BrdU ELISA Research Articles

Synergistic immunomodulatory effects of interferon-gamma and bone marrow mesenchymal stem cells

To investigate mesenchymal stem cells (MSCs) immunosuppressive activity in the presence of interferon-gamma (IFN-γ) to reveal synergistic immunomodulatory effects of IFN-γ and MSCs. ① MSCs were cultured in the presence or absence of IFN-γ（100 ng/ml）, the supernatants were collected for measurements of PGE2、HGF and TGF-β1 by ELISA kits. ② MSCs were cultured in the presence or absence of IFN-γ （100 ng/ml）for 48 h. The cDNA was analysed for the expression of human indoleamine 2, 3-dioxygenase（IDO）mRNA by semiquantitative RT-PCR. ③ Mononuclear cells (MNCs) were extracted from peripheral blood of healthy donors. The T cell proliferation was tested in the co-culture system added with MSCs, recombinant human IFN-γ (100 ng/ml) and anti-IFN-γ mAb (5 μg/ml) by BrdU ELISA kit. ①The immunosuppressive cytokines PGE2、HGF and TGF-β1 were detectable within 24-48 h in the supernatants. Their expressions were significantly up-regulated in the presence of IFN-γ. Concentrations of these cytokines were as of (1715.5±628.6) pg/ml vs (1344.5±709.4) pg/ml (P=0.001);(4031.8±1496.8) pg/ml vs (2452.4±1375.3) pg/ml（P=0.011）;(1753.5±413.8) pg/ml vs (1026.6±450.5) pg/ml（P<0.001）,respectively. ②The expression of IDO mRNA was undetectable when MSCs were cultured alone. In contrast, The IDO mRNA expression was remarkably enhanced in the presence of IFN-γ. ③Bone marrow-derived MSCs remarkably suppressed allogeneic T cell proliferation in vitro. Addition of exogenous IFN-γ had no significant effect on the inhibitory capacity of MSCs, the inhibitory ratios of T cell proliferation were （40.4±10.9）% vs（36.7±7.4）% (P=0.272). By contrast, the inhibitory ratio of T cell proliferation was significantly decreased in the presence of anti-IFN-γ mAb[(40.4±10.9)% vs (23.9±7.6)%,P=0.002]. ①Human MSCs constitutively expressed immunosuppressive concentrations of PGE2, HGF and TGF-β1, and their expressions were significantly up-regulated by IFN-γ. ②IFN-γ-induced expression of IDO on MSCs involved in tryptophan catabolism. ③MSCs notably suppressed allogeneic T cell proliferation in vitro. IFN-γ promoted the immunosuppressive capacity of human MSCs, indicating the synergistic immunomodulatory effect of IFN-γ and MSCs.

Gremlin induces cell proliferation and extra cellular matrix accumulation in mouse mesangial cells exposed to high glucose via the ERK1/2 pathway

BackgroundGremlin, a bone morphogenetic protein antagonist, plays an important role in the pathogenesis of diabetic nephropathy (DN). However, the specific molecular mechanism underlying Gremlin’s involvement in DN has not been fully elucidated. In the present study, we investigated the role of Gremlin on cell proliferation and accumulation of extracellular matrix (ECM) in mouse mesangial cells (MMCs), and explored the relationship between Gremlin and the ERK1/2 pathway.MethodsTo determine expression of Gremlin in MMCs after high glucose (HG) exposure, Gremlin mRNA and protein expression were evaluated using real-time polymerase chain reaction and western blot analysis, respectively. To determine the role of Gremlin on cell proliferation and accumulation of ECM, western blot analysis was used to assess expression of pERK1/2, transforming growth factor-β1 (TGF-β1) and connective tissue growth factor (CTGF). Cell proliferation was examined by bromodeoxyuridine (BrdU) ELISA, and accumulation of collagen IV was measured using a radioimmunoassay. This enabled the relationship between Gremlin and ERK1/2 pathway activation to be investigated.ResultsHG exposure induced expression of Gremlin, which peaked 12 h after HG exposure. HG exposure alone or transfection of normal-glucose (NG) exposed MMCs with Gremlin plasmid (NG + P) increased cell proliferation. Transfection with Gremlin plasmid into MMCs previously exposed to HG (HG + P) significantly increased this HG-induced phenomenon. HG and NG + P conditions up-regulated protein levels of TGF-β1, CTGF and collagen IV accumulation, while HG + P significantly increased levels of these further. Inhibition of Gremlin with Gremlin siRNA plasmid reversed the HG-induced phenomena. These data indicate that Gremlin can induce cell proliferation and accumulation of ECM in MMCs. HG also induced the activation of the ERK1/2 pathway, which peaked 24 h after HG exposure. HG and NG + P conditions induced overexpression of pERK1/2, whilst HG + P significantly induced levels further. Inhibition of Gremlin by Gremlin siRNA plasmid reversed the HG-induced phenomena. This indicates Gremlin can induce activation of the ERK1/2 pathway in MMCs.ConclusionCulture of MMCs in the presence of HG up-regulates expression of Gremlin. Gremlin induces cell proliferation and accumulation of ECM in MMCs. and enhances activation of the ERK1/2 pathway.

