calcimycin and Thymoma

The mechanisms by which trichothecene mycotoxins cause immunological effects in leukocytes such as cytokine up-regulation, aberrant IgA production, or apoptotic cell death are not fully understood. In the present study, mRNA differential display analysis was used to evaluate changes in gene expression induced by the trichothecene vomitoxin (VT or deoxynivalenol) in a T-cell model, the murine EL-4 thymoma, that was stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin (ION). Ten differentially expressed fragments of cDNA were isolated and sequenced and three of these were identified as the known genes GRP78/BiP, P58(IPK), and RAD17. Most notably, expression of GRP78/BiP (a 78-kDa glucose-regulated protein), a stress-response gene induced by agents or conditions that adversely affect endoplasmic reticulum (ER) function, was found to decrease in VT-exposed cells. Competitive RT-PCR analysis revealed that 250 ng/ml VT decreased GRP78/BiP mRNA expression in both unstimulated and PMA/ION-stimulated EL-4 cells at 6 and 24 h after VT treatment. Western blotting confirmed that VT (50 to 1000 ng/ml) also significantly diminished GRP/BiP protein levels in a dose-response manner in PMA/ION-stimulated cells. GRP78/BiP has been shown to play a role in regulation of protein folding and secretion, and to protect cells from apoptosis. When PMA/ION-stimulated cells were incubated with 50 to 1000 ng/ml VT for 24 h, 200-bp DNA laddering, a hallmark of apoptosis, increased in a dose-dependent manner. In addition to GRP78, mRNA expression of the cochaperone P58(IPK), which is the 58-kDa cellular inhibitor of the double-stranded RNA-regulated protein kinase (PKR), was also shown to be suppressed by VT-treatment. GRP78 and P58(IPK) are critical for maintenance of cell homeostasis and prevention of apoptosis. The down-regulation of these molecular chaperones by VT represent a novel observation and has the potential to impact immune function at multiple levels.

Topics: Animals; Blotting, Western; Calcimycin; Carrier Proteins; DNA Fragmentation; DNA, Complementary; Down-Regulation; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Enzyme Inhibitors; Heat-Shock Proteins; HSP40 Heat-Shock Proteins; Ionomycin; Ionophores; Mice; Molecular Chaperones; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; T-Lymphocytes; Tetradecanoylphorbol Acetate; Thapsigargin; Thymoma; Trichothecenes

Suppression of apoptosis appears to contribute to the development of various diseases, including autoimmune disorders and cancer. Numerous genes that encode activators and suppressors of apoptosis have been identified; however, such genes have not been shown to be expressed in all cell types. Furthermore, the sensitivity of different cell types to induction of apoptosis varies widely. We have employed a genetic approach using somatic cell hybridization to determine if apoptosis is a dominant or a recessive process in cells. These studies have utilized cell fusion partners with differing sensitivity to induction of apoptosis. The apoptosis-sensitive cells chosen were BW5147 murine thymoma cells. These cells readily undergo apoptosis in response to glucocorticoids and calcium ionophore. The resistant fusion partners were HTC rat hepatoma cells, which possess an intact glucocorticoid signal transduction pathway but are resistant to induction of apoptosis by either agent. Neither cell type expresses detectable Bcl-2 protein. Heterokaryons were identified by their retention of fluorescent cytosolic dyes and by nuclear morphology and cell size. The three types of heterokaryons observed were intratypic HTC/HTC and BW5147/BW5147 heterokaryons and intertypic BW5147/HTC heterokaryons. Glucocorticoid receptor was shown by immunohistochemistry to undergo hormone-dependent translocation to all nuclei in intertypic heterokaryons. BW5147/BW5147 heterokaryons die after treatment with glucocorticoid and calcium ionophore, whereas both HTC/ HTC and BW5147/HTC hybrids survive. The presence of multiple BW5147 cells fused to a single HTC cell did not affect this outcome. This demonstrates that HTC cells are able to dominantly suppress apoptosis in all BW5147/HTC heterokaryons. Thus, HTC cells contain activities that can suppress apoptosis in lymphocytes.

