sq-23377 and Burkitt-Lymphoma

We demonstrate here that selective activation of endogenous members of the caspase family and cleavage of substrates responsible for the maintenance of nuclear functional and structural integrity are major effectors of antigen receptor (AgR)- and ionomycin-triggered apoptosis in Ramos-Burkitt lymphoma (Ramos-BL) B cells. Ramos-BL B cells express significant proenzyme levels of caspase-2, -3, -7 and -8, low levels of caspase-6 and are caspase-1-negative. However, while anti-IgM and ionomycin trigger for significant activation of caspase-3, -7 and -8 at 12-16 h and at 4 h post-stimulation respectively, both anti-IgM and ionomycin fail to activate caspase-2 indicating that AgR- and ionomycin-triggered Ramos-BL B cell apoptosis is mediated by the selective activation of, at least, caspase-3, -7 and -8. Anti-IgM triggers for cleavage of the resident nuclear proteins poly(ADP-ribose) polymerase (PARP) at 8 h, lamins B1 and B2 from 12 to 16 h; likewise, ionomycin triggers for degradation of PARP at 2 h, lamins B1 and B2 at 4 h. Signal transduction through CD40 rescues Ramos-BL B cells from AgR- and ionomycin-triggered apoptosis at a very early stage of the apoptotic process by inhibiting both the early cleavage of PARP as well as the activation of caspase-3, -7 and -8 and cleavage of lamin B1; CD40-mediated rescue occurs upstream of CD40-induced expression of Bcl-2 and increased expression of Bcl-xL. In such cellular populations subject to regulation through apoptosis, dysregulation of the apoptotic mechanisms can have devastating consequences by contributing to the pathogenesis of malignancy as well as to lymphoproliferative and autoantibody disorders. An understanding of the role played by caspases in the execution of apoptosis may provide insight into the pathogenesis of these disease states and thereby provide targets for novel therapeutic strategy.

Topics: Apoptosis; B-Lymphocytes; bcl-X Protein; Burkitt Lymphoma; Caspase 1; Caspases; CD40 Antigens; Cell Cycle; Child; Enzyme Activation; Enzyme Inhibitors; Enzyme Precursors; HL-60 Cells; Humans; Immunoglobulin M; Ionomycin; Ionophores; Jurkat Cells; Lamin Type B; Male; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Receptors, Antigen, B-Cell

Ramos-Burkitt lymphoma (Ramos-BL) B cell line is a neoplastic model of normal B cell selection by apoptosis at the germinal center site during maturation of the humoral immune response and can be triggered into apoptosis by cross-linking their surface antigen receptor with antibodies directed against immunoglobulin (Ig)M (anti-IgM) or by treating with the calcium ionophore ionomycin. We have recently demonstrated that anti-IgM and ionomycin trigger significant activation of caspase-3, -7 and -8 and for cleavage of the resident nuclear proteins poly(ADP-ribose) polymerase (PARP) and lamin B1 in Ramos-BL B cells, suggesting that these caspases may be localized to the nucleus as well as to the cytoplasm of Ramos-BL B cells. In order to examine this hypothesis further, we fractionated Ramos-BL B cells into their cytosolic and nuclear components and examined for expression of the endogenous proform and active large subunit of caspase-3; procaspase-3 and its active p17 large subunit were identified in both the cytosolic and nuclear fractions of Ramos-BL B cells. Immunofluorescence staining together with ordinary and confocal microscopy confirmed the observations that procaspase-3 immunoreactivity was clearly identified in the cytoplasm and nucleus while Fas ligand staining was localized to the cell surface and PARP immunoactivity to the nucleus, which were used as controls; procaspase-3 exhibited granular nuclear immunoreactivity whereas PARP displayed diffuse nuclear immunoreactivity; both of which was more intense in the internucleolar regions. Taken together, we now present evidence that procaspases and their active large subunits are found in both the cytoplasm and the nucleus and that procaspases localized not only in the cytoplasm but also in the nucleus are activated following application of apoptotic stimulus in Ramos-BL B cells.