The role of norepinephrine in adult human articular chondrocytes

Norepinephrine (NE) belongs to the catecholamine family of tyrosine-derived neurotransmitters of the sympathetic nervous system. Tyrosine-hydroxylase positive sympathetic nerve fibers have been identified in bone marrow, in the periosteum, and in bone-adherent ligaments indicating that growth and metabolic activity of bone and joint tissues is regulated by sympathetic neurotransmitters. It is known that NE can regulate cell proliferation or apoptosis in several cell types, such as osteoblasts. It is further described that NE modulates inflammation during rheumatoid arthritis and gut inflammation. Here, we aim to understand the role of NE in human osteoarthritic chondrocytes with regard to inflammation and its impact on metabolic activity. Human chondrocytes were isolated from post-surgery discarded human osteoarthritic articular cartilage. Expression of adrenergic receptor on articular cartilage chondrocytes was tested with standard end point PCR and immunohistochemical analysis. Employing 3D cell cultures in fibrin gel, effects of NE on IL-1β induced gene expression of pro-inflammatory cytokines and matrix metalloproteinases (MMP) were analyzed with quantitative real-time PCR. The impact of NE on cell proliferation was determined in monolayer culture with BrdU ELISA and FACS analyses, the effect on apoptosis with Caspase 3/7 ELISA. To test the adrenergic receptor involved in apoptosis or proliferation, the α1-adrenergic receptor antagonist doxazosin, the α2-adrenergic receptor antagonist yohimbine, and the β1/2/3-adrenergic receptor antagonist nadolol were included in BrdU/caspase-3/7-ELISAs. Chondrocytes cultured in monolayer and in 3D under non- and inflammatory conditions expressed α1D-, α2A/B/C- and β2-adrenergic receptors. β2-Adrenergic receptors were detected on protein level in human osteoarthritic cartilage as well. Stimulation with NE has significantly reduced IL-1β induced gene expression of IL-8 and MMP-13 in human osteoarthritic chondrocytes cultured in 3D fibrin gel. Notably, we were unable to detect an impact on IL-1β induced gene expression of interleukin-6, MMP-2 and MMP-3. Furthermore, NE inhibits BrdU incorporation compared to the controls. This effect was reversed by nadolol. In cell cycle analyses, we showed that NE increased the G1/G0-phase population and decreased S-phase population. Additionally, we observed an increase of caspase-3/7 activity after NE treatment that was reversed after addition of α1-adrenergic receptor antagonist doxazosin. Neurotransmitters of the sympathetic nervous system like NE presumably mediate an anti-inflammatory/chondroprotective effect in human osteoarthritic chondrocytes via reducing IL-1β induced IL-8 and MMP-13. Furthermore, NE is able to modulate the metabolic activity of chondrocytes by a cell cycle slow-down via β2-adrenergic receptor signaling and by induction of apoptosis via α1D-adrenergic receptor signaling. We therefore assume a yet unknown function of catecholaminergic neurotransmitters in adult human cartilage that might have an impact on osteoarthritis pathology.

Rhinovirus has the unique ability to directly activate human T cells in vitro

Rhinovirus infection is a leading cause of exacerbation of airway diseases. We hypothesize that airway viruses activate inflammatory cells, inducing airway dysfunction. We have previously shown that airway viruses can induce eosinophil degranulation when cocultured with T cells and monocyte-derived dendritic cells (moDCs). These findings suggested that antigen presentation was important for T-cell activation. Given the clinical importance of rhinovirus, we sought to determine whether it had any unique abilities to activate inflammatory cells compared with another common virus, such as respiratory syncytial virus (RSV). We cocultured combinations of human leukocytes (T cells, moDCs, and eosinophils) with each virus. Using assays of BrdU incorporation, flow cytometry, and ELISA, we measured T-cell activation, rhinovirus expression, T-cell death, and eosinophil cysteinyl leukotriene release. In contrast to RSV, rhinovirus induced T-cell activation without the involvement of moDCs. Without moDCs, rhinovirus induced T-cell proliferation of both CD4 and CD8(+) cells, cytokine production, and ultimately, eosinophil stimulation. Although chloroquine inhibited RSV-induced activation of T cells through moDCs, rhinovirus was not inhibited; UV inactivation did block the rhinovirus effect. We also found that T cells could be infected by rhinovirus in vitro and within human nasal explant tissue. Although Toll-like receptors did not appear to be involved in T-cell activation, antagonists of Jun N-terminal kinase and nuclear factor κB did inhibit T-cell responses to rhinovirus. Rhinovirus has the unique ability to bypass antigen presentation and directly infect and activate human T cells. This could explain the strong association of rhinovirus with exacerbation of airway diseases.

Human Eb Peptide: Not just a By-product of Pre-pro-IGF1b Processing?

