Quantitative Flow Cytometry Assay Research Articles

MicroRNA-30b is a multifunctional regulator of aortic valve interstitial cells

Calcific aortic valve disease is an active process involving a wide range of pathologic changes. Valve interstitial cells are the most prevalent cells in the heart valve and maintain normal valve structure and function. MicroRNAs (miRNAs) are essential posttranscriptional modulators of gene expression, and miRNA-30b is a known repressor of bone morphogenetic protein 2-mediated osteogenesis. We hypothesized that miRNA-30b is a multifunctional regulator of aortic valve interstitial cells during calcification. To determine the role of miRNA-30b in calcific aortic valve disease, we evaluated miRNA expression in human calcific aortic valve leaflets obtained intraoperatively. Furthermore, human valve interstitial cells were evaluated with regard to miRNA-30b expression and osteogenesis by quantitative real-time polymerase chain reaction, Western blotting, flow cytometry, and alkaline phosphatase assays. In this study, we demonstrated that miRNA-30b attenuates bone morphogenetic protein 2-induced osteoblast differentiation by targeting Runx2, Smad1, and caspase-3. Transfection of a mimic of miRNA-30b led to decreases in alkaline phosphatase activity and expressions of Runx2, Smad1, and caspase-3. Furthermore, dual luciferase reporter assays confirmed that Runx2, Smad1, and caspase-3 are direct targets of miRNA-30b. We demonstrated a remarkable role of miRNA-30b in calcific aortic valve disease as a regulator of human aortic valvular calcification and apoptosis through direct targeting of Runx2, Smad1, and caspase-3. Targeting of miRNA-30b could serve as a novel therapeutic strategy to limit progressive calcification in aortic stenosis.

CD86 expression as a surrogate cellular biomarker for pharmacological inhibition of the histone demethylase lysine-specific demethylase 1

There is a lack of rapid cell-based assays that read out enzymatic inhibition of the histone demethylase LSD1 (lysine-specific demethylase 1). Through transcriptome analysis of human acute myeloid leukemia THP1 cells treated with a tranylcypromine-derivative inhibitor of LSD1 active in the low nanomolar range, we identified the cell surface marker CD86 as a sensitive surrogate biomarker of LSD1 inhibition. Within 24h of enzyme inhibition, there was substantial and dose-dependent up-regulation of CD86 expression, as detected by quantitative polymerase chain reaction, flow cytometry, and enzyme-linked immunosorbent assay. Thus, the use of CD86 expression may facilitate screening of compounds with putative LSD1 inhibitory activities in cellular assays.

Glucocorticoids Suppress T Cell Function by Up‐Regulating MicroRNA‐98

To identify microRNAs (miRNAs) in human T cells that can explain known antiinflammatory properties of steroids. Activated human CD4+ T cells from healthy donors were exposed to 1 μM methylprednisolone (MP) in vitro and then subjected to miRNA and messenger RNA microarray analyses. Changes in expression profiles were recorded. Using quantitative polymerase chain reaction (qPCR), flow cytometry, and enzyme-linked immunosorbent assay (ELISA), we confirmed the suppression of predicted targets, and through miRNA transfection experiments, we could suggest mechanistic links. We identified numerous steroid-responsive genes and miRNAs-many known and some novel-including multiple previously unknown proinflammatory genes suppressed by MP. Further studies using qPCR, flow cytometry, and ELISA demonstrated that methylprednisolone increased the expression of miRNA-98 (miR-98) and suppressed the levels of predicted targets, including interleukin-13 and 3 tumor necrosis factor receptors (TNFRs): Fas, FasL, and TNFR superfamily member 1B. Forced expression of miR-98 in T cells resulted in suppression of the same targets. The findings of this study demonstrate a link between miR-98 expression and the effects of MP and provide evidence suggesting that MP acts through miR-98 to inhibit specific proinflammatory targets. Identification of this antiinflammatory mechanism of glucocorticoids is important, since it may pave the way toward the elusive goal of dissociating adverse effects from therapeutic effects.