Topics: Animals; Apoptosis; Calcimycin; Cell Division; Cell Fusion; Dexamethasone; Dimethyl Sulfoxide; Hybrid Cells; Liver Neoplasms, Experimental; Lymphocytes; Mice; Rats; Thapsigargin; Thymoma; Tumor Cells, Cultured

In the WEHI7.2 thymoma cell line, cAMP, glucocorticoids, or increases in cytosolic Ca2+ concentration lead to cell death by apoptosis. In the present study, we examined the effects of these compounds on cAMP response element (CRE)-mediated gene expression. Thapsigargin and A23187 were employed to increase cytosolic Ca2+ levels and induce apoptosis. Both compounds enhanced transcription from a CRE preceding apoptotic death. Moreover, the transcriptional response to the combination of forskolin and either thapsigargin or A23187 was synergistic mirroring the effect on cell death. Importantly, dexamethasone treatment, which causes an efflux of Ca2+ from the ER, induced transcription from a CRE alone or in synergy with forskolin. The increase in CRE-controlled gene expression correlated with a decrease in cell viability. Following treatment with forskolin, thapsigargin, or dexamethasone, the CRE binding protein (CREB) was phosphorylated at levels correlating with the level of induced gene expression. These data suggest that transcriptional crosstalk between independent signaling pathways occurs in lymphocytes, and CREB may play a central role in the mediation of CRE-dependent transcription by these diverse set of apoptotic agents.

Topics: Animals; Apoptosis; Calcimycin; Calcium; Cell Division; Chloramphenicol O-Acetyltransferase; Colforsin; Cyclic AMP; Dexamethasone; Gene Expression Regulation, Neoplastic; Genes, Reporter; Glucocorticoids; Lymphocytes; Mice; Signal Transduction; Thapsigargin; Thymoma; Thymus Neoplasms; Transfection; Tumor Cells, Cultured

To investigate the human interleukin (IL)-5 gene promoter, we have constructed a plasmid with the firefly luciferase reporter gene linked to human IL-5 5' flanking sequence (nucleotides -507 to +44). We have used this plasmid to transfect the mouse EL4 T cell line, which can, under certain conditions, produce IL-5 transcripts. Phorbol 12-myristate 13-acetate, A23187 and N6, 2'-O-dibutyryl-adenosine 3':5'-cyclic monophosphate co-stimulation of EL4 cells transfected with the human IL-5/luciferase reporter gene construct resulted in maximal induction of the luciferase gene. Deletion analysis of the IL-5 promoter revealed the presence of negative regulatory elements between nucleotides -404 and -312 and two regions, located between nucleotides -312 and -227 and between nucleotides -80 and -35, that are involved in the positive regulation of the IL-5 promoter. Using electrophoretic mobility shift assays, we show that the positive element located between nucleotides -312 and -227 involves the binding of factors antigenically related to Oct1, Oct2A and Oct2B, to a perfect octamer motif located at position -244/-237. Introduction of three point mutations in the octamer motif of the IL-5/luciferase reporter gene plasmid, which results in the loss of competition for the factors binding to the IL-5 promoter sequence, reduced the production of luciferase from stimulated, transfected EL4 cells, by 90%. Octamer factors can also bind within the second positive regulatory region.

Topics: Animals; Base Sequence; Binding Sites; Bucladesine; Calcimycin; Consensus Sequence; Gene Expression Regulation; Genes; Genes, Reporter; Humans; Interleukin-5; Mice; Molecular Sequence Data; Mutagenesis, Site-Directed; Plasmids; Point Mutation; Promoter Regions, Genetic; Recombinant Fusion Proteins; Regulatory Sequences, Nucleic Acid; T-Lymphocytes; Tetradecanoylphorbol Acetate; Thymoma; Thymus Neoplasms; Transcription Factors; Transfection; Tumor Cells, Cultured