Several physiological activities have been assigned to E-peptides derived from pre-pro-insulin-like growth factor (IGF1) processing; however, the whole range of the E-peptides' functions is still unknown. The objective of this study was to investigate human Eb peptide (hEb) in terms of its bioactivity, cellular localization, and intracellular trafficking using human cancer cells. Human Eb fused with red fluorescence protein (RFP) or green fluorescence protein (GFP) localizes strongly to nucleoli and to a lesser extent to nuclei of HeLa and U2-OS cells. Mutagenesis of hEb nucleolus localization sequence (NoLS) leads to its partial delocalization from nuclei and nucleoli to cytoplasm of transfected cells. Thus, NoLS is not sufficient for the hEb to be localized in nucleoli of the cells and a different mechanism may be involved in hEb targeting. A BrdU ELISA showed that the proliferation index of cells expressing hEb hybrid proteins increased up to 28%. For comparison, the same assay was performed using HeLa cells treated extracellularly with synthetic hEb. A significant increase in the proliferation index was observed (41-58% for concentrations ranging from 10-100 nM, respectively). Additionally, a cell migration assay was performed using stable U2-OS cell lines expressing hEb fused with RFP or RFP alone as a negative control. The migration index of hEb expressing cells was 38.3% greater. The increase in cell proliferation index and in motile properties of hEb expressing cells demonstrate that hEb is more than a pre-pro-IGF1b processing product, and has intrinsic activity of biological significance.

Effects of TRAIL and taurolidine on apoptosis and proliferation in human rhabdomyosarcoma, leiomyosarcoma and epithelioid cell sarcoma

Soft tissue sarcomas (STS) are a heterogeneous group of malignant tumours representing 1% of all malignancies in adults. Therapy for STS should be individualised and multimodal, but complete surgical resection with clear margins remains the mainstay of therapy. Disseminated soft tissue sarcoma still represents a therapeutic dilemma. Commonly used chemotherapeutic agents such as doxorubicin and ifosfamide have proven to be effective in fewer than 30% in these cases. Therefore, we tested the apoptotic and anti-proliferative in vitro effects of TNF-related apoptosis-inducing ligand (TRAIL) and taurolidine (TRD) on rhabdomyosarcoma (A-204), leiomyosarcoma (SK-LMS-1) and epithelioid cell sarcoma (VA-ES-BJ) cell lines. Viability, apoptosis and necrosis were quantified by FACS analysis (propidium iodide/Annexin V staining). Gene expression was analysed by DNA microarrays and the results validated for selected genes by rtPCR. Protein level changes were documented by western blot analysis. Cell proliferation was analysed by BrdU ELISA assay. The single substances TRAIL and TRD significantly induced apoptotic cell death and decreased proliferation in rhabdomyosarcoma and epithelioid cell sarcoma cells. The combined use of TRAIL and TRD resulted in a synergistic apoptotic effect in all three cell lines, especially in rhabdomyosarcoma cells leaving 18% viable cells after 48 h of incubation (p<0.05). Analysis of the differentially regulated genes revealed that TRD and TRAIL influence apoptotic pathways, including the TNF-receptor associated and the mitochondrial pathway. Microarray analysis revealed remarkable expression changes in a variety of genes, which are involved in different apoptotic pathways and cross talk to other pathways at multiple levels. This in vitro study demonstrates that TRAIL and TRD synergise in inducing apoptosis and inhibiting proliferation in different human STS cell lines. Effects on gene expression differ relevantly in the sarcoma entities. These results provide experimental support for in vivo trials assessing the effect of TRAIL and TRD in STS and sustain the approach of individualized therapy.

Induced apoptosis in human prostate cancer cells by blocking of vascular endothelial growth factor by siRNA

Vascular endothelial growth factor (VEGF) regulates several cell functions including; proliferation, differentiation, permeability, vascular tone, and the production of vasoactive molecules. The purpose of this study was to evaluate the potency of specific short-interfering RNA (siRNA) to suppress human VEGF expression by siRNA and investigate the effects of VEGF down-regulation on the cell proliferation and apoptosis of the human prostate cancer cell lines DU-145. Transfection was performed using X-tremeGENE siRNA transfection reagent. At different time intervals, transfected cells were harvested and total RNA was extracted for RT-PCR. The VEGF content in supernatants were measured by ELISA. Inhibition of cell growth by hVEGF-siRNA was measured by using cell proliferation ELISA BrdU assay. Apoptotic cells were evaluated by using annexin-V-FITC apoptotic detection method. Transfection of hVEGF-siRNA resulted in statistically significant inhibition of hVEGF-mRNA that in turn caused a marked reduction in the expression of hVEGF. The cell growth was assessed every 24 h for 4 days after siRNA treatment resulted in a marked inhibition of cell proliferation as compared to scramble siRNA. The results of apoptosis showed that approximately 15 % of the cells treated with control-siRNA manifested evident apoptotic changes after 24 hpt, whereas DU-145 cells treated with hVEGF-siRNA significantly were positive, that is to say, 53 % at 72 hpt 23.9 ± 2.78 % (P < 0.001) and 13 ± 1.57 % at 96 hpt. Our findings indicate that siRNA are effective in eliciting the RNAi pathway in cancerous cells and that specific siRNA efficiently down-regulate VEGF expression. They could decrease VEGF production and induce apoptosis, which may also be linked to the inhibition of cancerous cell proliferation. Therefore, it can be concluded that siRNA-mediated suppression of VEGF represents a powerful tool against prostate cancer cell proliferation. VEGF down-regulation exerts a direct anti-apoptotic function in the DU-145 cell lines and promises the development of drugs for cancer therapy.

