sq-23377 and Lymphoma--T-Cell

Scopoletin is an antioxidant found in certain weedy plants. It exerts anti-inflammatory, anti-allergic, and anti-diabetic activities. It remains unknown whether scopoletin regulates T helper (Th) cells, including Th 1 and Th 2 cells. We found that scopoletin significantly inhibited phorbol myristate acetate (PMA)/ionomycin-induced interleukin-4 (IL-4), IL-5, and IL-10 production in EL-4 T cells. In addition, scopoletin significantly enhanced the inhibitory action of PMA/ionomycin on interferon-γ (IFN-γ) expression. In EL-4 T cells, PMA/ionomycin treatment markedly increased the expression of nuclear factor of activated T cells (NFAT) and GATA-3; in contrast, scopoletin significantly down-regulated expressions of these transcription factors. Furthermore, this downregulation depended on protein kinase C (PKC) attenuation. This leads us to suggest that the anti-allergic properties of scopoletin might be mediated by the downregulation of cytokine expression in Th 2 cells.

Topics: Animals; Cell Line, Tumor; Cytokines; Hypersensitivity; Interferon-gamma; Interleukin-1; Interleukin-10; Interleukin-4; Ionomycin; Lymphoma, T-Cell; Mice; Phytotherapy; Protein Kinase C; Scopoletin; Tetradecanoylphorbol Acetate; Th2 Cells

In transfection protocols, the expression levels of the transgene is important to define, still is difficult to obtain in certain cell lines such as those derived from T-lymphoma cells. In this study we evaluate transgene expression kinetics in the presence and absence of two well known transcription activators such as phorbol-12-myristate13-acetate (PMA) and Ionomicin (IO). Three murine T lymphoma cell lines (LBC, EL4 and BW5147) were transfected by electroporation using green fluorescent protein (GFP) as a reporter gene and analyzed by flow cytometry. Addition of PMA/IO resulted in a significant increase of the Mean Fluorescence Intensity but not in GFP-positive cell percentages, either in transient or stable transfected LBC and EL4 cells. Remarkable, BW5147 cells showed low GFP induction with a significant increment only in stable transfected cells. Our results demonstrated that CMV promoter activity can be enhanced in transfected lymphoma cells by PMA/IO suggesting that transgene expression levels can be optimized by means of the use of transcription activators.

Topics: Animals; Cell Line, Tumor; Electroporation; Flow Cytometry; Gene Expression; Green Fluorescent Proteins; Ionomycin; Lymphoma, T-Cell; Mice; Plasmids; Promoter Regions, Genetic; Tetradecanoylphorbol Acetate; Transfection; Transgenes

Traditional methods for identifying T cell-recognized tumor antigens (Ags) are laborious and time-consuming. In an attempt to simplify the procedure, a novel strategy, SING (SIgnal transduction molecule-mediated, NFAT-controlled, GFP expression) was established as a direct approach for cloning T cell-recognized tumor Ags. In the SING system, a mouse T cell line (BW5147) was transduced with a chimeric H-2Kb construct containing T cell-signaling domains and a green fluorescent protein (GFP) reporter gene under the transcriptional control of nuclear factor of activated T cells (NFAT). The resultant BW5147 cells were named BS cells. This cell line could "sense" TCR stimulation through the T cell-signaling domains after coculture with Ag-specific T cells and then become fluorescent (expressing green fluorescence protein, GFP+) in the presence of Ag peptides. The interaction between BS cells and Ag-specific T cells could be enhanced by addition of costimulatory signals. Currently, BS cells have been optimized to "sense" TCR stimulation after being pulsed with the relevant peptides at concentrations as low as 10(-9) M. Endogenous Ag-expressing BS cells could also become fluorescent after coculture with Ag-specific T cells. Our results provide a proof of principle for using the SING system to directly isolate Ag-expressing BS cells from BS cell repertoires, which are retrovirally transduced with tumor-derived cDNA libraries. Once tumor Ag-marked BS cells are identified, the sequences encoding tumor Ags could be easily retrieved by PCR amplification of the genomic DNA using vector-specific primers.