Lentivirus‐Mediated RNAi Knockdown of NUPR1 Inhibits Human Nonsmall Cell Lung Cancer Growth In Vitro and In Vivo

NUPR1 (nuclear protein 1) was found to play a key role in the development of several malignancies including pancreas, breast, and prostate cancers. However, the functional role of NUPR1 in nonsmall cell lung cancer (NSCLC) progression and development is little known. Here, lentivirus-mediated small interfering RNA (siRNA) was employed to downregulate endogenous NUPR1 expression to study the function of NUPR1 in growth of nonsmall cell lung cancer. A lentivirus-mediated RNAi technology was used to specifically knock down the expression of NUPR1 in H1299 cells. Quantitative real-time reverse transcriptase polymerase chain reaction, flow cytometry, western blot and cell count assays were studied to characterize NUPR1 expression in vitro. Furthermore, nonsmall cell lung cancer xenograft models in nude mice were established to investigate whether knockdown of NUPR1 reduces the tumor growth in vivo. We found that downregulation of NUPR1 expression significantly inhibited nonsmall cell lung cancer H1299 cells proliferation and colony formation in vitro. Moreover, the specific downregulation of NUPR1 arrested cells in G0 phase of cell cycle and increased apoptosis rate. Silencing of NUPR1 also suppressed tumor growth by tail vein injection of lentivirus encoded shRNA against NUPR1 in vivo. Our findings revealed that the NUPR1 gene represents a promising target for gene silencing therapy in nonsmall cell lung cancer.

MicroRNA-17∼92a upregulation by estrogen leads to Bim targeting and inhibition of osteoblasts apoptosis

Anti-apoptotic effects of estrogen on osteoblasts are very important in the etiology of estrogen protection of the adult skeleton against bone loss. The mechanisms of this process are still not fully understood. Recent studies implicated an important role of microRNAs in estrogen-mediated responses in various cellular processes, including cell apoptosis and proliferation. Therefore, we hypothesized that these regulatory molecules might be involved with estrogen in protecting osteoblasts from apoptosis. Western blotting, quantitative real-time PCR, flow cytometry and luciferase assays were employed to investigate the role of microRNAs in this process. The microRNA cluster miR-17-92a, a post-transcriptional regulator, was significantly reduced during dexamethasone, etoposide and tumor necrosis factor alpha (TNF-α)-induced osteoblasts apoptosis. The repression of miR-17-92a was significantly attenuated by estrogen. To delineate the role of miR-17-92a in apoptosis, we silenced and overexpressed miR-17-92a in osteoblasts. We found that miR-17-92a depletion significantly enhanced dexamethasone-induced apoptosis and overexpressing miR-17-92a remarkably increased the anti-apoptotic effects of estrogen on osteoblasts. Mechanistic studies showed that miR-17-92a inhibited Bim expression through a microRNA-17-92a-binding site within the 3'-untranslated region of Bim. The post-transcriptional repression of Bim was further confirmed by a luciferase reporter assay. These results showed that miR-17-92a, plays a significant role in the process of estrogen protection of osteoblasts against apoptosis, by regulating Bim expression.

A Novel FACS Assay for Absolute Quantification of CD20 on B-Cells and Determination of CD20 Occupancy in Whole Blood Samples Shows a Higher Binding Strength of Obinutuzumab (GA101) to CD20 on B-Lymphocytes