Topics: Antibodies, Anti-Idiotypic; Apoptosis; B-Lymphocytes; Burkitt Lymphoma; Caspase 3; Caspases; Cell Nucleus; Child, Preschool; Clonal Deletion; Cytoplasm; Enzyme Precursors; Histones; Humans; Ionomycin; Ionophores; Lamin Type B; Male; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Proteins

Depending on the microenvironment, murine gamma delta T cells differentiate into either Th1 (IFN-gamma-producing) or Th2 (IL-4-producing) cells. It is unclear, however, whether circulating human peripheral blood gamma delta T cells can be driven into Th1 or Th2 cells by modulation of the priming cytokine milieu. In this study, peripheral blood gamma delta T cells were stimulated by phosphoantigen (isopentenyl pyrophosphate) or Daudi lymphoma cells in the presence of Th1-priming (rIL-12, anti-IL-4 Ab) or Th2-priming (rIL-4, anti-IL-12 Ab) conditions. Single-cell analysis of cytokine secretion (IFN-gamma and IL-4) was performed by flow cytometry after 18 h and after restimulation of expanded gamma delta T cells. The early activation of resting gamma delta T cells was characterized by the induction of IFN-gamma. Priming under Th1 conditions induced a Th1 profile characterized by increased secretion of IFN-gamma and TNF-alpha, while Th2 conditions caused increased production of IL-4 (Th2 profile) by the gamma delta T cells. These results indicate that the major subset of human gamma delta T cells can be polarized into either Th1 or Th2 cytokine pattern depending on the cytokine milieu in which contact with antigen occurs.

Topics: Antibodies, Monoclonal; Antigens; Burkitt Lymphoma; Cell Differentiation; Cell Survival; Cells, Cultured; Flow Cytometry; Hemiterpenes; Humans; Immunophenotyping; Interferon-gamma; Interleukin-1; Interleukin-12; Interleukin-4; Ionomycin; Lymphocyte Activation; Organophosphorus Compounds; Receptors, Antigen, T-Cell, gamma-delta; Recombinant Proteins; T-Lymphocyte Subsets; Tetradecanoylphorbol Acetate; Th1 Cells; Th2 Cells; Tumor Cells, Cultured

Anti-CD20 monoclonal antibodies have been successfully employed in the clinical treatment of non-Hodgkin's lymphomas in both unmodified and radio-labeled forms. Previous publications have demonstrated that the antitumor effects of unmodified anti-CD20 mAb are mediated by several mechanisms including antibody-dependent cellular cytotoxicity, complement-mediated cell lysis, and induction of apoptosis by CD20 cross-linking. In this report, we demonstrate induction of apoptosis by three anti-CD20 monoclonal antibodies [1F5, anti-B1, and C2B8 (Rituximab)]. The magnitude of apoptosis induction was greater with the chimeric Rituximab antibody than with the murine 1F5 and anti-B1 antibodies. Apoptosis could be enhanced with any of the antibodies by cross-linking with secondary antibodies (or Fc-receptor-bearing accessory cells). The signaling events involved in anti-CD20-induced apoptosis were investigated, including activation of protein tyrosine kinases, increases in intracellular Ca2+ concentrations, caspase activation, and cleavage of caspase substrates. Our results indicate that anti-CD20-induced apoptosis can be attenuated by PP1, an inhibitor of protein tyrosine kinases Lck and Fyn, chelators of extracellular or intracellular Ca2+, and inhibitors of caspases, suggesting that anti-CD20-induced apoptosis may involve modulation of these signaling molecules. We also demonstrated that varying the expression of Bc1-2 did not affect the magnitude of anti-B1-induced apoptosis, possibly because of the sequestering effects of other Bc1-2 family members, such as Bad. These studies identify several of the signal-transduction events involved in the apoptosis of malignant B cells that transpire following ligation of CD20 by anti-CD20 antibodies in the presence of Fc-receptor-expressing cells or secondary goat anti-(mouse Ig) antibodies and which may contribute to the tumor regressions observed in mouse models and clinical trials.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Antigens, CD20; Apoptosis; B-Lymphocytes; bcl-Associated Death Protein; Burkitt Lymphoma; Calcium; Calcium Signaling; Carrier Proteins; Caspases; Chelating Agents; Cysteine Proteinase Inhibitors; Enzyme Activation; fas Receptor; Humans; Immunoglobulin Fc Fragments; Ionomycin; Lymphoma, B-Cell; Lymphoma, T-Cell; Mice; Phosphoprotein Phosphatases; Phosphorylation; Poly(ADP-ribose) Polymerases; Protein Processing, Post-Translational; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-bcl-2; Receptor Aggregation; Recombinant Fusion Proteins; Rituximab; Signal Transduction; Tumor Cells, Cultured

Owing to its lineage and differentiation stage-restricted expression, CD77 has been mooted as a therapeutic target in Burkitt lymphoma (BL). The recognition that the globotriaosyl moiety of this neutral glycosphingolipid is a receptor for Escherichia coli-derived Verotoxin-1 (Shiga-Like Toxin-1) offers a potential delivery system for the attack. Here we show that CD77-expressing Group I BL cells which are normally susceptible to activation-induced death on binding Verotoxin-1 B chain are protected in the presence of CD40 ligand. Ectopic expression of either bcl-2 or bcl-xL also afforded resistance to the actions of the B chain. In total contrast, neither of the survival genes nor a CD40 signal - even when acting in concert - protected against killing mediated by the holotoxin. These findings indicate that while therapeutic modalities for CD77-expressing B cell tumors (which include follicular lymphoma) based on the use of Verotoxin-1 B chain might be compromised by the activation of endogenous or exogenous survival pathways, those exploiting the holotoxin should be left unscathed.