Topics: 3T3 Cells; Animals; Antigen Presentation; Antigens, Neoplasm; Cell Line, Tumor; Cloning, Molecular; Coculture Techniques; DNA-Binding Proteins; Egg Proteins; Exocytosis; Genes, Reporter; Genetic Vectors; Green Fluorescent Proteins; H-2 Antigens; Hybridomas; Ionomycin; Luminescent Proteins; Lymphoma, T-Cell; Macrolides; Mice; NFATC Transcription Factors; Nuclear Proteins; Ovalbumin; Peptide Fragments; Receptors, Antigen, T-Cell; Recombinant Fusion Proteins; Signal Transduction; T-Lymphocytes, Cytotoxic; Tetradecanoylphorbol Acetate; Transcription Factors; Transcription, Genetic; Transduction, Genetic

IL-15 and IL-2 are two structurally and functionally related cytokines whose high affinity receptors share the IL-2R beta-chain and gamma-chain in association with IL-15R alpha-chain (IL-15R alpha) or IL-2R alpha-chain, respectively. Whereas IL-2 action seems restricted to the adaptative T cells, IL-15 appears to be crucial for the function of the innate immune responses, and the pleiotropic expression of IL-15 and IL-15R alpha hints at a much broader role for the IL-15 system in multiple cell types and tissues. In this report, using a highly sensitive radioimmunoassay, we show the existence of a soluble form of human IL-15R alpha (sIL-15R alpha) that arises from proteolytic shedding of the membrane-anchored receptor. This soluble receptor is spontaneously released from IL-15R alpha-expressing human cell lines as well as from IL-15R alpha transfected COS-7 cells. This release is strongly induced by PMA and ionomycin, and to a lesser extent by IL-1 beta and TNF-alpha. The size of sIL-15R alpha (42 kDa), together with the analysis of deletion mutants in the ectodomain of IL-15R alpha, indicates the existence of cleavage sites that are proximal to the plasma membrane. Whereas shedding induced by PMA was abrogated by the synthetic matrix metalloproteinases inhibitor GM6001, the spontaneous shedding was not, indicating the occurrence of at least two distinct proteolytic mechanisms. The sIL-15R alpha displayed high affinity for IL-15 and behaved as a potent and specific inhibitor of IL-15 binding to the membrane receptor, and of IL-15-induced cell proliferation (IC(50) in the range from 3 to 20 pM). These results suggest that IL-15R alpha shedding may play important immunoregulatory functions.

Topics: Animals; Cell Division; Cell Line, Tumor; Chlorocebus aethiops; COS Cells; Culture Media, Conditioned; Dipeptides; DNA, Complementary; Glioblastoma; Glycosylphosphatidylinositols; Humans; Interleukin-1; Interleukin-15; Ionomycin; Lymphoma, T-Cell; Molecular Weight; Protease Inhibitors; Protein Binding; Protein Subunits; Receptors, Interleukin-15; Receptors, Interleukin-2; Solubility; Tetradecanoylphorbol Acetate; Transfection; Tumor Necrosis Factor-alpha; U937 Cells

The CD40L expressed on activated CD4+ T cells delivers contact-dependent proliferative and anti-apoptotic signals to B lymphocytes. Little is known about molecular mechanisms of constitutive expression of CD40L on some non-Hodgkin's lymphomas, especially about involvement of two signal pathways regulating its expression in normal cells; one involving calcineurin, and the other protein kinase C. We analyzed by flow cytometry the effects of 6-hour stimulation of both pathways (stimuli: PMA and ionomycin) and their inhibitors: cyclosporin A and chelerythrine, on CD40L expression. Two Jurkat clones differing in CD40L surface expression: clone 217.6 (CD40L-) and 217.7 (CD40L+) were studied. Our experiments showed that high level of CD40L expression on the surface of 217.7 cells was reduced after stimulation with PMA. The same effect was observed for combination of PMA and chelerythrine or for PKC inhibitor alone. In 217.6 cells, only chelerythrine used alone induced low level of CD40L expression, while PMA and ionomycin were without effect. These results suggest that CD40L surface expression is mainly dependent on protein kinase C activity. By using PepTag Assay we have confirmed that in both Jurkat clones PKC activity is higher than in normal blood lymphocytes.

