sq-23377 and Thymoma

The T-box transcription factors T-bet and eomesodermin (Eomes) have been shown to regulate the lineage-specific expression of interferon-γ (IFN-γ). However, in contrast to T-bet, the role of Eomes in the expression of IFN-γ remains unclear. In this study, we investigated the Eomes-dependent expression of IFN-γ in the mouse thymoma BW5147 and EL4 cells, which do not express T-bet or Eomes. The ectopic expression of Eomes induced BW5147 and EL4 cells to produce IFN-γ in response to phorbol 12-myristate 13-acetate (PMA) and ionomycin (IM). In BW5147 cells, Eomes augmented luciferase activity driven by the Ifng promoter encoding from -2500 to +113 bp; however, it was not increased by a stimulation with PMA and IM. A chromatin immunoprecipitation assay showed that Eomes bound to the Ifng promoter and conserved noncoding sequence (CNS) -22 kb across the Ifng locus with high efficacy in BW5147 cells. Moreover, Eomes increased permissive histone modifications in the Ifng promoter and multiple CNSs. The stimulation with PMA and IM greatly augmented Eomes binding to CNS-54, CNS-34, CNS+19 and CNS+30, which was inhibited by FK506. These results indicated that Eomes bound to the Ifng promoter and multiple CNSs in stimulation-dependent and stimulation-independent manners.

Topics: Animals; Base Sequence; Cell Line, Tumor; Conserved Sequence; Humans; Interferon-gamma; Ionomycin; Mice; Promoter Regions, Genetic; T-Box Domain Proteins; Tetradecanoylphorbol Acetate; Thymoma; Thymus Neoplasms

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

Trichothecene mycotoxins have been reported to suppress or superinduce cytokine mRNA expression by leukocytes both in vitro and in vivo. Modulation of transcription factor activities may be critical for these observations. Here, the effect of trichothecene vomitoxin (VT, deoxynivalenol) on activator protein-1 (AP-1) activity was determined in the murine EL-4 thymoma. Electrophoretic mobility shift assay (EMSA) revealed that VT modulated AP-1 binding activity in a concentration- and time-dependent manner when using a synchronous model in which VT was added concurrently with phorbol 12-myristate 13-acetate (PMA) and ionomycin (ION) to EL-4 cells. Induction of AP-1 binding activity by PMA/ION was suppressed in the presence of VT for a short period (1 to 12 h), but was enhanced upon prolonged VT exposure (48 to 72 h). VT also enhanced AP-1 binding activity when added to the cell culture 12 h after PMA/ION activation (delayed synchronous model). Using specific antibodies against AP-1 complex proteins, it was demonstrated by gel supershift assay that VT preferentially affected phosphorylated c-Jun, Jun B, c-Fos, and Fra-2 binding activities, whereas it did not alter Jun D and Fra-1 binding. A transient transfection assay demonstrated that these increased binding activities are associated with enhanced AP-1 transactivation potential. Elevation of AP-1 activity may contribute to cytokine dysregulation and immunotoxic effects associated with exposure to trichothecene mycotoxins such as VT.

Topics: Animals; DNA-Binding Proteins; Down-Regulation; Fos-Related Antigen-2; Ionomycin; Kinetics; Mice; Phosphorylation; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Tetradecanoylphorbol Acetate; Thymoma; Transcription Factor AP-1; Transcription Factors; Transcriptional Activation; Trichothecenes; Tumor Cells, Cultured

We have previously shown that while spleen cells from untreated mice bearing a large MOPC-315 tumor are not cytotoxic in vitro for MOPC-315 tumor cells, spleen cells obtained from such mice on day 7 after low-dose melphalan (L-phenylalanine mustard); L-PAM therapy exert a substantial anti-MOPC-315 cytotoxicity [Mokyr et al. (1989) Cancer Res 49: 4597]. Here we show that this anti-MOPC-315 lytic activity is evident by day 5, and peaks on day 7 after the low-dose chemotherapy, at a time when the mice are actively engaged in tumor eradication. Short-term exposure of spleen cells from mice bearing a MOPC-315 tumor and treated with low-dose L-PAM (L-PAM TuB mice) to phorbol 12-myristate 13-acetate (PMA) was found to enhance greatly the ability of these spleen cells to lyse MOPC-315 tumor cells. The highest level of anti-MOPC-315 cytotoxicity was obtained when spleen cells from tumor-bearing mice that had received chemotherapy 7 days earlier were exposed to PMA at a concentration of 1-10 ng/ml. The exertion of the enhanced anti-MOPC-315 lytic activity by L-PAM TuB spleen cells exposed to PMA was found to require CD8+, but not CD4+, T cells. The apparent specificity of the lytic activity exerted by the PMA-stimulated L-PAM TuB spleen cells was illustrated not only by the inability of the spleen cells to lyse an allogeneic, antigenically unrelated thymoma (EL4), but also by their relatively weak lytic activity for two antigenically related syngeneic plasmacytomas. In addition, when EL4 target cells were admixed with MOPC-315 tumor cells, the lytic activity triggered in the L-PAM TuB spleen cells by the MOPC-315 tumor cells plus PMA was not effective in lysing the antigenically unrelated target cells. Moreover, even in the presence of the calcium-specific ionophore, ionomycin, L-PAM TuB spleen cells exposed to PMA were unable to lyse the EL4 target cells. Thus, fresh CD8+ splenic T cells from L-PAM TuB mice that are in the process of eradicating a large MOPC-315 tumor as a consequence of low-dose L-PAM therapy can be triggered with PMA to exert enhanced lytic activity against MOPC-315 tumor cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Animals; Antigens, Differentiation, T-Lymphocyte; CD8 Antigens; Cell Line; Cytotoxicity, Immunologic; Dose-Response Relationship, Drug; Drug Synergism; Female; In Vitro Techniques; Ionomycin; Lymphocyte Depletion; Melphalan; Mice; Mice, Inbred BALB C; Phagocytes; Plasmacytoma; T-Lymphocytes; Tetradecanoylphorbol Acetate; Thymoma; Time Factors

