Murine T-lymphoma Cell Line Research Articles

Regulator of Calcineurin 3 Ameliorates Autoimmune Arthritis by Suppressing Th17 Cell Differentiation

Regulator of calcineurin 3 (RCAN3), an endogenous regulator of the calcineurin-nuclear factor of activated T cells (NFAT) signaling pathway, inhibits the phosphatase activity of calcineurin, the nuclear translocation of NFAT, and the NFAT downstream pathway. To investigate the effects of RCAN3 on T-cell regulatory function and the development and progression of inflammatory arthritis, we studied the effects of RCAN3 transfection on regulation of Th17 cell differentiation in a murine T-lymphoma cell line and primary splenic CD4+ T cells. Overexpression of RCAN3 suppressed Th17 cell differentiation through the down-regulation of RAR receptor orphan receptor γT mRNA and up-regulation of forkhead box P3 mRNA. In mice with collagen-induced arthritis, injection of an RCAN3-overexpression vector controlled arthritis development invivo. Injection ofRCAN3 reduced the formation of osteoclasts and expression of inflammatory cytokines invivo. Antioxidants stimulated the expression of RCAN3 invitro, and combination therapy with pcDNA-RCAN3 had a synergistic suppressive effect on the development of arthritis. These data suggest that RCAN3 may be an effective treatment for rheumatoid arthritis.

P.1.f.007 Effects of the administration of lipopolysaccharide and a murine T-lymphoma cell line on brain activation and immune function in male Wistar rats

P.1.f.007 Effects of the administration of lipopolysaccharide and a murine T-lymphoma cell line on brain activation and immune function in male Wistar rats

Intracellular superoxide dismutase activity defines invasiveness of the murine T-lymphoma cell line L5187Y-ML25 in vitro and in vivo

Superoxide anion in the tumor microenvironment promotes a malignant phenotype. Here, we examined superoxide in a murine T-lymphoma cell line L5187Y-ML25. Clones with high and low intracellular superoxide dismutase (SOD) activities were obtained from parental L5187Y-ML25 cells and were subjected to assays determining cell invasiveness, motility, and in vivo dissemination. Cells with lower SOD activity exhibited higher invasiveness in vitro and in vivo. NADPH oxidase inhibitor suppressed intracellular free radical levels and cell motility, suggesting NADPH oxidase as a source of superoxides that stimulates cell motility. These results implicate superoxide as a potential anti-metastatic therapy for hematopoietic cell malignancies.

Tissue inhibitor of metalloproteinases‐1‐induced scattered liver metastasis is mediated by host‐derived urokinase‐type plasminogen activator

Paradoxically, not only proteinases but also their inhibitors can correlate with bad prognosis of cancer patients, underlining the evolving concept of the protease web as the complex interplay between proteinases, their inhibitors and effector molecules. Elevated levels of tissue inhibitor of metalloproteinases-1 (TIMP-1) render the liver more susceptible to metastasis by triggering urokinase plasminogen activator (uPA) expression as well as hepatocyte growth factor (HGF) signalling, thereby leading to the fatal scattered infiltration of metastasizing tumour cells throughout the parenchyma of the target organ. Here, we investigated whether host uPA is a crucial protagonist for the TIMP-1-induced modulation of a pro-metastatic microenvironment in the liver. Indeed, in livers of uPA-ablated mice elevated TIMP-1 levels did not trigger HGF signalling and did not promote metastasis of a murine T-lymphoma cell line. In contrast, lack of tumour cell-derived uPA induced by gene silencing did not interfere with this pro-metastatic pathway. Furthermore, host uPA was necessary for the recruitment of neutrophilic granulocytes and the associated increase of HGF in livers with elevated TIMP-1 levels. This newly identified co-operation between TIMP-1 and host uPA suggests that therapies, simultaneously interfering with pro- and anti-proteolytic pathways may be beneficial for patients with metastatic disease.