Topics: Alkaloids; Benzophenanthridines; Calcineurin; CD4-Positive T-Lymphocytes; CD40 Antigens; CD40 Ligand; Cyclosporine; Enzyme Inhibitors; Humans; Ionomycin; Jurkat Cells; Lymphoma, T-Cell; Phenanthridines; Phosphoric Monoester Hydrolases; Protein Kinase C; Signal Transduction; Tetradecanoylphorbol Acetate; Up-Regulation

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

To study the oncogenic activity of cyclin E in an in vivo system we generated transgenic mice expressing high levels of cyclin E in T-lymphocytes by using a construct containing the CD2 locus control region. These animals were neither predisposed to develop any tumors spontaneously nor showed an increased incidence when crossbred with Emu L-myc transgenic mice but developed hyperplasia in peripheral lymphoid organs at later age with an incidence of 27%. When treated with the DNA methylating carcinogen N-methylnitrosourea (MNU) that provokes the development of T-cell lymphomas, CD2-cyclin E transgenic animals came down with T-cell neoplasia showing a significant higher incidence (54%) than normal non transgenic controls (31%). In one of eight tumors that arose in normal MNU treated mice we could find an expected activating point mutation in the Ki-ras gene (12.5%). In contrast, the same mutation occurred in five of 16 tumors from CD2-cyclin E transgenic mice (31.2%). Whereas cyclin E overexpression alone did not lead to an increased CDK2 activity we observed in all tumors that emerged from either MNU treated normal mice or treated CD2-cyclin E transgenics a downregulation of p27KIP1 and a higher histone H1 kinase activity in CDK2 immunoprecipitates compared to normal tissue. These findings demonstrate that high level expression of cyclin E can predispose T-cells for hyperplasia and malignant transformation. However, the results also suggest that this activity of cyclin E is manifest only when other cooperating oncogenes in particular ras genes are present and activated. This would be consistent with our previous finding that cyclin E and Ha-Ras cooperate in focus formation assays in rat embryo fibroblasts.

Topics: Animals; Cell Transformation, Neoplastic; Cyclin E; Embryo, Mammalian; Fibroblasts; Genes, myc; Genes, ras; Hyperplasia; Ionomycin; Locus Control Region; Lymphoid Tissue; Lymphoma, T-Cell; Methylnitrosourea; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Transgenic; Point Mutation; Protamine Kinase; Rats; T-Lymphocytes; Tetradecanoylphorbol Acetate; Thymus Gland

Interleukin 2 (IL-2) and interleukin 4 (IL-4) secreted by activated but not by resting mature T cells are pleiotropic cytokines affecting growth and differentiation of diverse cell types, such as T cells, B cells, and mast cells. There is little information about the molecular basis for the constitutive repression of IL-2 and IL-4 gene expression in unstimulated T cells. We investigated the possibility that wild-type (wt) p53, a nuclear tumor suppressor protein, might serve to repress IL-2 and IL-4 gene expression in murine E14 T lymphoma and in human Jurkat cells. We transiently cotransfected these cells with constitutive simian virus 40 (SV 40) early promoter expression plasmids overproducing wt or mutant murine p53 and with appropriate luciferase (luc) reporter plasmids containing the promoter elements of murine IL-2 and IL-4 genes to evaluate the effect of various p53 species on these promoters. Murine wt p53 derived from pSG5p53cD strongly repressed the IL-2 and IL-4 promoters in both cell lines induced by the phorbol ester TPA and the Ca2+ ionophore ionomycin but not, however, in uninduced cells. In similar transient transfection experiments with lymphoma cells, overexpression of deletion mutant species of murine p53 revealed that the N-terminal and C-terminal domains are crucial for inhibition of both IL-2 and IL-4 gene expression. These parts of p53 comprise the transactivation domain at the amino terminal side, which has previously also been shown to interact with the TATA-box binding-protein TBP and the carboxy-terminal oligomerization domain. Additionally, it was shown that a previously described inhibitory protein, the high-mobility-group protein HMG-I/Y, does not functionally interact with p53. Cotransfection of expression plasmids for both p53 and HMG-I/Y did not alter the extent of inhibition by the individual proteins. These data suggest that p53 can downmodulate both IL-2 and IL-4 gene expression and that both the transactivation and oligomerization domains of the tumor suppressor protein are essential for this transcriptional repression.

