calcimycin and Burkitt-Lymphoma

Apoptosis and necrosis are two forms of cell death that are induced under different conditions and that differ in morphological and biochemical features. In this report, we show that, in the presence of oxidative stress, human B lymphoma cells are unable to undergo apoptosis and die instead by a form of necrosis. This was established using the chemotherapy drug VP-16 or the calcium ionophore A23187 to induce apoptosis in Burkitt's lymphoma cell lines and by measuring classical markers of apoptotic death, including cell morphology, annexin V binding, DNA ladder formation, and caspase activation. In the presence of relatively low levels of H2O2 (75-100 microM), VP-16 and A23187 were unable to induce apoptosis in these cells. Instead, the cells underwent non-apoptotic cell death with mild cytoplasmic swelling and nuclear shrinkage, similar to the death observed when they were treated with H2O2 alone. We found that H2O2 inhibits apoptosis by depleting the cells of ATP. The effects of H2O2 can be overcome by inhibitors of poly(ADP)-ribosylation, which also preserve cellular ATP levels, and can be mimicked by agents such as oligomycin, which inhibit ATP synthesis. The results show that oxidants can manipulate cell death pathways, diverting the cell away from apoptosis. The potential physiological ramifications of this finding will be discussed.

Topics: Adenosine Triphosphate; Annexin A5; Apoptosis; Benzamides; Burkitt Lymphoma; Calcimycin; Caspase Inhibitors; Caspases; DNA Fragmentation; Etoposide; Humans; Hydrogen Peroxide; Oligomycins; Oxidative Stress; Protein Binding; Quinazolines; Quinazolinones; Tumor Cells, Cultured

Bcl-2 is an oncogene that confers deregulated growth potential to B lymphocytes through its ability to inhibit apoptotic cell death. A specific molecular activity for the Bcl-2 protein has not been identified, but several lines of evidence have supported a role in protection of cells from oxidative stress. We investigated whether there is a correlation between expression of high levels of Bcl-2 and susceptibility of human Burkitt's lymphoma cell lines to H2O2-induced killing. The amount of H2O2 required to kill 50% of cells in 24 hours varied widely in the seven different lymphoma cell lines that were tested, ranging from 35 to 500 micromol/L H2O2. However, expression of high levels of endogenous Bcl-2 did not protect the cells from H2O2-induced killing, even though it was effective in protecting the cells from apoptosis induced by agents such as A23187. Thus, Bcl-2 was functional in preventing apoptosis but did not act in an antioxidant capacity. The results were confirmed using a Burkitt's lymphoma cell line overexpressing transfected bcl-2. The results may be explained by the observation that H2O2 was inefficient at inducing apoptosis in these mature B-cell lines. Nonapoptotic death induced by H2O2 was not prevented by Bcl-2.

Topics: Apoptosis; Burkitt Lymphoma; Calcimycin; Cell Death; Genes, p53; Herpesviridae Infections; Herpesvirus 4, Human; Humans; Hydrogen Peroxide; Neoplasm Proteins; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Recombinant Fusion Proteins; Transfection; Tumor Cells, Cultured; Tumor Virus Infections

Initiation of the Epstein-Barr virus (EBV) lytic cycle is dependent on the transcription of the BZLF1 gene. The BZLF1 gene promoter (Zp) was activated by crosslinking of cell surface immunoglobulin (Ig) with anti-Ig antibody in B cells, even in the absence of other viral genes. We identified several anti-Ig response elements within Zp, which were originally defined as 12-O-tetradecanoylphorbol-13-acetate (TPA) response elements (ZI repeats and ZII, an AP-1-like domain). Since anti-Ig crosslinking leads to activation of protein kinase C (PKC) and an increase in intracellular calcium level, Zp was tested for the response to these cellular factors. Treatment with calcium ionophore A23187 increased Zp activity. When the calcium ionophore was used in conjunction with TPA, a PKC activator, the Zp induction was synergistically enhanced. 1-(5-Isoquinolinyl sulfonyl)-2-methylpiperazine, an inhibitor of PKC, inhibited the anti-Ig inducibility of Zp. Calmodulin antagonists, compound R24571 and trifluoperazine, blocked the Zp activation with anti-Ig. These findings suggest that Zp responds directly to changes in the activity of both PKC and calcium/calmodulin-dependent protein kinase. Requirement of tyrosine kinase activation for the anti-Ig-mediated Zp activation was also demonstrated through the use of the tyrosine kinase inhibitor herbimycin. These cellular gene regulatory molecules induced with anti-Ig may cooperatively play an important part in achieving efficient EBV activation as seen with anti-Ig treatment in B cells.