Induction of apoptosis in murine lymphoma cells by cyclosporin A

The aim of this study was to investigate if CsA could induce apoptosis in the murine T-lymphoma cell line LBC, whose growth is inhibited by this immunosuppressive drug. CsA induced programmed cell death in LBC cells with typical features of apoptosis demonstrated by exposure of phosphatidyl serine residues on the cell membrane, the decrease of cell DNA content, chromatin condensation, and nuclear fragmentation. Apoptosis was evident within 12 h after CsA incubation, with a maximal effect at 48 h, in a time and dose-dependent fashion. In addition, the role of apoptosis inhibitors (Bcl-2 and Bcl-x) and the apoptosis inducer (Bax) in CsA induced-apoptosis was evaluated. The expression of Bcl-2 and Bax proteins were high in LBC cells and following CsA treatment the expression of these proteins as well as Bcl-XL decreased. In this work we demonstrated that cell growth inhibition following CsA treatment in LBC was paralleled by the induction of apoptosis thus providing an interesting animal model to identify the mechanism participating in the regulation of apoptotic genes by CsA in T-cell neoplasms and to assess preclinical in vivo trials of T-cell lymphoma-related disorders.

Beta-adrenergic receptor/cAMP-mediated signaling and apoptosis of S49 lymphoma cells.

beta-Adrenergic receptor (betaAR) activation and/or increases in cAMP regulate growth and proliferation of a variety of cells and, in some cells, promote cell death. In the current studies we addressed the mechanism of this growth reduction by examining betaAR-mediated effects in the murine T-lymphoma cell line S49. Wild-type S49 cells, derived from immature thymocytes (CD4(+)/CD8(+)) undergo growth arrest and subsequent death when treated with agents that increase cAMP levels (e.g., betaAR agonists, 8-bromo-cAMP, cholera toxin, forskolin). Morphological and biochemical criteria indicate that this cell death is a result of apoptosis. In cyc(-) and kin(-) S49 cells, which lack G(s)alpha and functional protein kinase A (PKA), respectively, betaAR activation of G(s)alpha and cAMP action via PKA are critical steps in this apoptotic pathway. S49 cells that overexpress Bcl-2 are resistant to cAMP-induced apoptosis. We conclude that betaAR activation induces apoptosis in immature T lymphocytes via G(s)alpha and PKA, while overexpression of Bcl-2 prevents cell death. betaAR/cAMP/PKA-mediated apoptosis may provide a means to control proliferation of immature T cells in vivo.

Evidence for Different Mechanisms of Growth Inhibition of T-cell Lymphoma by Phorbol Esters and Concanavalin A

Stimuli that are mitogenic for mature T-cells induce cell cycle arrest in some T-cell tumors and T-cell hybridomas. The molecular mechanism of this growth inhibition is poorly understood. In this report, we show that in EL4, a murine T-lymphoma cell line, stimulation with concanavalin A or treatment with phorbol 13-myristate 12-acetate (PMA) inhibit growth, due to cell cycle arrest at both the G1 and the G2/M phases. The block at the G1 phase is accompanied by the appearance of a hypophosphorylated form of the retinoblastoma protein (pRb), due to the inhibition of G1 cyclin-Cdk complexes. However, the molecular mechanisms leading to this G1 cell cycle arrest differ between concanavalin A and PMA: concanavalin A inhibits both cyclin E-Cdk2 and cyclin D-Cdk4 complexes, while PMA inhibits only cyclin E-Cdk2. We demonstrate that concanavalin A inhibits cyclin D-Cdk4 activity by decreasing the amount of cyclin D. The inhibition of cyclin E-Cdk2 by both concanavalin A and PMA is due to increased binding of the Cdk inhibitor p21 to this complex. However, while stimulation of the cells with concanavalin A did not result in an evident increase of the total level of p21, treatment of the cells with PMA increased p21 levels significantly. Our results indicate, furthermore, that the G2/M block results from the inhibition of cyclin A- and cyclin B1-associated kinase activities. As for cyclin E-Cdk2, the inhibition of the cyclin A-Cdk2 complex is due to increased binding of the p21 inhibitor.