Configuration of electrofusion-derived human insulin-secreting cell line as pseudoislets enhances functionality and therapeutic utility

Formation of pseudoislets from rodent cell lines has provided a particularly useful model to study homotypic islet cell interactions and insulin secretion. This study aimed to extend this research to generate and characterize, for the first time, functional human pseudoislets comprising the recently described electrofusion-derived insulin-secreting 1.1B4 human β-cell line. Structural pseudoislets formed readily over 3-7 days in culture using ultra-low-attachment plastic, attaining a static size of 100-200 μm in diameter, corresponding to ~6000 β cells. This was achieved by decreases in cell proliferation and integrity as assessed by BrdU ELISA, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, and lactate dehydrogenase assays. Insulin content was comparable between monolayers and pseudoislets. However, pseudoislet formation enhanced insulin secretion by 1·7- to 12·5-fold in response to acute stimulation with glucose, amino acids, incretin hormones, or drugs compared with equivalent cell monolayers. Western blot and RT-PCR showed expression of key genes involved in cell communication and the stimulus-secretion pathway. Expression of E-Cadherin and connexin 36 and 43 was greatly enhanced in pseudoislets with no appreciable connexin 43 protein expression in monolayers. Comparable levels of insulin, glucokinase, and GLUT1 were found in both cell populations. The improved secretory function of human 1.1B4 cell pseudoislets over monolayers results from improved cellular interactions mediated through gap junction communication. Pseudoislets comprising engineered electrofusion-derived human β cells provide an attractive model for islet research and drug testing as well as offering novel therapeutic application through transplantation.

Nicotine stimulated bone marrow-derived dendritic cells could augment HBV specific CTL priming by activating PI3K-Akt pathway

Our previous studies have revealed that nicotine-treated immature dendritic cells (imDCs) have anti-tumor effects in murine lymphoma models. The present study is to explore HBV-specific CTL priming and its cytolytic activities of nicotine-treated murine DCs, the mechanism of α7 nicotinic acetylcholine receptor (nAChR) up-regulation by nicotine and the efficiency of nicotine with other cytokines. To address these hypotheses, bone marrow-derived imDCs were stimulated by nicotine and expression of α7 nAChR was firstly determined by flow cytometry and Western blot. Then, DCs-dependent HBV-specific T cell proliferation and IL-12 secretion were secondly determined by BrdU cell proliferation assay and ELISA, respectively. The HBV-specific CTL priming and its activities were further explored by intraperitoneal transfer of nicotine treated imDCs. The mechanism of nicotine up-regulating α7 nAChR was finally explored by Western blot. The results showed that: first, the maximal activation of PI3K and Akt was reached at 30 and 60–120min respectively after nicotine stimulation. Nicotine up-regulated the expression of α7 nAChR by activating PI3K-Akt pathway in murine DCs; secondly, nicotine stimulation could enhance DCs’ ability of HBV-specific T cell proliferation and IL-12 secretion; thirdly, adoptive transfer of nicotine stimulated DCs could induce HBV specific CTL priming in vivo and those CTL had cytolytic activities; fourthly, nicotine had equal efficiencies to 2ng/ml IFN-γ in DCs-mediated T cell proliferation. All these data presented here indicated that nicotine treated imDCs might be considered as a potential candidate for HBV immunotherapy.

Inhibitory effects of ZSTK474, a phosphatidylinositol 3-kinase inhibitor, on adjuvant-induced arthritis in rats

We examined the effects of ZSTK474, a phosphatidylinositol 3-kinase (PI3K) inhibitor, on adjuvant-induced arthritis (AIA). AIA was induced in Lewis rats by subcutaneous administration of Freund's complete adjuvant at the base of the tail on day 0. ZSTK474 was orally administered once daily from day 10. The severity of AIA was assessed by measuring the hind paw volume. The number of lymphocytes in inguinal lymph nodes (ILN) was determined by flow cytometry. The in vitro effects of ZSTK474 on the cell proliferation, and the cytokines and prostaglandin E(2) (PGE(2)) production were evaluated by BrdU method, ELISA and cytometric beads array. ZSTK474 ameliorated the progression of AIA. The temporary increases in the number of T cells in ILN, which occurred along with the appearance of arthritis, were inhibited in the ZSTK474-treated groups. In vitro studies revealed that ZSTK474 inhibited the production of IFNγ and IL-17 in concanavalin A-activated T cells. In vitro studies further revealed that ZSTK474 inhibited the proliferation and PGE(2) production by fibroblast-like synovial cells (FLS). ZSTK474 demonstrated prophylactic efficacy in a rat model of rheumatoid arthritis (RA) through inhibition of T cell and FLS functions. It was suggested that the inhibitors of PI3K have therapeutic potential for RA.