Abstract 4611 Introduction.The absolute quantification of CD20 on malignant B-lymphocytes on patients who already received anti-CD20 MAbs is technically challenging, but may play a role in identifying patients who will respond to subsequent treatment or help define patients that are rituximab-refractory due to decreased expression of CD20. We have developed a new quantitative flow cytometry assay to scrutinize the absolute number of receptors on cell lines and B-lymphocytes in whole blood (WB) samples as well as target occupancy. This assay allows us to accurately determine the number of human MAbs bound per target cell on various cell subsets. CD20 density on B-lymphocytes in WB and binding properties of the type II CD20 antibody obinutuzumab (GA101) and the classical Type I CD20 antibody Rituximab were tested on samples from healthy volunteers. Acidic striping of bound human anti-CD20 MAbs was explored to enable the set up of a trustworthy CD20 occupancy assay. Methods.Binding of anti-CD20 mAbs rituximab (RTX) and GA101 to CD20 on normal and malignant B-cells was assessed using the Human IgG Calibrator (BioCytex, F) on EDTA anticoagulated WB samples and cell lines to determine the CD20 density. In parallel, the murine MAb (Clone B9E9) was tested using Cellquant Calibrator (BioCytex, F). CD20 density and apparent Kd, a relative measure of affinity, for each MAb was calculated from the data obtained with whole blood samples. Both the total number of CD20 molecules and the CD20 bound fraction were appraised by spiking the sample with anti-CD20 MAbs at saturation and acid striping. Results.The absolute number of anti-CD20 MAbs bound to CD20 on B-cells in WB samples was between 58,000 to 123,000 molecules per cell as determined by GA101 and RTX, respectively. The apparent CD20 quantitation on cell lines was approx. 2- to 3-fold lower for GA101 which is a characteristic and reported property of type II CD20 antibodies. The apparent Kd of GA101 (4.1 to 9.9 nM) was approximately 2-fold lower than that of RTX (10.5 to 22.7 nM) on several different lymphoma-derived cell lines (Daudi, Raji, Ramos and MEC-1). On B-lymphocytes from healthy volunteers the Kd were 1.2 and 3.5 nM for GA101 and RTX, respectively. Furthermore, CD20-bound GA101 could only be detached from its target CD20 using acid-striping assay at pH < 2.0 and appropriate ionic strength whereas RTX could be stripped at pH 2.5. Binding of anti-CD20 MAbs to acid-treated B-lymphocytes was found unaffected. Conclusion.The density of RTX bound to CD20 on B-lymphocytes in whole blood is similar to previous published data obtained from radioimmunoassays and using 125I-MAb (Teeling et al, Blood, 2006). In contrast the number of the Type II CD20 antibody GA101 bound to B-lymphocytes in whole blood was approximately half in comparison to RTX as previously reported (Moessner et al, Blood, 2010). The data further confirmed a higher apparent binding affinity of GA101 for CD20 with Kd values comparable to those reported previously using 125I-MAb. In summary, the new Human IgG Calibrator is a fast and accurate technique that can be used to evaluate the CD20 density and occupancy using human MAbs in complex matrix including whole blood. The test can be implemented in pharmacodynamic clinical trials and may allow for further understanding of complex pharacodynamic relationships. Disclosures:Beroual:BioCytex: Employment. Boulay-Moine:BioCytex: Employment. Badoil:BioCytex: Employment. Klein:Roche: Employment, Equity Ownership, Patents & Royalties. Moulard:BioCytex: Consultancy, Employment.

Azithromycin May Inhibit Interleukin‐8 Through Suppression of Rac1 and a Nuclear Factor‐Kappa B Pathway in KB Cells Stimulated With Lipopolysaccharide