Expression of the mdr1 P-glycoprotein gene: a mechanism of escape from glucocorticoid-induced apoptosis.

Glucocorticoid hormones cause apoptosis in the murine T-lymphoma cell line WEHI-7. Glucocorticoid receptors in these cells are cytoplasmic proteins that translocate to the nucleus upon binding hormone. Thus, regulation of cytoplasmic glucocorticoid concentrations controls the level of activated receptors and sensitivity to steroid-induced apoptosis. We found that expression of the mdr1 P-glycoprotein gene produces a reduced accumulation of dexamethasone in WEHI-7 cells. Concomitantly, there is a suppression of dexamethasone-induced changes in transcription and a decrease in steroid sensitivity. P-glycoproteins are known to cause an outward, ATP-dependent transport of a variety of unrelated hydrophobic drugs across the plasma membrane. Our results indicate that glucocorticoid transport by P-glycoproteins depends upon the presence of an hydroxyl group at position 11 of corticosteroids and is enhanced by hydroxyl groups at the positions 16, 17, and 21. The antiprogestin RU486, which contains a dimethyl aminophenyl substitution at the position 11, is not transported by the mdr1 P-glycoprotein. We have found that RU486 is an inhibitor of P-glycoprotein function, indicating that steroid analogs could be useful chemosensitizers in patients undergoing chemotherapy.

Amplification of an adenylosuccinate synthetase gene in alanosine-resistant murine T-lymphoma cells. Molecular cloning of a cDNA encoding the “non-muscle” isozyme.

Adenylosuccinate synthetase (EC 6.3.4.4) catalyzes the initial step in the conversion of IMP to AMP. Two isoforms of this enzyme have been observed in vertebrates. A muscle isozyme is highly abundant in cardiac and skeletal muscle tissue and is thought to play a role in muscle energy metabolism. The non-muscle isozyme, which is present at low levels in most tissues, likely functions in de novo AMP biosynthesis. The analysis of the non-muscle isozyme has been hampered by its low abundance and instability during purification. In this study a genetic selection scheme was used to generate a murine T-lymphoma cell line which was at least 100-fold enriched for the non-muscle isozyme, as a result of amplification of the non-muscle synthetase gene. This cell line made possible the purification of the non-muscle isozyme, and the subsequent isolation of isozyme-specific peptides. Based on peptide sequence information a degenerate oligonucleotide probe was designed and used to screen a mouse kidney cDNA library. A 1.5-kilobase cDNA encoding the non-muscle isozyme was cloned and found to contain an open reading frame of 1368 base pairs encoding 456 amino acids. Gene transfer experiments showed that the cDNA encoded a 50-kDa protein, the size expected for mammalian synthetases, that correlated with the presence of high levels of synthetase activity. The deduced amino acid sequence of the mouse non-muscle synthetase is approximately 75% identical to the previously reported mouse muscle synthetase. Southern blot analysis of mouse genomic DNA with the isozyme-specific cDNA probes revealed that the synthetase isozymes are encoded by separate genes. The non-muscle gene is expressed in most tissues but is virtually undetectable in striated muscle tissues. Three different transcripts (1.7, 2.8, and 3.4 kilobases) are detected for the non-muscle isozyme which show a similar tissue distribution. The availability of a cDNA for the non-muscle isozyme of adenylosuccinate synthetase will facilitate further comparative analyses with the previously cloned muscle isozyme.

Cell-retained and secreted proteoglycans synthesized by murine T-lymphoma cell line, RDM-4

Cell-retained and secreted proteoglycans synthesized by murine T-lymphoma cell line, RDM-4

Inhibition of T-cell antigen receptor-mediated transmembrane signaling by protein kinase C activation.