Elevation of cAMP can cause gene-specific inhibition of interleukin 2 (IL-2) expression. To investigate the mechanism of this effect, we have combined electrophoretic mobility shift assays and in vivo genomic footprinting to assess both the availability of putative IL-2 transcription factors in forskolin-treated cells and the functional capacity of these factors to engage their sites in vivo. All observed effects of forskolin depended upon protein kinase A, for they were blocked by introduction of a dominant negative mutant subunit of protein kinase A. In the EL4.E1 cell line, we report specific inhibitory effects of cAMP elevation both on NF-kappa B/Rel family factors binding at -200 bp, and on a novel, biochemically distinct "TGGGC" factor binding at -225 bp with respect to the IL-2 transcriptional start site. Neither NF-AT nor AP-1 binding activities are detectably inhibited in gel mobility shift assays. Elevation of cAMP inhibits NF-kappa B activity with delayed kinetics in association with a delayed inhibition of IL-2 RNA accumulation. Activation of cells in the presence of forskolin prevents the maintenance of stable protein-DNA interactions in vivo, not only at the NF-kappa B and TGGGC sites of the IL-2 enhancer, but also at the NF-AT, AP-1, and other sites. This result, and similar results in cyclosporin A-treated cells, imply that individual IL-2 transcription factors cannot stably bind their target sequences in vivo without coengagement of all other distinct factors at neighboring sites. It is proposed that nonhierarchical, cooperative enhancement of binding is a structural basis of combinatorial transcription factor action at the IL-2 locus.

Topics: Base Sequence; Binding Sites; Calcimycin; Cell Line; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclosporine; DNA-Binding Proteins; Gene Expression; Interleukin-2; Kinetics; NF-kappa B; RNA, Messenger; T-Lymphocytes; Tetradecanoylphorbol Acetate; Thymoma; Thymus Neoplasms; Transcription Factors; Transcription, Genetic; Transfection; Tumor Cells, Cultured

Uncouplers CCCP (2-4 microM) or DNP (200-400 microM) when added to EL-4 thymoma or Ehrlich carcinoma ascites cells initially stimulated endogenous respiration about 2-fold but then inhibited it to a first-order rate 20-25% of controls. This inhibition was accelerated by intracellular acidification or by A23187, a Ca2+/H(+)-antiporter (i.e. when mitochondrial Ca2+ efflux was stimulated) whereas Ruthenium red, an inhibitor of uniporter-driven Ca2+ efflux, significantly slowed down the effect of uncouplers. The respiratory inhibition was associated with NAD(P)H oxidation and was partially reversed by exogenous substrates (glutamine or glucose). In the permeabilized cells, endogenous and glutamine-supported respiration was inhibited by EGTA, while succinate-supported respiration was Ca2+ independent. It is suggested that mitochondrial Ca2+ is necessary for NADH-dependent respiration of tumor cells, and uncouplers inhibit it by activation of mitochondrial Ca2+ efflux.

Topics: 2,4-Dinitrophenol; Adenosine Triphosphate; Animals; Calcimycin; Calcium; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Carcinoma, Ehrlich Tumor; Dinitrophenols; Egtazic Acid; Hydrogen-Ion Concentration; Mitochondria; NAD; NADP; Oxygen Consumption; Ruthenium Red; Thymoma; Tumor Cells, Cultured; Uncoupling Agents

The WEHI7.2 thymoma cell line undergoes apoptotic cell death when exposed to glucocorticoids and agents that increase intracellular cAMP. Several lines of evidence indicate that calcium may play an important role in events culminating in lymphocyte apoptosis. In these studies, calbindin-D28K was stably overexpressed in WEHI7.2 cells to determine if increasing the Ca(2+)-binding capacity of the cell interferes with the apoptotic pathway. Indeed, stable expression of calbindin-D28K decreased the apoptotic effects of dexamethasone and forskolin, and the level of resistance to these agents correlated with the relative amount of calbindin expressed in each line. Overexpression of calbindin also increased cell survival in the presence of the calcium ionophore A23187. The stably expressed calcium-binding protein appeared to exert its protective effect subsequent to transcriptional activation, since glucocorticoid- and cAMP-induced gene expression were not affected. These data support the proposal that calcium fluxes are involved in apoptosis and suggest that high level expression of proteins that buffer calcium fluxes can effectively suppress death in apoptosis-susceptible cells.