Topics: Antibodies; B-Lymphocytes; Base Sequence; Burkitt Lymphoma; Calcimycin; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; DNA-Binding Proteins; Drug Synergism; Enzyme Activation; Gene Expression Regulation, Viral; Herpesvirus 4, Human; Humans; Models, Genetic; Molecular Sequence Data; Promoter Regions, Genetic; Protein Kinase C; Protein-Tyrosine Kinases; Receptors, Antigen, B-Cell; Second Messenger Systems; Tetradecanoylphorbol Acetate; Trans-Activators; Tumor Cells, Cultured; Viral Proteins

The promoter of the human gene encoding the stress-responsive protein polypeptide-binding protein/78 kDa glucose-regulated protein (BiP/GRP78) was isolated from Burkitt's lymphoma cells by PCR. This promoter DNA segment (termed BiP670) or one of its 5' deletion derivatives was fused to the bacterial chloramphenicol acetyltransferase gene and introduced into HeLa cells for transient expression. BiP670 retained transcriptional activity at both the basal and Ca2+ ionophore A23187-inducible levels. However, there was no significant increase in promoter activity following a 5 h induction with 7 microM-A23187, and less than 5-fold induction at 15 h. In contrast, the steady-state mRNA level was induced by 18-fold at 5 h. The in vivo transactivation assays with BiP670 5' deletion derivatives indicate that the putative A23187-inducible element is located within a 70 bp DNA segment (i.e. spanning -39 to -107 bp upstream of the transcriptional initiation site). Using an in vitro gel mobility shift assay, A23187-inducible nuclear factors were identified from HeLa cell extracts. DNA-binding competition experiments also suggest that the 70 bp DNA segment contains a potential sequence motif for the binding of the A23187-inducible nuclear factors.

Topics: Base Sequence; Binding, Competitive; Burkitt Lymphoma; Calcimycin; Carrier Proteins; Chloramphenicol O-Acetyltransferase; Cloning, Molecular; DNA; Endoplasmic Reticulum Chaperone BiP; Gene Expression Regulation; Heat-Shock Proteins; HeLa Cells; Humans; Molecular Chaperones; Molecular Sequence Data; Oligonucleotides; Polymerase Chain Reaction; Promoter Regions, Genetic; RNA; Transcriptional Activation; Tumor Cells, Cultured

The c-fos proto-oncogene is found to be overexpressed at least 30-fold in SMS-SB, a pre-B leukemic cell line compared to other cell types. No gross alteration of the c-fos gene structure in SMS-SB cells can be detected by karyotypic or Southern analyses. C-fos in SMS-SB cells can still be induced by serum, TPA and the calcium ionophore, A23187, and superinduced by the combination of serum and cycloheximide. The elevated levels of c-fos transcripts are not due merely to an increased life span of mRNA, as the half-life of steady state c-fos mRNA in SMS-SB cells is about 40 min. Nuclear run-on transcription assays demonstrate that the transcription rate of c-fos in SMS-SB cells is up-regulated about 30-fold as compared to another pre-B leukemic cell line, NALM-6. In order to determine whether this is a cis- or transacting effect, we sequenced the 550 base pairs upstream of the SMS-SB c-fos coding region and found no mutations upstream of the mRNA CAP site. However, two point mutations at positions +23 and +98, respectively, were found in the 5' noncoding region of the first exon. Consistent with the overexpression of c-fos mRNA, the 55 kD c-fos protein is present in SMS-SB cells through detection by immunoprecipitation, but could not be detected in NALM-6 cells. SMS-SB cells however, do not appear to contain more abundant AP-1 DNA binding activity than NALM-6 cells.