Topics: Animals; Gene Expression Regulation, Neoplastic; Genes, p53; High Mobility Group Proteins; HMGA1a Protein; Humans; Interleukin-2; Interleukin-4; Ionomycin; Ionophores; Leukemia-Lymphoma, Adult T-Cell; Lymphoma, T-Cell; Mice; Promoter Regions, Genetic; Recombinant Fusion Proteins; Sequence Deletion; Tetradecanoylphorbol Acetate; Transcription Factors; Transcription, Genetic; Transfection; Tumor Cells, Cultured; Tumor Suppressor Protein p53

The immunosuppressant cyclosporin A (CsA) exerts its pharmacologic actions by inhibiting calcineurin function. Here, we investigated the effect of CsA on the DNA-binding activity of the transcription factor, AP-1, in YAC-1 cells. We found that elevation of intracellular Ca2+ by ionomycin increased AP-1 DNA-binding activity in these cells. CsA treatment upregulated the ionomycin-induced, but not the basal AP-1 DNA-binding activity. In contrast, a CsA analog, MeVal4CsA, that does not inhibit calcineurin, failed to enhance ionomycin-induced AP-1 DNA-binding activity. This activity was shown to involve c-Fos, c-Jun and JunB. CsA consistently augmented ionomycin-induced c-fos mRNA expression and more variably that of JunB. Therefore, calcineurin negatively regulates Ca(2+)-stimulated AP-1 activity principally at the c-fos induction level. By inhibiting calcineurin, CsA shifts the balance between positive and negative AP-1 regulation. Since AP-1 controls the transcription of many genes, this finding may have implications for both the immunosuppressive and toxic effects of CsA.

Topics: Animals; Calcium; Cyclosporine; DNA-Binding Proteins; Drug Interactions; Immunosuppressive Agents; Ionomycin; Ionophores; Lymphoma, T-Cell; Mice; Protein Binding; Proto-Oncogene Proteins c-fos; RNA, Messenger; T-Lymphocytes; Transcription Factor AP-1; Tumor Cells, Cultured

The calmodulin-stimulated phosphatase calcineurin plays a critical role in calcium-dependent T-lymphocyte activation pathways. Here, we report the identification of a missense mutation in the calcineurin A alpha gene expressed by EL4 T-lymphoma cells. This mutation changes an evolutionarily conserved aspartic acid to asparagine within the autoinhibitory domain of the calcineurin A alpha protein. A comparison of wild-type and mutant autoinhibitory peptides indicates that this amino acid substitution greatly reduces inhibition of calcineurin phosphatase activity. Additional peptide inhibition studies support a pseudosubstrate model of autoinhibitory function, in which the conserved aspartic acid residue may serve as a molecular mimic of either phosphoserine or phosphothreonine. Expression of the mutant calcineurin appears to affect cellular signal transduction pathways, as EL4 cells can be activated by suboptimal concentrations of calcium ionophore in the presence of phorbol esters. Moreover, this phenotype can be transferred to Jurkat T cells by transfection of the mutated calcineurin gene. These findings implicate a conserved aspartic acid in the mechanism of calcineurin autoinhibition and suggest that mutation of this residue is associated with aberrant calcium-dependent signaling in vivo.

Topics: Amino Acid Sequence; Animals; Asparagine; Aspartic Acid; Base Sequence; Calcineurin; Calcium; Calmodulin; Calmodulin-Binding Proteins; Cloning, Molecular; Dose-Response Relationship, Drug; Interleukin-2; Ionomycin; Lymphoma, T-Cell; Mice; Molecular Mimicry; Molecular Sequence Data; Mutation; Peptide Fragments; Phosphoprotein Phosphatases; Recombinant Proteins; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Signal Transduction; Structure-Activity Relationship; Tetradecanoylphorbol Acetate