RACK1 downregulation suppresses migration and proliferation of neuroblastoma cell lines

Neuroblastoma is the most common extracranial solid tumor in childhood, whose molecular mechanism on clinically heterogeneous behavior is still unclear. Receptor for activated C-kinase 1 (RACK1) has been shown to be involved in the regulation of growth and migration in many types of cells. Two types of human neuroblastoma cell lines, SK-N-SH and SK-N-BE (2), were used to explore the role of RACK1 in neuroblastoma cell migration and proliferation. Cell migration and proliferation were detected by a transwell system and colorimetric bromodeoxyuridine (BrdU) ELISA, respectively. The expressions of RACK1, N-Myc, phospho-Src(Tyr416), phospho-Src(Tyr527), phospho-Akt, phospho-ERK1/2 and phospho-p38 were analyzed by Western blotting. The expression of RACK1 was then repressed by target RNA interference (RNAi) in both types of neuroblastoma cells, and the expression of signaling molecules, migration and proliferation of cells was analyzed. Compared with SK-N-SH, the migration and proliferation of SK-N-BE(2) cells was remarkably higher which was accompanied by higher expression of RACK1 and phospho-Src(Tyr416). RACK1 RNAi repressed cell migration and proliferation, and decreased the expression of phospho-Src(Tyr416) in both cell lines. In summary, RACK1 was expressed in the neuroblastoma cells and positively regulated cell migration and proliferation probably via modulating the activation of Src on Tyr416 residue.

MicroRNA Profiling Reveals Aberrant MicroRNA Expression in Adult ETP-ALL and Functional Studies Demonstrate a Role of Mir-222 in Acute Leukemia,

Abstract 3458 Introduction:Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) has been identified as high-risk subgroup in pediatric acute T-lymphoblastic leukemia (T-ALL). ETP-ALLs originate from early thymic progenitors that retain stem cell features and are characterized by a specific immunophenotype and by a specific gene expression profile. Recently, we have characterized ETP-ALL as subgroup of early T-ALL in adults. To unravel the underlying mechanisms for the aberrant and distinct gene expression profile in ETP-ALL we now explored the expression of microRNAs (miRNAs) as gene regulators in ETP-ALL and further investigated their functional role in acute leukemia. Patients and methods: We screened expression levels of 667 miRNAs in newly diagnosed T-ALL patients (n=14: ETP-ALL n=8, non-ETP early T-ALL n=3, thymic T-ALL n=3) and 2 healthy controls (CD3 selected T cells) using a Taqman Low Density Array (TLDA; Applied Biosystems). The expression levels of miR-221 and miR-222 were validated in a large cohort of adult T-ALL (n=178: ETP-ALL n=66 and typical T-ALL n=112 including early-, thymic-, and mature T-ALLs) and healthy controls (n=6) by real-time-PCR (Taqman microRNA assays; Applied Biosystems). These patients were enrolled on the GMALL study 07/03. Functional analysis was performed by AMAXA transfection of pSUPER vector containing the miR-221 and miR-222 DNA sequences in Jurkat (T-lineage ALL) and KG1a (AML) cells. Viability was determined by cell proliferation reagent WST-1, and BrdU ELISA. Apoptosis was measured by annexin-V/7AAD staining with subsequent flow cytometric analysis. Results: In our screen 229 (34%) of the 667 miRNAs represented by the TLDA were expressed at detectable levels in at least two-thirds of the T-ALL samples and the controls. Of these we identified 55 miRNAs to be differentially expressed in T-ALL samples compared to healthy controls: 49 miRNAs were upregulated and 6 miRNAs were downregulated in the patient samples. Of these, miR-221 and miR-222 were the only miRNAs significantly upregulated in the ETP-ALL compared to the combined early and thymic T-ALL subgroups (8.0-fold, P<0.01; 5.0-fold, P<0.01, respectively). Therefore, we further validated miR-221 and miR-222 expression in a larger set of 178 T-ALL samples and observed a 3.3-fold higher expression of miR-221 (P<0.0001) and 3.9-fold higher expression of miR-222 (P<0.0001) in ETP-ALL (n=66) compared to typical T-ALL (n=112). For the correlation of miR-221 and miR-222 expression with molecular features, samples were divided into groups (low and high) according to the median of the miRNA expression levels. High miR-222 expression was associated with an immature phenotype of early T-ALL (P<0.01) and cell surface expression of CD34 (mean: 32%) compared to low miR-222 expressers (mean: 8%; P<0.01). Moreover, high miR-222 expression was associated with expression of myeloid markers CD33 (mean: 23% compared to 4% in low miRNA expressers, P<0.01) and CD13 (mean: 18% vs. 4%, P<0.01). Similar results were obtained for miR-221. In vitro studies revealed that overexpression of miR-222 inhibited proliferation of Jurkat (58% reduction of proliferation compared to cells transfected with the empty vector, P<0.001) and KG1a cells (50% reduction of proliferation compared to control cells, P<0.01) determined by WST-1. A reduction of the DNA synthesis was detected by BrdU incorporation after miR-222 overexpression in Jurkat (37% reduction compared to control cells) and in KG1a cells (54% reduction compared to control cells). Overexpression of miR-222 induced a 1.3- (P=0.02) and 3.0-fold (P<0.01) increase in apoptosis in Jurkat and KG1a cells, respectively. No significant changes were observed for miR-221 transfected cells in these functional assays. Conclusion: In summary, our study revealed aberrant expression of miRNAs in ETP-ALL, with miR-221 and miR-222 as the most overexpressed miRNAs. Functional analysis demonstrated that miR-222 impaired the proliferation and induced apoptosis, indicating a potential role for miR-222 in acute leukemia. Importantly, miR-222 targets such as the proto-oncogene c-KIT and the ETS1oncogene are contained in the gene expression profile of ETP-ALL. Thus, aberrant expression of miR-222 may directly impact leukemogenesis by altering expression of key oncogenes in acute leukemia. Disclosures:Goekbuget:Micromet: Consultancy.