Topics: Antibodies, Monoclonal; B-Lymphocytes; bcl-X Protein; Burkitt Lymphoma; CD40 Ligand; Cell Death; Cell Line; DNA; Escherichia coli; Humans; Ionomycin; Protein Subunits; Proto-Oncogene Proteins c-bcl-2; Receptors, Cell Surface; Shiga Toxin 1; Signal Transduction; Trihexosylceramides

The new and growing family of interleukin-1beta-converting enzyme (ICE) cysteine proteases are now recognised to be major effectors of cellular death by apoptosis. Like other members of this family, the CPP32/Yama proform is activated by processing to its active heterodimeric enzyme or apopain when it likely contributes to the process of apoptosis by cleaving poly(ADP-ribose) polymerase (PARP) and thereby inhibiting much of its DNA repair activity. Apoptosis plays a fundamental role in the regulation of the immune system where it is involved in the selection of both T and B lymphocytes bearing antigen receptor (AgR) for non-self. Cells of the Ramos Epstein-Barr virus (EBV)-genome-negative Burkitt lymphoma (BL) B cell line (Ramos-BL) can be triggered into growth arrest and apoptosis by treating with the calcium ionophore ionomycin or by crosslinking their surface AgR with antibodies directed against immunoglobulin (Ig)M (anti-IgM). Ionomycin- and AgR-triggered growth arrest and apoptosis are arrested by signals transduced through the surface CD40 of Ramos-BL B cells. Both ionomycin and anti-IgM trigger activation of CPP32 and cleavage of PARP prior to the onset of apoptosis; this process is abrogated by treatment with anti-CD40 and is independent of Bcl-2 expression. A tripeptide inhibitor of ICE family cysteine proteases, Z-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) inhibits ionomycin- and AgR-triggered CPP32 activation, PARP cleavage and apoptosis, but not growth arrest, in Ramos-BL B cells. Thus, in this report we demonstrate that in a physiological system, activation of endogenous members of the ICE family, including CPP32, and cleavage of the death substrate PARP act as major effectors of apoptotic death.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antibodies; Apoptosis; B-Lymphocytes; Burkitt Lymphoma; Calcium; Caspase 3; Caspases; CD40 Antigens; Cell Line; Child, Preschool; Cysteine Endopeptidases; Humans; Immunoglobulin M; Ionomycin; Ionophores; Male; Mice; Poly(ADP-ribose) Polymerases; Protease Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Receptors, Antigen, B-Cell; Sheep; Time Factors

Using a Burkitt lymphoma cell line to model human B-cell apoptosis in vitro, we observed that crosslinking, by antibody, of cell surface immunoglobulin induced G1 growth-arrest followed by apoptosis. By contrast, cells treated with the Ca(2+)-ionophore, ionomycin, generated apoptotic signals in G2/M as well as in G1. Both ionomycin and anti-immunoglobulin treatment induced rapid dephosphorylation of Rb prior to apoptosis. Apoptosis was repressed following exposure to CD40-ligand and was accompanied by hyperphosphorylation of Rb and cell-cycle progression but not Bcl-2 expression. Expression of Bcl-2 protein in stable bcl-2-transfectants, also resulted in repression of apoptosis and anti-immunoglobulin-treated cells no longer underwent growth-arrest. In Bcl-2-expressing cells in which apoptosis was repressed, Rb remained hyperphosphorylated, even during G1-arrest induced by ionomycin. TGF beta treatment of Bcl-2-expressing cells induced G1-arrest, de-phosphorylation of Rb and apoptosis. These results suggest that the functional activity of Bcl-2 in B-lymphoma cells is dependent upon, or leads to, sustained hyperphosphorylation of Rb and that Rb hyperphosphorylation can be uncoupled from cell-cycle progression.

