The dexamethasone-induced inhibition of proliferation, migration, and invasion in glioma cell lines is antagonized by macrophage migration inhibitory factor (MIF) and can be enhanced by specific MIF inhibitors.

Glioblastomas (GBMs) are the most frequent and malignant brain tumors in adults. Glucocorticoids (GCs) are routinely used in the treatment of GBMs for their capacity to reduce the tumor-associated edema. Few in vitro studies have suggested that GCs inhibit the migration and invasion of GBM cells through the induction of MAPK phosphatase 1 (MKP-1). Macrophage migration inhibitory factor (MIF), an endogenous GC antagonist is up-regulated in GBMs. Recently, MIF has been involved in tumor growth and migration/invasion and specific MIF inhibitors have been developed on their capacity to block its enzymatic tautomerase activity site. In this study, we characterized several glioma cell lines for their MIF production. U373 MG cells were selected for their very low endogenous levels of MIF. We showed that dexamethasone inhibits the migration and invasion of U373 MG cells, through a glucocorticoid receptor (GR)- dependent inhibition of the ERK1/2 MAPK pathway. Oppositely, we found that exogenous MIF increases U373 MG migration and invasion through the stimulation of the ERK1/2 MAP kinase pathway and that this activation is CD74 independent. Finally, we used the Hs 683 glioma cells that are resistant to GCs and produce high levels of endogenous MIF, and showed that the specific MIF inhibitor ISO-1 could restore dexamethasone sensitivity in these cells. Collectively, our results indicate an intricate pathway between MIF expression and GC resistance. They suggest that MIF inhibitors could increase the response of GBMs to corticotherapy.

The winged-helix transcription factor Trident is expressed in actively dividing lymphocytes.

We recently identified the winged-helix transcription factor Trident and described its expression pattern in synchronized fibroblasts. We have now studied Trident expression in cell lines, differentiating thymocytes and in lymphocytes derived from peripheral blood. During T cell differentiation, expression peaked in the actively dividing immature single positive cells. In peripheral blood lymphocytes, expression of Trident mRNA was absent, but could be induced upon stimulation with mitogens in vitro. These observations imply a function for Trident in dividing lymphocytes.

Heterogeneity of the induction of HLA-DR expression by human immune interferon on glioma cell lines and their clones.

The modulation of HLA-DR and HLA-A, -B, and -C by human recombinant immune interferon (IFN-gamma) was studied on 10 malignant glioma cell lines established in our laboratory, on 8 clones or subclones derived from these lines, and on a fetal astrocyte cell line. Comparative studies were performed with recombinant leukocyte interferon (IFN-alpha). The results not only confirmed the selective activity of IFN-gamma on the modulation of HLA-DR expression, as opposed to that of IFN-alpha, but also demonstrated a marked heterogeneity in the response of glioma cell lines and their clones to the two types of IFN tested. For example, all 3 clones of an inducible cell line could be modulated to express HLA-DR, whereas only 2 of 5 clones derived from a noninducible line were modulated. This heterogeneity did not seem to be due to the absence of the receptor for IFN-gamma on the surface of these cells, since almost all of the cell lines or clones tested (17 of 19) responded to IFN-gamma by the induction or enhancement of the expression for either HLA-DR or HLA-A, -B, and -C (or both). The heterogeneity of induction was also demonstrated between clones derived from a glioma line that did not express HLA-DR after IFN-gamma treatment. The production of HLA-DR by one of the clones was abundant enough to be confirmed by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis.

The cooperative induction of hypoxia-inducible factor-1 alpha and STAT3 during hypoxia induced an impairment of tumor susceptibility to CTL-mediated cell lysis.

Hypoxia is an essential component of tumor microenvironment. In this study, we investigated the influence of hypoxia (1% PO(2)) on CTL-mediated tumor cell lysis. We demonstrate that exposure of target tumor cells to hypoxia has an inhibitory effect on the CTL clone (Heu171)-induced autologous target cell lysis. Such inhibition correlates with hypoxia-inducible factor-1alpha (HIF-1alpha) induction but is not associated with an alteration of CTL reactivity as revealed by granzyme B polarization or morphological change. Western blot analysis indicates that although hypoxia had no effect on p53 accumulation, it induced the phosphorylation of STAT3 in tumor cells by a mechanism at least in part involving vascular endothelial growth factor secretion. We additionally show that a simultaneous nuclear translocation of HIF-1alpha and phospho-STAT3 was observed. Interestingly, gene silencing of STAT3 by small interfering RNA resulted in HIF-1alpha inhibition and a significant restoration of target cell susceptibility to CTL-induced killing under hypoxic conditions by a mechanism involving at least in part down-regulation of AKT phosphorylation. Moreover, knockdown of HIF-1alpha resulted in the restoration of target cell lysis under hypoxic conditions. This was further supported by DNA microarray analysis where STAT3 inhibition resulted in a partly reversal of the hypoxia-induced gene expression profile. The present study demonstrates that the concomitant hypoxic induction of phospho-STAT3 and HIF-1alpha are functionally linked to the alteration of non-small cell lung carcinoma target susceptibility to CTL-mediated killing. Considering the eminent functions of STAT3 and HIF-1alpha in the tumor microenvironment, their targeting may represent novel strategies for immunotherapeutic intervention.

