their interaction with the cytoskeletal matrix. The dissociation of IFN-alpha from IFN-sensitive and IFN-resistant cells can be resolved into fast and slow components. IFN-alpha dissociates more slowly from IFN-sensitive cells than from IFN-resistant cells. Very slow dissociation of IFN-alpha from Triton-insoluble complexes correlates with this difference. These observations suggest that IFN-receptor complexes become coupled to the cytoskeletal matrix in IFN-sensitive but not in IFN-resistant cells, and that such interaction is an important element in the mechanism of the antiproliferative action of IFN-alpha on Daudi cells.

Topics: Burkitt Lymphoma; Cell Line; Cell Membrane; Cytochalasin B; Cytoskeleton; Demecolcine; Humans; Interferon Type I; Kinetics; Receptors, Immunologic; Receptors, Interferon

The interaction between polymer particles with an immune ligand on their surfaces and normal (hepatocytes) and malignant (Raji) cells with a corresponding membrane receptor was investigated by scanning and transmission electron microscopy. The influence of time, temperature, and anti-metabolites on this process was studied. The results indicate that when particles with an immunoligand are added to cells with a corresponding membrane receptor, three successive steps are initiated, the first two of which are not dependent on the temperature or the metabolic state of the cells. The first step in this process is the binding of particles to the cell surface through the immunoligand on the particle and the receptor on the cell surface. The initial binding formed by immunoligand and corresponding receptor is followed by a stronger interaction between cell and particle. This second step seems to be governed by a successive antibody-antigen interaction: the zipper mechanism. A third step in the particle-to-cell interaction is characterized by movements of the cellular surface, as revealed by membrane folds, pseudopods, and lamellae extending from the surface and enveloping large parts of the particle.

Topics: Antigen-Antibody Reactions; Antigens, Neoplasm; Antigens, Surface; Burkitt Lymphoma; Cell Line; Cell Membrane; Cytochalasin B; Demecolcine; Humans; Liver; Magnetics; Microscopy, Electron, Scanning; Polymers; Temperature

Topics: Autoradiography; Burkitt Lymphoma; Cell Line; Cell Membrane; Cell Nucleus; Chromatin; Cytoplasm; Demecolcine; Humans; Microscopy, Electron; Microtubules