Topics: Antibodies, Anti-Idiotypic; Apoptosis; B-Lymphocytes; Burkitt Lymphoma; CD40 Ligand; Cell Cycle; G1 Phase; Humans; Ionomycin; Ionophores; Membrane Glycoproteins; Phosphorylation; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Retinoblastoma Protein; Signal Transduction; Tumor Cells, Cultured

The B cell response to ligation of surface immunoglobulin (sIg) and CD40 is dependent on the stage of cellular differentiation of the population studied. Cross-linking sIg promotes proliferation of follicular mantle (FM) B cells, rescues germinal center (GC) B cells from spontaneous apoptosis but induces apoptosis in susceptible Burkitt lymphoma (BL) B cells; signals transduced through CD40 induce resting FM B cells to enter cell cycle while promoting GC and BL B cell survival. This study investigates whether the 3', 5'-cyclic adenosine monophosphate (cAMP)-dependent second-messenger pathway plays a role in the regulation of these sIg- and CD40-promoted B cell responses, using prostaglandin E2 (PGE2) and forskolin to artificially increase intracellular levels of cAMP. The Epstein-Barr virus (EBV)-genome-negative BL B cell line Ramos is susceptible to growth arrest and apoptosis triggered by calcium ionophore, anti-IgM and forskolin but not by PGE2; forskolin does not affect the outcome of anti-IgM treatment. Anti-CD40 rescues Ramos-BL B cells from ionophore- and anti-IgM-triggered but not forskolin-triggered growth arrest and apoptosis; moreover, forskolin and anti-CD40 act additively and independently for enhanced growth inhibition. By contrast, both forskolin and PGE2 potentiate the proliferative response of FM B cells cultured with anti-Ig and anti-CD40 together but not individually. Forskolin and PGE2 fail to affect the spontaneous apoptosis and anti-Ig- and anti-CD40-promoted survival of GC B cells. Thus, the cAMP-dependent second messenger pathway can differentially influence the BL, FM, and GC B cell functional response to signals transduced through sIg and CD40.

Topics: Adjuvants, Immunologic; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Apoptosis; B-Lymphocytes; Burkitt Lymphoma; CD40 Antigens; Cell Line, Transformed; Colforsin; Cyclic AMP; Dinoprostone; Growth Inhibitors; Herpesvirus 4, Human; Humans; Immunoglobulin D; Immunoglobulin M; Ionomycin; Palatine Tonsil; Receptors, Antigen, B-Cell; Second Messenger Systems; Tumor Cells, Cultured

We have previously shown that interferon-alpha (IFN-alpha) can repress apoptosis in Burkitt lymphoma (BL) cells. In this study, we have compared this protective response with a further, well-established effect of IFN-alpha on BL cells, that of growth arrest. Of a panel of BL lines comprising (i) EBV-positive and -negative lines that retain the phenotype of the parental tumour cells and (ii) the prototype IFN-alpha-growth-inhibited line, Daudi, only Daudi cells were found to undergo substantial growth inhibition in response to the cytokine. By contrast, all lines, with the notable exception of Daudi, were protected by IFN-alpha from high-rate apoptosis initiated by the Ca2+ ionophore ionomycin. Ionomycin failed to elicit an IFN-alpha-repressible apoptotic response in either wild-type Daudi cells or IFN-resistant sublines that were refractory to the growth-arresting effects of the cytokine. Analysis of c-myc protein levels confirmed previous observations that repression of apoptosis in IFN-alpha-rescuable BL cells was associated with an early inhibition of myc that was followed by a return to high-level expression. Significantly, ionomycin alone induced a comparable transient inhibition of myc protein in Daudi cells. In Daudi cells, but not in IFN-alpha-rescuable BL cells, renewed expression of myc observed after the early, transient down-regulation was followed by sustained down-regulation of the protein, which paralleled growth arrest. Our results indicate that long-term growth arrest and repression of apoptosis in BL are distinct cellular responses to IFN-alpha.

Topics: Apoptosis; B-Lymphocytes; Burkitt Lymphoma; Cell Division; Cell Line; DNA, Neoplasm; Dose-Response Relationship, Drug; Gene Expression; Humans; Interferon-alpha; Ionomycin; Kinetics; Proto-Oncogene Proteins c-myc; Thymidine; Tumor Cells, Cultured

Chromosomal translocation and subsequent de-regulation of the c-myc proto-oncogene are considered to be critical events in the multi-stage evolution of Burkitt lymphoma (BL). It is widely accepted that Myc protein functions as a competence factor for proliferation. However, recent studies indicate that it can also act in some cell types as a regulator of apoptosis. BL cell populations display a high frequency of apoptosis in vivo, a property which is also readily demonstrable in vitro in group I BL cell lines. Such lines are known to retain the cell surface marker characteristics of the parental tumour cells and, in the case of Epstein-Barr virus-positive tumours, their restricted viral protein expression. We have shown previously that apoptosis in a group I BL cell line is inhibited by interferon (IFN)-alpha. Here we show that IFN-alpha-mediated suppression of apoptosis in group I BL cells corresponds temporally with inhibition of Myc protein levels. Furthermore, inhibition of Myc expression following treatment with c-myc anti-sense oligonucleotides markedly enhanced survival of group I BL cells. These results indicate that, whilst c-myc may facilitate cycling of tumour cells in which it is de-regulated, it also stimulates their apoptosis.