The activity of a new 2-amino-1,3,4-thiadiazole derivative 4ClABT in cancer and normal cells

The 2-amino-5-(2,4-dihydroxyphenyl)-1,3,4-thiadiazole set are well known compounds with interesting in vitro and in vivo anti-cancer profiles. The aim of this study was an in vitro evaluation of the anti-cancer activity of a new synthesized aminothiadiazole derivative 2-(3-chlorophenyloamino)-5-(2,4-dihydroxyphenyl)- -1,3,4-thiadiazole 4ClABT. The effect on tumor cell proliferation, motility and morphology, DNA synthesis as well as the influence on normal cells was assessed. The antiproliferative activity of 4ClABT in tumor cells derived from peripheral cancers including breast carcinoma (T47D), colon carcinoma (HT-29), thyroid carcinoma (FTC-238), teratoma (P19), and T-cell leukemia (Jurkat E6.1), as well as cancers of the nervous system including rhabdomyosarcoma/medulloblastoma (TE671), brain astrocytoma (MOGGCCM) and glioma (C6) was studied by means of MTT assay. DNA synthesis level was determined in BrdU ELISA test. Wound assay model was applied for tumor cell motility assessment. Morphological changes induced by 4ClABT in cancer and normal cells were analyzed in HE staining specimens. Moreover, the influence of 4ClABT on normal cells including skin fibroblasts (HSF), hepatocytes (Fao), astroglia and neurons was studied by means of LDH assay. The tested compound inhibited the proliferation of tumor cells in dose-dependent fashion. The anti-cancer effect was attributed to decreased DNA synthesis, prominent changes in tumor cell morphology as well as reduced cell motility. In antiproliferative concentrations, 4ClABT was not toxic to normal cells. Our study showed prominent anti-cancer effects of the tested aminothiadiazole derivative in the absence of toxicity in normal cells. The obtained results confirmed the promising anti-cancer profile of previously tested 2-(monohalogenphenylamino)- -5-(2,4-dihydroxyphenyl)-1,3,4-thiadiazole derivatives (ClABT - chlorophenyl derivative, FABT and 3FABT - fluorophenyl derivatives and 4BrABT - bromophenyl derivative). The molecular mechanisms and the in vivo activity of aminothiadiazole derivatives will be the subject of further studies.

ShRNA targeting β1-integrin suppressed proliferative aspects and migratory properties of airway smooth muscle cells

Dysfunction of airway smooth muscle (ASM) is an essential feature of airway remodeling in chronic asthma. However, the precise mechanisms of this pathological process have not been well studied. In previous study, we found that β1-integrin, which was dramatically upregulated in ASM cells in an asthmatic mouse model, was associated with the cell proliferation. In this study, we employed short hairpin RNA (shRNA) targeting β1-integrin to assess the effect of down-regulation of this receptor on the proliferative aspects and migratory properties of ASM cells in vitro. The cells were treated with shRNA expression vectors directed against β1-integrin, control vectors that included the blank control, empty vector without shRNA, and mismatched shRNA, respectively. The mRNA and protein expressions of β1-integrin were determined by real-time PCR and Western blotting. Cell proliferation was measured by BrdU ELISA and cell cycle by fluorescence-activated cell sorter. Cell apoptosis was detected by Annexin V-PE/7-AAD staining. Cell migration assays were evaluated by transwell assay and expression of IL-6 and IL-8 by ELISA. The results revealed that shRNA targeting β1-integrin significantly decreased the mRNA and protein expressions of β1-integrin, enhanced the proportion of cells in G0/G1 phase, decreased the proportion in S phase, promoted cell apoptosis, inhibited cell proliferation, migration, IL-6 and IL-8 secretion in vitro. In conclusion, the overexpression of β1-integrin in ASM cells is essential for airway dysfunction development because it promotes proliferative aspects and migratory properties of ASM cells. Importantly, shRNA targeting β1-integrin may provide a new approach to preventing airway remodeling in chronic asthma.