The murine T-lymphoma cell line LBRM-33 is known to require synergistic signals delivered through the antigen receptor (Ti-CD3) complex, together with interleukin 1 (IL-1), for activation of IL-2 gene expression and IL-2 production. Although 12-O-tetradecanoylphorbol-13-acetate (TPA) was capable of replacing IL-1 as an activating stimulus under certain conditions, biologic studies indicated that TPA failed to synergize with Ti-CD3-dependent stimuli under conditions in which IL-1 was clearly active. Acute exposure to TPA and other active phorbol esters resulted in a concentration-dependent inhibition of the increases in phosphoinositide hydrolysis and intracellular free Ca2+ concentration stimulated by phytohemagglutinin or anti-Ti antibodies. TPA treatment induced no direct alteration of phospholipase C enzymatic activities in LBRM-33 cells. In contrast, both Ti-CD3 cross-linkage and TPA rapidly stimulated the phosphorylation of identical CD3 complex polypeptides, presumably via activation of protein kinase C. Exposure of LBRM-33 cells to TPA resulted in a time-dependent, partial down-regulation of surface Ti-CD3 expression. Thus, TPA treatment inhibited the responsiveness of LBRM-33 cells to Ti-CD3-dependent stimuli by inducing an early desensitization of Ti-CD3 receptors, followed by a decrease in membrane receptor expression. These studies indicate that phorbol esters deliver bidirectional signals that both inhibit Ti-CD3-dependent phosphoinositide hydrolysis and augment IL-2 production in LBRM-33 cells.

Inhibition of T-cell antigen receptor-mediated transmembrane signaling by protein kinase C activation.

The murine T-lymphoma cell line LBRM-33 is known to require synergistic signals delivered through the antigen receptor (Ti-CD3) complex, together with interleukin 1 (IL-1), for activation of IL-2 gene expression and IL-2 production. Although 12-O-tetradecanoylphorbol-13-acetate (TPA) was capable of replacing IL-1 as an activating stimulus under certain conditions, biologic studies indicated that TPA failed to synergize with Ti-CD3-dependent stimuli under conditions in which IL-1 was clearly active. Acute exposure to TPA and other active phorbol esters resulted in a concentration-dependent inhibition of the increases in phosphoinositide hydrolysis and intracellular free Ca2+ concentration stimulated by phytohemagglutinin or anti-Ti antibodies. TPA treatment induced no direct alteration of phospholipase C enzymatic activities in LBRM-33 cells. In contrast, both Ti-CD3 cross-linkage and TPA rapidly stimulated the phosphorylation of identical CD3 complex polypeptides, presumably via activation of protein kinase C. Exposure of LBRM-33 cells to TPA resulted in a time-dependent, partial down-regulation of surface Ti-CD3 expression. Thus, TPA treatment inhibited the responsiveness of LBRM-33 cells to Ti-CD3-dependent stimuli by inducing an early desensitization of Ti-CD3 receptors, followed by a decrease in membrane receptor expression. These studies indicate that phorbol esters deliver bidirectional signals that both inhibit Ti-CD3-dependent phosphoinositide hydrolysis and augment IL-2 production in LBRM-33 cells.

Regulation of cyclic AMP accumulation in lymphoid cells.