Topics: Animals; Apoptosis; Calbindin 1; Calbindins; Calcimycin; Colforsin; Cyclic AMP; Depression, Chemical; Dexamethasone; Lymphocytes; Mice; Neoplastic Stem Cells; Recombinant Fusion Proteins; S100 Calcium Binding Protein G; Thymoma; Thymus Neoplasms; Tumor Cells, Cultured

Glucocorticoid-induced lymphocyte cell death is a programmed process which is thought to involve the calcium-dependent degradation of DNA into multiples of 180 basepairs, characteristic of internucleosomal degradation. We have used the glucocorticoid-sensitive mouse lymphoma cell line S49.1 [wild-type (wt)] and the glucocorticoid-resistant cell line S49.22r (nt-) to evaluate the role of both glucocorticoid receptors and calcium in the regulation of internucleosomal DNA degradation and expression of calcium-dependent deoxyribonuclease activity. DNA was isolated from untreated (control) and dexamethasone (dex)-treated viable cells and analyzed for internucleosomal DNA degradation by agarose gel electrophoresis, followed by ethidium bromide staining. Glucocorticoid treatment resulted in substantial internucleosomal DNA degradation in wt cells, but not in nt- cells. This effect was inhibited by coincubation of cells with dex and the glucocorticoid receptor antagonist RU486. In contrast to the glucocorticoid response, administration of either of two calcium ionophores, ionomycin or A23187, produced internucleosomal degradation of DNA in both wt and nt- cells, although the latter were less sensitive to ionophore treatment. Interestingly, A23187 treatment also resulted in a loss of cell viability in HeLa S3 cells, a cell line that does not exhibit glucocorticoid-induced apoptosis. No internucleosomal DNA degradation was detected in HeLa S3 cells killed by A23187. To determine whether similar nucleases are associated with this internucleosomal DNA degradation resulting from both glucocorticoid and calcium ionophore treatment, 0.3 M NaCl nuclear protein extracts were prepared from control and treated cells and analyzed for protein composition or nuclease activity. To assay for nuclease activity, nuclear extracts were electrophoresed in sodium dodecyl sulfate-polyacrylamide gels impregnated with [32P]DNA. Nuclease activity was detected by removal of sodium dodecyl sulfate from the gel, activation with calcium, and subsequent visualization of the loss of [32P]DNA by autoradiography. Dex treatment of wt cells resulted in the appearance of several proteins within the mol wt range of 12-18 kDa, only one of which (16-18 kDa) exhibited calcium-dependent nuclease activity. The appearance of these proteins in nuclear extracts was inhibited by coincubation of glucocorticoid-treated cells with RU 486. Glucocorticoid treatment did not result in the appearance of nuclease acti

Topics: Animals; Calcimycin; Calcium; Cell Death; Deoxyribonucleases; DNA; Glucocorticoids; Ionomycin; Lymphocytes; Mice; Mifepristone; Receptors, Glucocorticoid; Thymoma; Thymus Neoplasms; Tumor Cells, Cultured

Activated macrophages synthesize and release numerous tumoricidal soluble factors that can be divided into receptor- or nonreceptor-dependent agents. Tumor necrosis factor (TNF) would be an example of the former. In our experimental model the killing of EL4 thymoma cells by syngeneic activated macrophages involves, but not exclusively, TNF. Our results show that approximately 50% of the anti-EL4 activity expressed by macrophages can be specifically inhibited with rabbit anti-mouse TNF antibody. EL4 variants resistant to the lytic activity of TNF were still susceptible to macrophage-mediated lysis. A tumor-promoting phorbol ester, TPA, rendered TNF-sensitive and -insensitive EL4 cells resistant to M phi-mediated lysis. However, TPA down-regulated TNF-specific binding sites on both TNF-sensitive and -resistant cell surface membranes, suggesting that resistance to TNF involves postligand:receptor events. Tumor cell G-protein involvement (ADP-ribosylation), as a result of TNF-TNF receptor interactions, was investigated. The results showed that pertussis toxin was cytotoxic against TNF-sensitive and -resistant EL4 cells but not against TPA-treated target cells. Inhibitors of ADP-ribosyltransferase inhibited pertussis toxin cytotoxicity and macrophage-mediated lysis but did not interfere with recombinant TNF lytic activity.