Topics: Base Sequence; Blotting, Southern; Burkitt Lymphoma; Calcimycin; Cell Line; Cyclic GMP; DNA; Gene Expression Regulation, Neoplastic; Humans; Molecular Sequence Data; Polymerase Chain Reaction; Proto-Oncogene Mas; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fos; RNA; Tetradecanoylphorbol Acetate; Transcription, Genetic

A preparation of three C-terminal fragments of the platelet protein beta-thromboglobulin was previously described to have immunomodulatory properties on phagocytic cells. One of the components is obviously identical to the recently described neutrophil-activating peptide 2 (NAP-2). In further investigations on this protein preparation (called factor C) we are able to show an additional influence on the tumour-cytolytic activities of mononuclear cells. Total neutralization of the factor C effect, by treating a factor C preparation with specific monoclonal antibody C24 prior to application in cell culture, proved that the effect is really restricted to factor C proteins. If factor C is given in combination with natural interleukin-2 (IL-2) a dose-dependent suppression of IL-2-mediated natural killer lymphokine-activated killer activity can be measured, which is first detectable 72 h after addition of factor C. Suppression does not occur if the both factors are added within a time interval of more than 12 h. Depletion of monocytes from mononuclear cells has no effect on factor-C-mediated cytotoxicity, demonstrating that factor C acts directly on lymphoid cells.

Topics: Amino Acid Sequence; Antibodies, Monoclonal; Arthropod Proteins; beta-Thromboglobulin; Burkitt Lymphoma; Calcimycin; Cells, Cultured; Cytotoxicity, Immunologic; Drug Synergism; Enzyme Precursors; Humans; Immunity, Cellular; Immunotherapy; Interleukin-2; Killer Cells, Lymphokine-Activated; Killer Cells, Natural; Leukocytes, Mononuclear; Lymphocyte Activation; Lymphocytes; Molecular Sequence Data; Serine Endopeptidases; Stimulation, Chemical; Tetradecanoylphorbol Acetate; Time Factors; Tumor Cells, Cultured

Anti-IgG treatment activated latent EBV genomes in 50 to 70% of the cells of the Burkitt's lymphoma cell line Akata. The EBV-activating role of intracellular Ca2+, as potentiated by diacylglycerol (DAG) and suppressed by cAMP, was analyzed in the cells through effects of agonists and antagonists of these second messenger pathways. Early Ag (EA) was induced in 10% of cells with the calcium ionophore A23187 (A23187). EA induction with anti-IgG or A23187 was blocked by a calmodulin antagonist, trifluoperazine. The DAG pathway had a potentiating but not direct effect on EBV activation because: 1) the DAG analog, dioctanoylglycerol (diC8), an agonist for protein kinase C, alone induced only 2% EA-positive cells, 2) diC8 synergized with A23187 for EA induction, and 3) the protein kinase C antagonist, staurosporine, almost completely inhibited EA induction by anti-IgG. When cells were reincubated in medium with fresh diC8 and A23187 at 3, 6, 9, and 12 h, EA induction at 24 h reached the levels seen with anti-IgG stimulation. A cAMP-mediated pathway suppressed EBV activation because dibutyryl cAMP or 8-bromo-cAMP, plus blockage of phosphodiesterase by theophylline, or use of forskolin, inhibited EA induction with anti-IgG. Although the principal stimulatory role in EBV activation of a Ca2(+)-mediated, second messenger pathway, as synergized by DAG and inhibited by cAMP, was established, we did not explain the significant lag in EA induction by A23187 and diC8 as compared with anti-IgG induction of EA. We conclude that EBV genome activation with anti-IgG is mediated by Ca2+/calmodulin and DAG pathways in Akata cells, that the cAMP pathway suppresses EA induction by anti-IgG, and that a mechanism regulating the speed of EA induction remains unexplained.

Topics: Alkaloids; Antibodies, Anti-Idiotypic; B-Lymphocytes; Burkitt Lymphoma; Calcimycin; Cyclic AMP; Diglycerides; Herpesvirus 4, Human; Humans; Immunoglobulin G; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Peptides; Protein Kinase C; Receptors, Antigen, B-Cell; Signal Transduction; Staurosporine; Trifluoperazine; Tumor Cells, Cultured; Virus Replication; Wasp Venoms

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