Topics: Apoptosis; Base Sequence; Burkitt Lymphoma; Cell Division; Humans; Interferon-alpha; Ionomycin; Molecular Sequence Data; Oligonucleotides, Antisense; Proto-Oncogene Mas; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; Tumor Cells, Cultured

Activation of lymphocytes with 10 microM ionomycin leads to a rapid increase in the concentration of free cytoplasmic calcium ([Ca2+]i) and, at a slower rate, also to an increase in the cytoplasmic free magnesium concentration ([Mg2+]i). The ionomycin-induced Mg(2+)-mobilization response is dependent on the influx of extracellular Ca2+. After receptor-mediated lymphocyte activation, induced by mitogens or anti-receptor antibodies, a Mg(2+)-mobilization response does occur in a small fraction of the cells. Simultaneous measurement of [Ca2+]i and [Mg2+]i in individual cells showed that the receptor-triggered Mg(2+)-mobilization response is restricted to cells that have a high [Ca2+]i. It can therefore be concluded that a high [Ca2+]i induces the release into the cytoplasm of Mg2+ from intracellular stores.

Topics: Burkitt Lymphoma; Calcium; Cells, Cultured; Cytoplasm; Flow Cytometry; Fluorescent Dyes; Humans; Indoles; Ionomycin; Kinetics; Lymphocyte Activation; Magnesium; T-Lymphocytes; Time Factors; Tumor Cells, Cultured

We examined the response in the free intracellular calcium concentration ([Ca2+]i) of Daudi (human lymphoblastoid) cells to antibodies against human immunoglobulins (anti-Ig), and the relationship of [Ca2+]i to anti-Ig-induced capping. At 80 microM intracellular quin-2 (a fluorescent probe for [Ca2+]i), anti-Ig (10 micrograms/ml) caused a rapid increase in [Ca2+]i from 100 to 600 nM; the signal returned to baseline with approximately 1 min. At 450 microM intracellular quin-2, [Ca2+]i rose to only approximately 250 microM, and the signal declined gradually, returning to base line after greater than 7 min. In subsequent experiments, the lower concentrations of quin-2 were employed. Plots of the amplitude of the [Ca2+]i transients and of the binding of 125I-anti-Ig to Daudi cells versus the concentrations of anti-Ig showed similar saturation kinetics, with half-saturation occurring at 2-3 micrograms/ml. Part of the calcium in the transient is derived from the extracellular medium, and part from the nonmitochondrial intracellular stores. Caffeine (4 mM) and 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate HCl (0.5 mM) suppressed the release of calcium from internal stores and the entry of calcium from outside the cells, but permitted capping in more than half of the cells. Phorbol esters (1-2 nM) inhibited both capping and the anti-Ig-induced decrease in [Ca2+]i. None of these agents blocked the binding of anti-Ig to the cells. It appears that receptor capping is not dependent on the anti-Ig-induced transient increase in calcium concentration.

Topics: Aminoquinolines; Antibodies, Anti-Idiotypic; Burkitt Lymphoma; Caffeine; Calcium; Cell Line; Cytosol; Ethers; Humans; Immunoglobulins; Interferons; Ionomycin; Kinetics; Spectrometry, Fluorescence; Tetradecanoylphorbol Acetate

In many viral infections the host cell carries the viral genome without producing viral particles, a phenomenon known as viral latency. The cellular mechanisms by which viral latency is maintained or viral replication is induced are not known. The modulation of intracellular calcium concentrations by calcium ionophores induced Epstein-Barr viral antigens in lymphoblastoid cell lines that carry the virus. When calcium ionophores were used in conjunction with direct activators of protein kinase C (12-O-tetradecanoyl phorbol-13-acetate and a synthetic diacylglycerol), a greater induction of viral antigens was observed than with either agent alone. Activation of protein kinase C may be required for the expression of the viral genome.

Topics: Aminoquinolines; Burkitt Lymphoma; Calcimycin; Calcium; Cell Line; Cell Transformation, Viral; Culture Media; Ethers; Fluorescent Dyes; Genes, Viral; Herpesvirus 4, Human; Humans; Ionomycin; Kinetics; Tetradecanoylphorbol Acetate