We have examined several features of the regulation of cyclic AMP accumulation in lymphoid cells isolated from peripheral blood of human subjects and in the murine T-lymphoma cell line, S49, S49 cells are unique because of the availability of variant clones with lesions in the pathway of cyclic AMP generation and response. We found that human lymphoid cells prepared at 4 degrees C showed substantially greater cyclic AMP accumulation in response to histamine and the beta-adrenergic agonist isoproterenol than did cells prepared at ambient temperature. The muscarinic cholinergic agonist carbamylcholine and peptide hormone somatostatin failed to inhibit cyclic AMP accumulation in human lymphoid cells and treatment with pertussis toxin (which blocks function of Gi, the guanine nucleotide binding protein that mediates inhibition of adenylate cyclase) only minimally increased cyclic AMP levels in these cells. Thus the Gi component of adenylate cyclase appears to play only a small role in modulating cyclic AMP levels in this mixed population of lymphoid cells. Incubation of whole blood with isoproterenol desensitized human lymphocytes to subsequent stimulation with beta agonist. This desensitization was associated with a redistribution of beta-adrenergic receptors such that a substantial portion of the receptors in intact cells could no longer bind a hydrophilic antagonist. Wild-type S49 lymphoma cells showed a similar redistribution of beta-adrenergic receptors after a few minutes' incubation with agonist. Based on studies in S49 variants, this redistribution is independent of components distal to receptors in the adenylate cyclase/cyclic AMP pathway. By contrast, a more slowly developing, agonist-mediated down-regulation of beta-adrenergic receptors was blunted in variants with defective interaction between receptors and Gs, the guanine nucleotide binding protein that mediates stimulation of adenylate cyclase. Unlike results in human lymphoid cells, S49 cells show a prominent inhibition of cyclic AMP accumulation mediated by Gi; this inhibition is promoted by somatostatin and blocked by pertussis toxin. Inhibition by Gi is unable to account for the marked decrease in ability of the diterpene forskolin to maximally stimulate adenylate cyclase in S49 variants having defective Gs. These results emphasize that both Gs and Gi component are important in modulating cyclic AMP accumulation and receptors linked to adenylate cyclase in S49 lymphoma cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of antigen receptor-associated structures on murine T cells.

The specific antigen receptor on human and murine T lymphocytes is a heterodimer of relative molecular mass (Mr) 80,000-90,000 (80-90K) composed of two 40-50K disulphide-linked glycoprotein subunits. Peptide map analysis of the alpha- and beta-chains of receptor isolated from distinct tumour cell lines suggests the presence of both constant and variable regions. Unlike the antigen receptor on B lymphocytes (that is, surface immunoglobulin), the human T-cell antigen receptor seems to be non-covalently associated with another invariant structure recognized by monoclonal antibodies to the cell-surface antigens T3 and Leu 4 (refs 4, 5, 9, 12). Meuer et al. have demonstrated comodulation of the T3 structure and T-cell antigen receptor using anti-clonotypic and anti-T3 monoclonal antibodies. Furthermore, immunoprecipitation with anti-T3 weakly co-precipitates a small amount of the 80-90K heterodimer in certain conditions. The murine homologue of the Leu 4/T3 structure has not been identified, although Gunter et al. have suggested that Thy-1 may be the counterpart of Leu 4/T3 (ref. 13). Here we describe a Leu 4/T3-like structure, distinct from Thy-1, associated with the T-cell receptor of a murine T-lymphoma cell line.

Effect of an anti-murine transferrin receptor-ricin A conjugate on bone marrow stem and progenitor cells treated in vitro

A monoclonal antibody with specificity for murine transferrin receptor was conjugated with the toxic A subunit of ricin. The dose range, specificity, and kinetics of inhibition of protein synthesis of the conjugate were determined on the murine T-lymphoma cell line, BW5147. When toxin was present throughout the period of culture, in vitro myeloid (CFUc) and erythroid (CFUe and BFUe) bone marrow colonies were inhibited by doses of conjugate comparable to those that inhibit protein synthesis in murine cell lines (IC 50 of 5 × 10 −11 M). Bone marrow exposed briefly (30 min to 6 h) to anti-transferrin receptor antibody-ricin A conjugate was assayed for myeloid (CFUc) and erythroid (CFUe and BFUe) progenitors in vitro and for in vivo spleen colony formation (CFUs). Only CFUe were depleted by this pulse exposure, consistent with the higher frequency of proliferating cells and transferrin receptor expression in the CFUe population relative to other progenitors.