Topics: Animals; Antibodies; Benzamides; Calcimycin; Cytotoxicity, Immunologic; Cytotoxins; Down-Regulation; Genetic Variation; Macrophage Activation; Mice; Niacinamide; Pertussis Toxin; Poly(ADP-ribose) Polymerase Inhibitors; Recombinant Proteins; Tetradecanoylphorbol Acetate; Thymoma; Thymus Neoplasms; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Virulence Factors, Bordetella

We produced the T cell hybridoma D9C1.12.17 by fusing an IL-4-producing T cell clone D9.1Hi with the AKR thymoma BW5147. The resulting hybridoma produced IL-2 as well as IL-4 even though none of the parental cells produced IL-2 after stimulation with Con A. The production of IL-2 was confirmed at the mRNA level by using an S1 nuclease protection assay. Further analysis indicated that Con A-induced IL-2 production was a common phenomenon among T cell hybridomas derived from this fusion. Although BW5147 does not produce detectable lymphokines after Con A stimulation, this line was able to produce IL-2, granulocyte-macrophage colony stimulating factor, and small amounts of IL-3 and IFN-gamma when stimulated with calcium ionophore and phorbol ester. The latter agents are thought to mimic the activating signal(s) delivered through the Ag:MHC TCR. This observation indicates that BW5147 has the ability to produce lymphokines but may lack component(s) which couple the extracellular signal to lymphokine production, and suggests that in T cell hybridomas, part of the spectrum of lymphokines produced may be contributed by BW5147.

Topics: Animals; Calcimycin; Cell Fusion; Cell Line; Colony-Stimulating Factors; Granulocyte-Macrophage Colony-Stimulating Factor; Growth Substances; Hybridomas; Interferon-gamma; Interleukin-2; Interleukin-3; Lymphokines; Mice; Mice, Inbred AKR; T-Lymphocytes; Tetradecanoylphorbol Acetate; Thymoma; Tumor Cells, Cultured

The present study was performed in an attempt to understand the mechanism involved in the inhibition of interleukin 2 (IL-2) synthesis by lipoxygenase (LO) pathway inhibitors. Using the two IL-2-producing lymphoid cell lines, (Jurkat and EL4 cells), we showed first that the inhibitory effect of the phenolic compounds tested (NDGA, BHA and caffeic acid) acted on lymphoid cells themselves and not on eventual monocytic or granulocytic contaminant cells. Secondly, these inhibitors were demonstrated as exerting their effect on two levels: they affected the events controlled by both second messengers implicated in T cell activation, namely rise of intracellular free calcium concentration [( Ca++]i) and protein kinase C (PKC) activation. For this purpose, LO inhibitor effects have been compared: (a) on IL-2 production by the two different lines: Jurkat cells, which need both signals, and EL4 cells, which require only PKC activation for the induction of this production; and (b) on the events induced by the different ways of Jurkat cell activation: PHA (or anti-CD3 monoclonal antibody) versus calcium ionophore. These results are discussed with respect to an eventual involvement of arachidonic acid [AA] derivatives in IL-2 synthesis.

Topics: Animals; Butylated Hydroxyanisole; Calcimycin; Calcium; Cell Line; Enzyme Activation; Humans; Interleukin-2; Kinetics; Lipoxygenase Inhibitors; Masoprocol; Mice; Phenols; Protein Kinase C; Tetradecanoylphorbol Acetate; Thymoma; Thymus Neoplasms

The Ca2+ ionophore A23187 increases intracellular calcium content in normal thymic cells, while it is without effect on the corresponding neoplastic cell (Ascites thymoma) and on Ehrlich ascites tumour cells. The A23187-induced total cell calcium increase in normal thymocytes takes place both in control and energy-depleted cells, while it is lacking in neoplastic cells. In addition the ionophore stimulates aerobic glycolysis of normal thymocytes, whereas it is ineffective on neoplastic cells. The study of intracellular calcium exchange properties reveals that in normal cells the ionophore A23187 provokes a 60% increase of the exchangeable pool together with a more significant, 4-fold enlargement of the unexchangeable pool. These effects are lacking in cancer cells. The data give rise to interesting considerations concerning the regulation and compartmentalization of calcium in neoplastic cells. The results will be also discussed in relation to the models that predict altered cell calcium metabolism as a cause of cancer cell high aerobic glycolysis and uncontrolled growth.

Topics: Animals; Anti-Bacterial Agents; Calcimycin; Calcium; Carcinoma, Ehrlich Tumor; Cell Membrane Permeability; Cell Transformation, Neoplastic; Mice; Neoplasms, Experimental; Rats; Thymoma; Thymus Gland; Thymus Neoplasms