Transforming growth factor beta 1 (TGF-beta 1) is a multifunctional cytokine whose potent immunomodulatory activity is well documented. To explore the mechanisms of this activity we examined the effect of TGF-beta 1 on the production of IFN-gamma measured at the mRNA and protein levels in the YAC-1 T cell lymphoma. In previous studies, this model proved useful to characterize the mode of action of the immunosuppressant rapamycin (RAP). Here, we found that when induced by IL-1 or IL-1 + PMA, the production of IFN-gamma is suppressed by both TGF-beta 1 (ED50 = 1.9 pM) and RAP (ED50 = 0.2 nM). In contrast, when induced by the calcium ionophore ionomycin, in the absence or in the presence of PMA, this production is enhanced up to 10-fold by TGF-beta 1 (ED50 = 1.8 pM) and 1.5-3-fold by RAP. Therefore, in YAC-1 cells, TGF-beta 1 exerts opposite effects on IFN-gamma production depending on the mode of activation, and these effects parallel those of RAP. To further analyze the mode of action of TGF-beta 1 in this system, we used okadaic acid (OA), an inhibitor of serine/threonine protein phosphatases. Treatment with OA rendered the expression of IFN-gamma mRNA induced by IL-1 insensitive to TGF-beta 1 or RAP, indicating that activation of a phosphatase may play a role in the suppressive effect of both agents. However, OA did not prevent the augmentation of ionomycin-mediated induction of IFN-gamma mRNA by either TGF-beta 1 or RAP. Hence, the up-regulation of IFN-gamma production by TGF-beta 1 and RAP may involve a different biochemical mechanism than that mediating their suppressive action. These observations also favor the hypothesis that the two agents act on the same regulatory pathways. This was further supported by the finding that TGF-beta 1 and RAP modulate IFN-gamma production in an additive rather than synergistic fashion. However, their effects could be dissociated in mutants of YAC-1 cells selected for resistance to the inhibition of IL-1-mediated IFN-gamma induction by RAP. Moreover, the IFN-gamma modulatory action of RAP in YAC-1 cells was accompanied by an antiproliferative effect, whereas TGF-beta 1 failed to alter the growth of these cells. Therefore, the immunomodulatory action of TGF-beta 1 may result from the disruption of biochemical processes related to, although distinct from, those affected by RAP.

Topics: Animals; Cell Division; Drug Interactions; Drug Synergism; Ethers, Cyclic; Immunosuppressive Agents; Interferon-gamma; Interleukin-1; Ionomycin; Lymphoma, T-Cell; Mice; Okadaic Acid; Phosphoprotein Phosphatases; Polyenes; RNA, Messenger; Sirolimus; Swine; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta

Cyclosporin A (CsA) and FK-506 inhibit lymphokine gene activation in T-cells. In the present study, we investigated the effects of these immunosuppressants on the regulation of a non-lymphokine molecule, the Ly-6E surface antigen, in the YAC-1 T-cell lymphoma. These cells do not normally express Ly-6E mRNA or Ly-6E surface molecules but are induced to do so upon treatment with IFN-gamma. At submicromolar concentrations, CsA or FK-506 did not alter this induction. However, at higher concentrations (1-12 microM), they both increased the induction of Ly-6E mRNA expression. Cyclosporin A or FK-506 also markedly affected Ly-6E induction when the cultures were co-treated with the calcium ionophore, ionomycin. In the absence of CsA or FK-506, ionomycin suppressed Ly-6E induction by IFN-gamma. Both immunosuppressants reversed this inhibitory effect and increased Ly-6E mRNA and Ly-6E surface expression to levels that were 2- to 3-fold higher than in cells induced with IFN-gamma alone. In this system, the two immunosuppressants were active at pharmacologically relevant concentrations, similar to those inhibiting normal T-cell activation, with FK-506 being 30- to 50-fold more potent than CsA. The ability of CsA analogs to enhance Ly-6E induction in the presence of ionomycin also correlated with their immunosuppressive activity. Therefore, through mechanisms apparently related to those involved in their immunosuppressive action, both CsA and FK-506 convert the negative effect of ionomycin on IFN-gamma-mediated Ly-6E induction into an overall positive effect. The YAC-1 cell model, described here, provides a unique example of upregulation of gene expression by these two immunosuppressants.

Topics: Animals; Antigens, Ly; Cyclosporine; Gene Expression Regulation, Neoplastic; In Vitro Techniques; Interferon-gamma; Ionomycin; Lymphoma, T-Cell; Mice; Mice, Inbred BALB C; T-Lymphocytes; Tacrolimus; Transcriptional Activation; Tumor Cells, Cultured; Up-Regulation