Ex Vivo Expansion of Conjunctival and Limbal Epithelial Cells Using Cord Blood Serum–Supplemented Culture Medium

Conventional cell culture methods use fetal bovine serum (FBS) as a growth supplement. The purpose of this study was to develop a xenobiotic-free culture system using umbilical cord blood serum (CBS) as an alternative growth supplement for the cultivation of human conjunctival and limbal epithelial cells. Human conjunctival and limbal epithelial cells were cultivated in varying concentrations of CBS-supplemented medium and compared with FBS-supplemented medium. Bromodeoxyuridine (BrdU) ELISA proliferation assay, colony-forming efficiency (CFE), and a number of cell generations were analyzed. Cytokeratin expression of cultured cells was evaluated (K3, K4, K12, K13, K14, K15, K19, and PanCK). The authors compared the cytokine and growth factor levels in CBS, FBS, and adult serum using antibody array assays. Conjunctival and limbal cells cultivated in 0.25% CBS- and 0.5% CBS-supplemented culture media demonstrated the highest proliferative capacity in terms of BrdU proliferation assay, CFE, and number of cell generations. These results were comparable to FBS-supplemented medium. Cultured epithelial cells retained their normal cytokeratin expression. Cytokines brain-derived neurotrophic factor, growth-related oncogene, and leptin and growth factors EGF, HGF, FGF-6, IGF-1, PDGF, and IGFBP were present in higher concentrations in CBS than in FBS and adult serum. CBS-supplemented culture medium supported the proliferation and differentiation of conjunctival and limbal epithelial cells. CBS contained a higher concentration of growth factors and cytokines than FBS and adult serum. CBS may be a viable and safer alternative to FBS as a growth supplement in the culture medium for culturing epithelial cells, which may have important clinical implications when bioengineering tissues for clinical use.

Exendin-4 upregulates the expression of Wnt-4, a novel regulator of pancreatic β-cell proliferation

The Wnt-signaling pathway regulates β-cell functions. It is not known how the expression of endogenous Wnt-signaling molecules is regulated in β-cells. Therefore, we investigated the effect of antidiabetic drugs and glucose on the expression of Wnt-signaling molecules in β-cells. Primary islets were isolated and cultured. The expression of Wnt-signaling molecules (Wnt-4, Wnt-10b, Frizzled-4, LRP5, TCF7L2) and TNFα was analyzed by semiquantitative PCR and Western blotting. Transient transfections were carried out and proliferation assays of INS-1 β-cells performed using [(3)H]thymidine uptake and BrdU ELISA. Insulin secretion was quantified. A knockdown (siRNA) of Wnt-4 in β-cells was carried out. Exendin-4 significantly increased the expression of Wnt-4 in β-cells on the mRNA level (2.8-fold) and the protein level (3-fold) (P < 0.001). The effect was dose dependent, with strongest stimulation at 10 nM, and it was maintained after long-term stimulation over 4 wk. Addition of exd-(9-39), a GLP-1 receptor antagonist, abolished the effect of exendin-4. Treatment with glucose, insulin, or other antidiabetic drugs had no effect on the expression of any of the examined Wnt-signaling molecules. Functionally, Wnt-4 antagonized the activation of canonical Wnt-signaling in β-cells. Wnt-4 had no effect on glucose-stimulated insulin secretion or insulin gene expression. Knocking down Wnt-4 decreased β-cell proliferation to 45% of controls (P < 0.05). In addition, Wnt-4 and exendin-4 treatment decreased the expression of TNFaα mRNA in primary β-cells. These data demonstrate that stimulation with exendin-4 increases the expression of Wnt-4 in β-cells. Wnt-4 modulates canonical Wnt signaling and acts as regulator of β-cell proliferation and inflammatory cytokine release. This suggests a novel mechanism through which GLP-1 can regulate β-cell proliferation.

Impact of lenalidomide on the antiproliferative effect of gemcitabine/carboplatin (GC) and gemcitabine/cisplatin (GP) against urothelial carcinoma (UC) cells in vitro.

e15164 Background:Although urothelial carcinoma (UC) is a chemosensitive malignancy, responses are not durable and most patients with metastatic UC die from their disease. New strategies are needed. Both pre-clinical and clinical data support targeting angiogenesis in UC and other solid tumors. Lenalidomide is an anti-angiogenic and a potent immunomodulatory agent that has demonstrated activity in hematological malignancies. There are no in vitro data of single agent direct activity against solid tumor cells and few data regarding the combination of lenalidomide and standard of care chemotherapeutic agents, such as cisplatin (P), carboplatin (C) and gemcitabine (G). We explored in vitro combinations of lenalidomide, GC and GP to improve possible treatment options for UC. Methods: The effects of lenalidomide alone and in combination with GC or GP, on UC cell proliferation were studied. UC T24 cells were exposed to various concentrations of lenalidomide (0, 0.5uM, 1uM) for 24 hours. This was followed by treatment with a combination of G (10nM) and C (1nM and10nM), or G (10nM) and P (10ug/ml) for 48 hours. As a control, each drug was treated for 48 hours alone. Cell proliferation was assessed by an ELISA BrdU assay. Results: Lenalidomide alone had no effect on the proliferation of UC T24 cells, whereas G, C, and P had moderate anti-proliferative activity. With the combination of lenalidomide with GC or GP there was a clear and dose-dependent enhancement of anti-proliferative activity in T24 cell lines. Conclusions: These studies support the potential utility of lenalidomide in combination with GC or GP in the treatment of patients with solid tumors including UC. A phase I trial of lenalidomide in combination with GC in metastatic UC has is currently under way. The mechanism by which lenalidomide affects UC cells and the enhancement of chemotherapy cytotoxicity remains to be elucidated.