Recent studies have shown that the 15-member macrolide antibiotic azithromycin (AZM) not only has antibacterial activity, but also results in the role of immunomodulator. Interleukin (IL)-8 is an important inflammatory mediator in periodontal disease. However, there have been no reports on the effects of AZM on IL-8 production from human oral epithelium. Therefore, we investigated the effects of AZM on IL-8 production in an oral epithelial cell line. KB cells were stimulated by Escherichia coli or Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) lipopolysaccharide (LPS) with or without AZM. IL-8 mRNA and protein expression and production in response to LPS were analyzed by quantitative polymerase chain reaction, flow cytometry, and enzyme-linked immunosorbent assay. The activation of nuclear factor-kappa B (NF-κB) and Rac1, which is important for IL-8 expression, was analyzed by enzyme-linked immunosorbent assay and Western blotting, respectively. IL-8 mRNA expression, IL-8 production, and NF-κB activation in LPS-stimulated KB cells were inhibited by the addition of AZM. LPS-induced Rac1 activation was also suppressed by AZM. This study suggests that AZM inhibits LPS-induced IL-8 production in an oral epithelial cell line, in part caused by the suppression of Rac1 and NF-κB activation. The use of AZM might provide possible benefits in periodontal therapy, with respect to both its antibacterial action and apparent anti-inflammatory effect.

RNAi-mediated silencing of VEGF-C inhibits non-small cell lung cancer progression by simultaneously down-regulating the CXCR4, CCR7, VEGFR-2 and VEGFR-3-dependent axes-induced ERK, p38 and AKT signalling pathways

Vascular endothelial growth factor C (VEGF-C) expression is associated with the malignant tumour phenotype making it an attractive therapeutic target. We investigated the biological roles of VEGF-C in tumour growth, migration, invasion and explored the possibility of VEGF-C as a potential therapeutic target for the treatment of non-small cell lung cancer (NSCLC). A lentivirus-mediated RNA interference (RNAi) technology was used to specifically knockdown the expression of VEGF-C in A549 cells. Quantitative reverse transcriptase-polymerase chain reaction, flow cytometry, Western blot, immunohistochemistry, cellular growth, migration, invasion and ELISA assays were used to characterise VEGF-C expression in vitro. A lung cancer xenograft model in nude mice was established to investigate whether knockdown of VEGF-C reduced tumour growth in vivo. Silencing of VEGF-C suppressed tumour cell growth, migration and invasion in vitro; suppressed tumour growth, angiogenesis and lymphangiogenesis by tail vein injection of lentivirus encoded shRNA against VEGF-C in vivo. More importantly, silencing of VEGF-C also trapped the VEGFR-2, VEGFR-3, CXCR4, CCR7-dependent axes, and down-regulated the AKT, ERK and p38 signalling pathways. These results suggest that VEGF-C has a multifaceted role in NSCLC growth, migration and invasion; that VEGF-C-mediated autocrine loops with their cognate receptors and chemokine receptors are significant factors affecting tumour progression; and that RNAi-mediated silencing of VEGF-C represents a powerful therapeutic approach for controlling NSCLC growth and metastasis.

Measuring human T cell responses in blood and gut samples using qualified methods suitable for evaluation of HIV vaccine candidates in clinical trials

The next generation of candidate HIV vaccines include replicating vectors selected for tropism to mucosal sites, where an efficacious T cell response will be required to limit T cell replication and HIV associated CD4 T cell loss. To fully assess immunogenicity of such candidates, there is a need to develop robust quality controlled analysis of gut derived HIV specific CD8+ T-cell responses. Despite obvious challenges in obtaining sufficient amounts of tissue, the highly compartmentalised nature of the mucosal immune responses, requires the assessment of CD8 T cells isolated directly from local tissue before any conclusions regarding the induction of mucosal responses are made. Here we describe the optimisation and subsequent qualification of a qualitative and quantitative polychromatic flow cytometry assay to assess antigen specific CD8+ T cells isolated from the gut, using samples from HIV positive and negative volunteers. Internal quality controls monitored over time, combined with the use of quality gating and standard operating procedures were used to demonstrate the generation of robust and reliable data.

