96 research outputs found
Cover to Volume 3
The fibroblast mitogen platelet-derived growth factor -BB (PDGF-BB) induces a transient expression of the orphan nuclear receptor NR4A1 (also named Nur77, TR3 or NGFIB). The aim of the present study was to investigate the pathways through which NR4A1 is induced by PDGF-BB and its functional role. We demonstrate that in PDGF-BB stimulated NIH3T3 cells, the MEK1/2 inhibitor CI-1040 strongly represses NR4A1 expression, whereas Erk5 downregulation delays the expression, but does not block it. Moreover, we report that treatment with the NF-ΞΊB inhibitor BAY11-7082 suppresses NR4A1 mRNA and protein expression. The majority of NR4A1 in NIH3T3 was found to be localized in the cytoplasm and only a fraction was translocated to the nucleus after continued PDGF-BB treatment. Silencing NR4A1 slightly increased the proliferation rate of NIH3T3 cells; however, it did not affect the chemotactic or survival abilities conferred by PDGF-BB. Moreover, overexpression of NR4A1 promoted anchorage-independent growth of NIH3T3 cells and the glioblastoma cell lines U-105MG and U-251MG. Thus, whereas NR4A1, induced by PDGF-BB, suppresses cell growth on a solid surface, it increases anchorage-independent growth
DMA, a Bisbenzimidazole, Offers Radioprotection by Promoting NFΞΊB Transactivation through NIK/IKK in Human Glioma Cells
BACKGROUND: Ionizing radiation (IR) exposure often occurs for human beings through occupational, medical, environmental, accidental and/or other sources. Thus, the role of radioprotector is essential to overcome the complex series of overlapping responses to radiation induced DNA damage. METHODS AND RESULTS: Treatment of human glioma U87 cells with DMA (5- {4-methylpiperazin-1-yl}-2-[2'-(3, 4-dimethoxyphenyl)-5'-benzimidazolyl] in the presence or absence of radiation uncovered differential regulation of an array of genes and proteins using microarray and 2D PAGE techniques. Pathway construction followed by relative quantitation of gene expression of the identified proteins and their interacting partners led to the identification of MAP3K14 (NFΞΊB inducing kinase, NIK) as the candidate gene affected in response to DMA. Subsequently, over expression and knock down of NIK suggested that DMA affects NFΞΊB inducing kinase mediated phosphorylation of IKKΞ± and IKKΞ² both alone and in the presence of ionizing radiation (IR). The TNF-Ξ± induced NFΞΊB dependent luciferase reporter assay demonstrated 1.65, 2.26 and 3.62 fold increase in NFΞΊB activation at 10, 25 and 50 Β΅M DMA concentrations respectively, compared to control cells. This activation was further increased by 5.8 fold in drug + radiation (50 Β΅M +8.5 Gy) treated cells in comparison to control. We observed 51% radioprotection in control siRNA transfected cells that attenuated to 15% in siRNA NIK treated U87 cells, irradiated in presence of DMA at 24 h. CONCLUSIONS: Our studies show that NIK/IKK mediated NFΞΊB activation is more intensified in cells over expressing NIK and treated with DMA, alone or in combination with ionizing radiation, indicating that DMA promotes NIK mediated NFΞΊB signaling. This subsequently leads to the radioprotective effect exhibited by DMA
Functional Characterization of FLT3 Receptor Signaling Deregulation in Acute Myeloid Leukemia by Single Cell Network Profiling (SCNP)
Molecular characterization of the FMS-like tyrosine kinase 3 receptor (FLT3) in cytogenetically normal acute myeloid leukemia (AML) has recently been incorporated into clinical guidelines based on correlations between FLT3 internal tandem duplications (FLT3-ITD) and decreased disease-free and overall survival. These mutations result in constitutive activation of FLT3, and FLT3 inhibitors are currently undergoing trials in AML patients selected on FLT3 molecular status. However, the transient and partial responses observed suggest that FLT3 mutational status alone does not provide complete information on FLT3 biological activity at the individual patient level. Examination of variation in cellular responsiveness to signaling modulation may be more informative.Using single cell network profiling (SCNP), cells were treated with extracellular modulators and their functional responses were quantified by multiparametric flow cytometry. Intracellular signaling responses were compared between healthy bone marrow myeloblasts (BMMb) and AML leukemic blasts characterized as FLT3 wild type (FLT3-WT) or FLT3-ITD. Compared to healthy BMMb, FLT3-WT leukemic blasts demonstrated a wide range of signaling responses to FLT3 ligand (FLT3L), including elevated and sustained PI3K and Ras/Raf/Erk signaling. Distinct signaling and apoptosis profiles were observed in FLT3-WT and FLT3-ITD AML samples, with more uniform signaling observed in FLT3-ITD AML samples. Specifically, increased basal p-Stat5 levels, decreased FLT3L induced activation of the PI3K and Ras/Raf/Erk pathways, decreased IL-27 induced activation of the Jak/Stat pathway, and heightened apoptotic responses to agents inducing DNA damage were observed in FLT3-ITD AML samples. Preliminary analysis correlating these findings with clinical outcomes suggests that classification of patient samples based on signaling profiles may more accurately reflect FLT3 signaling deregulation and provide additional information for disease characterization and management.These studies show the feasibility of SCNP to assess modulated intracellular signaling pathways and characterize the biology of individual AML samples in the context of genetic alterations
Random PCR mutagenesis screening of secreted proteins by direct expression in mammalian cells.
Unique CD40-mediated biological program in B cell activation requires both type 1 and type 2 NF-ΞΊB activation pathways
B lymphocytes can be activated by many different stimuli. However, the mechanisms responsible for the signaling and functional specificities of individual stimuli remain to be elucidated. Here, we have compared the contribution of the type 1 (p50-dependent) and type 2 (p52-dependent) NF-ΞΊB activation pathways to cell survival, proliferation, homotypic aggregation, and specific gene regulation of murine primary B lymphocytes. Whereas lipopolysaccharide (LPS) and B cell activation factor (BAFF) mainly activate the type 1 or type 2 pathways, respectively, CD40 ligand (CD40L) strongly activates both. Rescue of spontaneous apoptosis is diminished in p52(β/β) B cells after BAFF stimulation and in p50(β/β)c-Rel(β/β) B cells after LPS stimulation. Interestingly, significant CD40-induced B cell survival is still observed even in p50(β/β)c-Rel(β/β)p65(β/+) B cells, which is correlated with the ability of CD40L to up-regulate Bcl-x(L) expression in these cells. CD40L- and LPS-induced B cell proliferation, as well as up-regulation of proliferation-related genes, however, are greatly reduced in c-Rel(β/β) and p50(β/β)c-Rel(β/β) B cells but are normal in p52(β/β) B cells. We have further demonstrated that both c-Rel and p52 are required for CD40-mediated B cell homotypic aggregation, which explains well why neither LPS nor BAFF has this function. Overall, our studies suggest that both type 1 and type 2 NF-ΞΊB pathways contribute to the gene expression and biological program unique for CD40 in B cell activation
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