A soluble form of B cell maturation antigen, a receptor for the tumor necrosis factor family member APRIL, inhibits tumor cell growth.

A proliferation-inducing ligand (APRIL) is a ligand of the tumor necrosis factor (TNF) family that stimulates tumor cell growth in vitro and in vivo. Expression of APRIL is highly upregulated in many tumors including colon and prostate carcinomas. Here we identify B cell maturation antigen (BCMA) and transmembrane activator and calcium modulator and cyclophilin ligand (CAML) interactor (TACI), two predicted members of the TNF receptor family, as receptors for APRIL. APRIL binds BCMA with higher affinity than TACI. A soluble form of BCMA, which inhibits the proliferative activity of APRIL in vitro, decreases tumor cell proliferation in nude mice. Growth of HT29 colon carcinoma cells is blocked when mice are treated once per week with the soluble receptor. These results suggest an important role for APRIL in tumorigenesis and point towards a novel anticancer strategy.

P2Y1 receptor-evoked glutamate exocytosis from astrocytes: control by tumor necrosis factor-alpha and prostaglandins.

ATP, released by both neurons and glia, is an important mediator of brain intercellular communication. We find that selective activation of purinergic P2Y1 receptors (P2Y1R) in cultured astrocytes triggers glutamate release. By total internal fluorescence reflection imaging of fluorescence-labeled glutamatergic vesicles, we document that such release occurs by regulated exocytosis. The stimulus-secretion coupling mechanism involves Ca2+ release from internal stores and is controlled by additional transductive events mediated by tumor necrosis factor-alpha (TNFalpha) and prostaglandins (PG). P2Y1R activation induces release of both TNFalpha and PGE2 and blocking either one significantly reduces glutamate release. Accordingly, astrocytes from TNFalpha-deficient (TNF(-/-)) or TNF type 1 receptor-deficient (TNFR1(-/-)) mice display altered P2Y1R-dependent Ca2+ signaling and deficient glutamate release. In mixed hippocampal cultures, the P2Y1R-evoked process occurs in astrocytes but not in neurons or microglia. P2Y1R stimulation induces Ca2+ -dependent glutamate release also from acute hippocampal slices. The process in situ displays characteristics resembling those in cultured astrocytes and is distinctly different from synaptic glutamate release evoked by high K+ stimulation as follows: (a) it is sensitive to cyclooxygenase inhibitors; (b) it is deficient in preparations from TNF(-/-) and TNFR1(-/-) mice; and (c) it is inhibited by the exocytosis blocker bafilomycin A1 with a different time course. No glutamate release is evoked by P2Y1R-dependent stimulation of hippocampal synaptosomes. Taken together, our data identify the coupling of purinergic P2Y1R to glutamate exocytosis and its peculiar TNFalpha- and PG-dependent control, and we strongly suggest that this cascade operates selectively in astrocytes. The identified pathway may play physiological roles in glial-glial and glial-neuronal communication.

Reactivity of antiglioma monoclonal antibodies for a large panel of cultured gliomas and other neuroectoderm derived tumors.

We produced three monoclonal antibodies, BF7, GE2 and CG12, against cultured human glioma cells. Their specificity was tested by an indirect antibody-binding radioimmunoassay on a panel of glial and non-glial tumor cell lines. BF7 and GE2 react preferentially with glioma cells and, except for one colon carcinoma line, they do not bind to the control non-neuroectodermal cells; they appear to be directed against common malignant glioma associated antigens. CG12, the third monoclonal antibody, binds to the great majority of tumor cell lines of neuroectodermal origin and does not bind to any other cell lines tested.

Experimental analysis and computer prediction of CTF/NFI transcription factor DNA binding sites.

Accurate prediction of transcription factor binding sites is needed to unravel the function and regulation of genes discovered in genome sequencing projects. To evaluate current computer prediction tools, we have begun a systematic study of the sequence-specific DNA-binding of a transcription factor belonging to the CTF/NFI family. Using a systematic collection of rationally designed oligonucleotides combined with an in vitro DNA binding assay, we found that the sequence specificity of this protein cannot be represented by a simple consensus sequence or weight matrix. For instance, CTF/NFI uses a flexible DNA binding mode that allows for variations of the binding site length. From the experimental data, we derived a novel prediction method using a generalised profile as a binding site predictor. Experimental evaluation of the generalised profile indicated that it accurately predicts the binding affinity of the transcription factor to natural or synthetic DNA sequences. Furthermore, the in vitro measured binding affinities of a subset of oligonucleotides were found to correlate with their transcriptional activities in transfected cells. The combined computational-experimental approach exemplified in this work thus resulted in an accurate prediction method for CTF/NFI binding sites potentially functioning as regulatory regions in vivo.