Inhibition of F-Actin Cycling in Rbl Mast Cells Prevents Endosome Acidification but not Internalization of Antigen-Crosslinked IgE Receptor Complexes

Previous studies have implicated roles for the actin cytoseleton in endocytosis via clathrin-coated pits, as well as in antigen-stimulated endocytosis of immunoglobulin-E (IgE) receptors (Ra et al., Eur. J. Immunol. 19:1771, 1989). To monitor endocytosis in real time, we use a fluorescence assay that reports pH-dependent quenching of FITC-IgE. We find that micromolar concentrations of either cytochalasin D or latrunculin A, inhibitors of actin polymerization, as well as jasplakinolide, a stabilizer of F-actin polymerization, all prevent this antigen-stimulated FITC quenching due to endosomal acidification. Under these conditions, crosslinked IgE receptors appear localized in endosomes by confocal imaging, despite the lack of acid-dependent FITC quenching. A quantitative flow cytometry assay for surface-available IgE shows that greater than 60% of these IgE receptor complexes are internalized in response to antigen crosslinking in the presence or absence of these inhibitors of actin polymerization cycling. After 20 min at 37oC with antigen, we observe partial co-localization of IgE receptor-containing endosomes with a lysosomal marker, indicating normal endosomal trafficking of these complexes in the presence or absence of these agents. Addition of latrunculin A or cytochalasin D after antigen stimulated endocytosis causes rapid, partial reversal of endosomal acidification monitored by FITC-IgE, suggesting dynamic regulation of this process by the actin cytoskeleton. In contrast, secretory lysosomes labeled with FITC-dextran by fluid-phase pinocytosis maintain normal acidification in cells treated with these agents. In ongoing experiments we are investigating the molecular mechanism for cytoskeletal regulation of endosomal acidification.

Underestimation of the expression of cellular prion protein on human red blood cells

Recent transmissions of variant Creutzfeldt-Jakob disease by blood transfusion emphasize the need for the development of prion screening tests. The detection of prions in blood is complicated by the presence of poorly characterized cellular prion protein (PrP(C) ) in both plasma and blood cells. According to published studies, most of PrP(C) in blood cells resides in platelets (PLTs) and white blood cells. To clarify conflicting reports about the quantity of PrP(C) associated with human red blood cells (RBCs), quantitative flow cytometry, Western blot (WB), and enzyme-linked immunosorbent assay (ELISA) were used to measure protein levels in healthy donors. RBCs expressed 290 ± 140 molecules of PrP(C) per cell, assuming equimolar binding of monoclonal antibody (MoAb) 6H4 to PrP(C). Binding of alternate PrP(C) MoAbs, FH11 and 3F4, was substantially lower. WB estimated the level of PrP(C) per cell on RBCs to be just four times lower than in PLTs. A similar level of PrP(C) was detected using ELISA. The weak binding of commonly used MoAb 3F4 was not caused by PrP(C) conformation, truncation, or glycosylation, suggesting a covalent modification, likely glycation, of the 3F4 epitope. Taken together, human RBCs express low but significant amounts of PrP(C) /cell, which makes them, due to high RBC numbers, major contributors to the pool of cell-associated PrP(C) in blood. Previous reports utilizing MoAb 3F4 may have underestimated the amount of PrP(C) in RBCs. Likewise, screening tests for the presence of the abnormal prion protein in blood may be difficult if the abnormal protein is modified similar to RBC PrP(C).

Transforming growth factor-β1 upregulates the expression of CXC chemokine receptor 4 (CXCR4) in human breast cancer MCF-7 cells