Development of a Cellular Tau Enzyme-Linked Immunosorbent Assay Method for Screening GSK-3β Inhibitors

Glycogen synthase kinase-3β (GSK-3β), a serine/threonine kinase also known as tau protein kinase I, has been implicated in the pathogenic conditions of Alzheimer's disease. Many investigators have focused on GSK-3 inhibitor as a therapeutic drug. In this study, we established a cell-based assay for the screening of novel GSK-3β inhibitors. For this purpose, four-repeat tau cDNAs were stably expressed in human embryonic kidney 293 (HEK293) cells (HEK293-Tau). The proliferation of HEK293-Tau cells was no different from that of HEK293 cells, as measured by the bromodeoxyuridine enzyme-linked immunosorbent assay (BrdU ELISA). The concentration-dependent reduction of tau phosphorylation by GSK-3 inhibitors, LiCl, Chir98023, and SB415286, was examined by immunoblot analysis and Tau ELISA (in situ ELISA). Highly consistent data were obtained, suggesting that this novel ELISA method is highly reproducible. Using this ELISA strategy, we isolated a few candidate compounds, including compounds 114 and 149, from several hundreds of synthetic agents and demonstrated that such candidates protect nerve growth factor-differentiated PC12 cells against amyloid-β-induced cell death. These data indicate that this Tau ELISA method in HEK293-Tau cells may be a suitable cell-based assay system to screen for GSK-3β inhibitors.

The effect of hypoxia and cycloxygenase inhibitors on nasal polyp derived fibroblasts

Background and Purpose The pathogenesis of chronic rhinosinusitis with nasal polyposis is unknown. Chronic inflammation along with local tissue hypoxia may effect polyp's growth. Activation of Cycloxygenases may also be involved. COX-2 up-regulates in response to different stimuli including hypoxia. Its activation is associated with enhanced cell proliferation. Histologically, besides inflammatory cells, increased stromal fibrosis is seen in nasal polyposis. The aims of this study were to test whether hypoxia amplifies nasal polyp fibroblasts proliferation, whether treatment with various COX inhibitors could influence fibroblasts, and whether this effect may be modulated in response to different oxygenation conditions. Materials and Methods Polyp fibroblasts were incubated under hypoxic or normoxic conditions with or without NSAIDs at different concentrations for 12 or 24 hours. Cell proliferation was quantified using BrdU ELISA. Metabolic activity was evaluated using MTT assay. Cell death was measured using Annexin V staining and FACS scan. Results No significant difference was found between proliferation of fibroblasts treated under hypoxia or normoxia. Cells incubated with indomethacin proliferated in a slightly enhanced manner compared with non-treated cells. Celecoxib inhibited fibroblast proliferation ( P < .001) but did not influence cell survival. Metabolic activity of cells treated with celecoxib was significantly reduced ( P < .003), unlike cells treated with indomethacin or rofecoxib. Conclusion Hypoxia does not affect fibroblasts proliferation. It may contribute to nasal polyposis pathogenesis in other ways. The anti-proliferative effect of celecoxib may be associated with cell cycle arrest rather than with pro-apoptotic activity. Celecoxib may be considered for treating nasal polyposis.

The AhR is constitutively activated and affects granulosa cell features in the human cell line KGN

A well-balanced activity of the aryl hydrocarbon receptor (AhR) is necessary for normal ovarian function. As known from murine AhR knock-out (KO) models, the AhR is involved in folliculogenesis, gonadotrophin receptor expression, proliferation of granulosa cells and intraovarian estrogen signalling. Highly potent, non-physiological ligands such as the dioxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) lead to a blockade of ovulation, estrogen receptor degradation and reduction of estrogen levels. Estrogen synthesis is a typical function of granulosa cells and essential for normal cyclicity and fertility. We employed the human granulosa cell line KGN to further characterize AhR signalling and AhR function in granulosa cell physiology. Real-time PCR quantification of the target genes Cyp1a1 and Cyp1b1 and reporter gene assays after stimulation with TCDD or beta-naphthoflavone (BNF) or inhibition with alpha-naphthoflavone (ANF) or 3'-methoxy-4'-nitroflavone (3,4-MNF) of the AhR demonstrated constitutive activity and functionality of AhR pathway in KGN granulosa cells. In untreated KGN cells, AhR protein was exclusively detected in the nuclear fraction. TCDD stimulation affected the gonadotrophin receptor but not estrogen receptor β (ERβ) protein expression. Additionally, the constitutively activated AhR suppressed aromatase expression and estrogen synthesis (enzyme-linked immunoassay, ELISA) and enhanced proliferation [Bromodeoxyuridine (BrdU) ELISA] of KGN cells. Activation of the AhR by BNF did not override this inhibitory effect on estrogen synthesis or proliferation. In conclusion, the AhR pathway is constitutively activated and functional in human KGN granulosa cells. It is a potential target for endocrine disruption by exogenous ligands and subsequent dysfunction of granulosa cells.