T-cell hybridoma specific for a cytochrome c peptide: specific antigen binding and interleukin 2 production.

T-cell hybridomas were obtained after fusion of BW 5147 thymoma and long-term cultured T cells specific for cytochrome c peptide 66-80 derivatized with a 2,4-dinitroaminophenyl (DNAP) group. The resulting hybridomas were selected for their capacity to specifically bind to soluble radiolabeled peptide antigen. One T-cell hybrid was positive for antigen binding. This hybrid T cell exhibits surface phenotypic markers of the parent antigen-specific T cells. The binding could be inhibited either by an excess of unlabeled homologous antigen or by cytochrome c peptide 11-25 derivatized with a 2-nitrophenylsulfenyl group. Several other peptide antigens tested failed to inhibit binding of the radioactive peptide. This suggests that a specific amino acid sequence, modified by a DNAP group, is the antigenic structure recognized by the putative T-cell receptor. In addition, direct interaction of DNAP-66-80 peptide with the hybridoma cell line induced production of the T-cell growth factor interleukin 2. Furthermore, supernatants derived from syngeneic macrophages pulsed with the relevant peptide also induced the antigen-specific hybridoma to produce interleukin 2.

An SH2 domain-containing 5' inositolphosphatase inhibits insulin-induced GLUT4 translocation and growth factor-induced actin filament rearrangement.

Tyrosine kinase receptors lead to rapid activation of phosphatidylinositol 3-kinase (PI3 kinase) and the subsequent formation of phosphatidylinositides (PtdIns) 3,4-P2 and PtdIns 3,4, 5-P3, which are thought to be involved in signaling for glucose transporter GLUT4 translocation, cytoskeletal rearrangement, and DNA synthesis. However, the specific role of each of these PtdIns in insulin and growth factor signaling is still mainly unknown. Therefore, we assessed, in the current study, the effect of SH2-containing inositol phosphatase (SHIP) expression on these biological effects. SHIP is a 5' phosphatase that decreases the intracellular levels of PtdIns 3,4,5-P3. Expression of SHIP after nuclear microinjection in 3T3-L1 adipocytes inhibited insulin-induced GLUT4 translocation by 100 +/- 21% (mean +/- the standard error) at submaximal (3 ng/ml) and 64 +/- 5% at maximal (10 ng/ml) insulin concentrations (P < 0.05 and P < 0.001, respectively). A catalytically inactive mutant of SHIP had no effect on insulin-induced GLUT4 translocation. Furthermore, SHIP also abolished GLUT4 translocation induced by a membrane-targeted catalytic subunit of PI3 kinase. In addition, insulin-, insulin-like growth factor I (IGF-I)-, and platelet-derived growth factor-induced cytoskeletal rearrangement, i.e., membrane ruffling, was significantly inhibited (78 +/- 10, 64 +/- 3, and 62 +/- 5%, respectively; P < 0.05 for all) in 3T3-L1 adipocytes. In a rat fibroblast cell line overexpressing the human insulin receptor (HIRc-B), SHIP inhibited membrane ruffling induced by insulin and IGF-I by 76 +/- 3% (P < 0.001) and 68 +/- 5% (P < 0.005), respectively. However, growth factor-induced stress fiber breakdown was not affected by SHIP expression. Finally, SHIP decreased significantly growth factor-induced mitogen-activated protein kinase activation and DNA synthesis. Expression of the catalytically inactive mutant had no effect on these cellular responses. In summary, our results show that expression of SHIP inhibits insulin-induced GLUT4 translocation, growth factor-induced membrane ruffling, and DNA synthesis, indicating that PtdIns 3,4,5-P3 is the key phospholipid product mediating these biological actions.

A nuclear factor I-like activity and a liver-specific repressor govern estrogen-regulated in vitro transcription from the Xenopus laevis vitellogenin B1 promoter.

A hormone-controlled in vitro transcription system derived from Xenopus liver nuclear extracts was exploited to identify novel cis-acting elements within the vitellogenin gene B1 promoter region. In addition to the already well-documented estrogen-responsive element (ERE), two elements were found within the 140 base pairs upstream of the transcription initiation site. One of them, a negative regulatory element, is responsible for the lack of promoter activity in the absence of the hormone and, as demonstrated by DNA-binding assays, interacts with a liver-specific transcription factor. The second is required in association with the estrogen-responsive element to mediate hormonal induction and is recognized by the Xenopus liver homolog of nuclear factor I.