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

In order to further characterize the action of transforming growth factor beta (TGF-beta) on lymphoid cells, we investigated the effects of porcine TGF-beta 1 and -2 on the IL-1 sensitive EL4/6.1 thymoma cell line. The proliferation of EL4/6.1 thymoma cells was inhibited by TGF-beta 1 and TGF-beta 2 (1 ng/ml) to a similar degree, the population doubling time was increased by 50-60%, total inhibition was not achieved. This decrease of proliferation was associated with an increase of the number of cells in the G0/G1 compartment of the cell cycle. TGF-beta-mediated inhibition could not be overcome by adding exogenous rIL-1 nor was the binding capacity for IL-1 reduced. In addition, TGF-beta did not interfere with the induction of IL-2 receptors by a combination of Ionomycin+PMA+IL-1. The data suggest that TGF-beta mediated inhibition of thymocyte/lymphocyte proliferation is not associated with an inhibition of the expression or the induction of expression of IL-2 or IL-1 receptors.

Topics: Animals; Cell Division; Gene Expression Regulation, Neoplastic; Interleukin-1; Ionomycin; Mice; Receptors, Immunologic; Receptors, Interleukin-1; Receptors, Interleukin-2; Recombinant Proteins; Tetradecanoylphorbol Acetate; Thymoma; Thymus Neoplasms; Transforming Growth Factor beta; Tumor Cells, Cultured

The present study compared the role of two protein kinase C (PK-C) activating agents, the phorbol ester phorbol-12-acetate-13-myristate (PMA) and the membrane-permeating diacylglycerol dioctanoyl-sn-glycerol (DiC8) in the activation of EL4/6.1 thymoma cells. These cells have been shown to express interleukin-2 receptors (IL-2R) upon stimulation with optimal amounts of PMA (10 ng/ml); also, suboptimal amounts of PMA (1 ng/ml) synergized with the Ca2+ ionophore ionomycin and recombinant interleukin-1 (rIL-1) (Lowenthal et al., 1986). Comparing PMA and DiC8 led to the following results: PMA at 10 ng/ml induced IL-2R; in contrast, DiC8 (30-3 micrograms/ml) alone was unable to induce IL-2R, although it did synergize with ionomycin (0.5 micrograms/ml) and rIL-1. Bihourly additions of DiC8 did not change this pattern. The addition of DiC8 together with rIL-2 also resulted in no IL-2R expression. Furthermore, DiC8 (10 micrograms/ml) effectively translocated PK-C. Therefore, the differences observed between PMA and DiC8 do not seem to be due to differences in metabolism or to an inability to translocate PK-C. Analysis of messenger (m) RNA produced in stimulated EL4/6.1 cells revealed that DiC8 was also unable to induce mRNA for IL-2R. Our data suggest that PMA, especially at "optimal" concentrations, might have effects that cannot be mimicked by diacylglycerol. Furthermore, it seems that the deficient activity of diacylglycerols can be compensated for by a Ca2+ ionophore and, depending on the cellular system, by further signals such as IL-1.

Topics: Animals; Diglycerides; Ethers; Flow Cytometry; Glycerides; Interleukin-1; Ionomycin; Lymphocyte Activation; Mice; Protein Kinase C; Receptors, Interleukin-2; RNA, Messenger; Tetradecanoylphorbol Acetate; Thymoma; Tumor Cells, Cultured

The effect of the Ca2+ ionophore ionomycin on neoplastic thymocytes in comparison to its effect on normal thymus cells was studied. Ionomycin increases intracellular Ca2+ in normal lymphocytes but fails to increase Ca2+ in neoplastic thymocytes. In these cells the ionophore causes a transient increase in cytosolic free Ca2+. The lack of effect of ionomycin reproduces that of A23187, but it does not depend on reduced availability of intracellular Mg2+ to exchange with Ca2+; it appears to depend on the strong activity of the plasma membrane Ca2+-extruding pump that counteracts ionomycin permeabilization and that can be partly inhibited by the calmodulin inhibitor R24571 (calmidazolium). Neoplastic thymocytes show a high content of magnesium, the intracellular binding of which is efficiently regulated by endogenous ATP. The data show also an interesting correlation between the regulation of energy metabolism (aerobic glycolysis) and cation homeostasis in the neoplastic cells studied.

Topics: Animals; Calcium; Ethers; Glycolysis; Imidazoles; In Vitro Techniques; Iodoacetates; Iodoacetic Acid; Ionomycin; Kinetics; Lymphoma; Magnesium; Mice; Mice, Inbred BALB C; Reference Values; Thymoma; Thymus Gland; Thymus Neoplasms