Regulation of neurotrophic factors and energy metabolism by antidepressants in astrocytes.

There is growing evidence that astrocytes are involved in the neuropathology of major depression. In particular, decreases in glial cell density observed in the cerebral cortex of individuals with major depressive disorder are accompanied by a reduction of several astrocytic markers suggesting that astrocyte dysfunction may contribute to the pathophysiology of major depression. In rodents, glial loss in the prefrontal cortex is sufficient to induce depressive-like behaviors and antidepressant treatment prevents the stress-induced reduction of astrocyte number in the hippocampus. Collectively, these data support the existence of a link between astrocyte loss or dysfunction, depressive-like behavior and antidepressant treatment. Astrocytes are increasingly recognized to play important roles in neuronal development, neurotransmission, synaptic plasticity and maintenance of brain homeostasis. It is also well established that astrocytes provide trophic, structural, and metabolic support to neurons. In this article, we review evidence that antidepressants regulate energy metabolism and neurotrophic factor expression with particular emphasis on studies in astrocytes. These observations support a role for astrocytes as new targets for antidepressants. The contribution of changes in astrocyte glucose metabolism and neurotrophic factor expression to the therapeutic effects of antidepressants remains to be established.

Clonal acquisition of the Ly49A NK cell receptor is dependent on the trans-acting factor TCF-1.

Families of clonally expressed major histocompatibility complex (MHC) class I-specific receptors provide specificity to and regulate the function of natural killer (NK) cells. One of these receptors, mouse Ly49A, is expressed by 20% of NK cells and inhibits the killing of H-2D(d) but not D(b)-expressing target cells. Here, we show that the trans-acting factor TCF-1 binds to two sites in the Ly49A promoter and regulates its activity. Moreover, we find that TCF-1 determines the size of the Ly49A NK cell subset in vivo in a dosage-dependent manner. We propose that clonal Ly49A acquisition during NK cell development is regulated by TCF-1.

Formation of virus-like clusters is an intrinsic property of the tumor necrosis factor family member BAFF (B cell activating factor).

The oligomeric state of BAFF (B cell activing factor), a tumor necrosis factor (TNF) family cytokine that plays a critical role in B cell development and survival, has been the subject of recent debate. Myc-tagged BAFF starting at residue Gln136 was previously reported to crystallize as trimers at pH 4.5, whereas a histidine-tagged construct of BAFF, starting at residue Ala134, formed a virus-like cluster containing 60 monomers when crystallized at pH 9.0. The formation of the BAFF 60-mer was pH dependent, requiring pH &gt;or= 7.0. More recently, 60-mer formation was suggested to be artificially induced by the histidine tag, and it was proposed that BAFF, like all other TNF family members, is trimeric. We report here that a construct of BAFF with no amino-terminal tag (Ala134-BAFF) can form a 60-mer in solution. Using size exclusion chromatography and static light scattering to monitor trimer to 60-mer ratios in BAFF preparations, we find that 60-mer formation is pH-dependent and requires histidine 218 within the DE loop of BAFF. Biacore measurements established that the affinity of Ala134-BAFF for the BAFF receptor BAFFR/BR3 is similar to that of myc-Gln136-BAFF, which is exclusively trimeric in solution. However, Ala134-BAFF is more efficacious than myc-Gln136-BAFF in inducing B cell proliferation in vitro. We additionally show that BAFF that is processed and secreted by 293T cells transfected with full-length BAFF, or by a histiocytic lymphoma cell line (U937) that expresses BAFF endogenously, forms a pH-dependent 60-mer in solution. Our results indicate that the formation of the 60-mer in solution by the BAFF extracellular domain is an intrinsic property of the protein, and therefore that this more active form of BAFF may be physiologically relevant.

BAFF binds to the tumor necrosis factor receptor-like molecule B cell maturation antigen and is important for maintaining the peripheral B cell population.

The tumor necrosis factor (TNF) family member B cell activating factor (BAFF) binds B cells and enhances B cell receptor-triggered proliferation. We find that B cell maturation antigen (BCMA), a predicted member of the TNF receptor family expressed primarily in mature B cells, is a receptor for BAFF. Although BCMA was previously localized to the Golgi apparatus, BCMA was found to be expressed on the surface of transfected cells and tonsillar B cells. A soluble form of BCMA, which inhibited the binding of BAFF to a B cell line, induced a dramatic decrease in the number of peripheral B cells when administered in vivo. Moreover, culturing splenic cells in the presence of BAFF increased survival of a percentage of the B cells. These results are consistent with a role for BAFF in maintaining homeostasis of the B cell population.