To investigate whether rhTGF-beta1 or a recombinant vector encoding a fusion protein comprising an extracellular domain of TGF-beta receptor II and an IgG Fc fragment) affects the regulation of CXC chemokine receptor 4 (CXCR4) expression in MCF-7 human breast cancer cells. MCF-7 breast cancer cells were treated with rhTGF-beta1 or transfected with a recombinant vector, pIRES2-EGFP-TbetaRII-Fc. Expression of CXCR4 in these cells was then analyzed at the mRNA and protein levels by quantitative RT-PCR and flow cytometry assay, respectively. A transwell assay was used to measure the chemotactic response of these cells to SDF-1alpha. CXCR4 mRNA and protein expression were upregulated in TGF-beta1-treated MCF-7 cells. These cells also demonstrated an enhanced chemotactic response to SDF-1alpha. In MCF-7 cells transiently transfected with pIRES2-EGFP-TbetaRII-Fc, a fusion protein named TbetaRII-Fc (approximately 41 kDa) was produced and secreted. In these transfected cells, there was a reduction in CXCR4 expression and in the SDF-1alpha-mediated chemotactic response. TGF-beta1 upregulated CXCR4 expression in MCF-7 cells, which subsequently enhanced the SDF-1alpha-induced chemotactic response. The results suggest a link between TGF-beta1 and CXCR4 expression in MCF-7 human breast cancer cells, which may be one of the mechanisms of TGF-beta1-mediated enhancement of metastatic potential in breast cancer cells.

CX3CR1 Is Expressed by Human B Lymphocytes and Meditates CX3CL1 Driven Chemotaxis of Tonsil Centrocytes

BackgroundFractalkine/CX3CL1, a surface chemokine, binds to CX3CR1 expressed by different lymphocyte subsets. Since CX3CL1 has been detected in the germinal centres of secondary lymphoid tissue, in this study we have investigated CX3CR1 expression and function in human naïve, germinal centre and memory B cells isolated from tonsil or peripheral blood.Methodology/Principal FindingsWe demonstrate unambiguously that highly purified human B cells from tonsil and peripheral blood expressed CX3CR1 at mRNA and protein levels as assessed by quantitative PCR, flow cytometry and competition binding assays. In particular, naïve, germinal centre and memory B cells expressed CX3CR1 but only germinal centre B cells were attracted by soluble CX3CL1 in a transwell assay. CX3CL1 signalling in germinal centre B cells involved PI3K, Erk1/2, p38, and Src phosphorylation, as assessed by Western blot experiments. CX3CR1+ germinal centre B cells were devoid of centroblasts and enriched for centrocytes that migrated to soluble CX3CL1. ELISA assay showed that soluble CX3CL1 was secreted constitutively by follicular dendritic cells and T follicular helper cells, two cell populations homing in the germinal centre light zone as centrocytes. At variance with that observed in humans, soluble CX3CL1 did not attract spleen B cells from wild type mice. OVA immunized CX3CR1−/− or CX3CL1−/− mice showed significantly decreased specific IgG production compared to wild type mice.Conclusion/SignificanceWe propose a model whereby human follicular dendritic cells and T follicular helper cells release in the light zone of germinal centre soluble CX3CL1 that attracts centrocytes. The functional implications of these results warrant further investigation.

Human antibody titers to Epstein–Barr Virus (EBV) gp350 correlate with neutralization of infectivity better than antibody titers to EBV gp42 using a rapid flow cytometry-based EBV neutralization assay

Measurement of neutralizing antibodies to Epstein-Barr virus (EBV) is important for evaluation of candidate vaccines. The current neutralization assay is based on antibody inhibition of EBV transformation of B cells and requires 6 weeks to perform. We developed a rapid, quantitative flow cytometry assay and show that neutralizing antibody titers measured by the new assay strongly correlate with antibody titers in the standard transformation-based assay. Antibodies to EBV gp350 and gp42 have been shown to block infection of B cells by EBV. Using new assays to quantify antibodies to these glycoproteins, we show for the first time that human plasma contains high titers of antibody to gp42; these titers correlate with neutralization of EBV infectivity or transformation. Furthermore, we show that antibody titers to EBV gp350 correlate more strongly with neutralization than antibody titers to gp42. These assays should be useful in accessing antibody responses to candidate EBV vaccines.

Formyl Peptide Receptor-Like 2 Is Expressed and Functional in Plasmacytoid Dendritic Cells, Tissue-Specific Macrophage Subpopulations, and Eosinophils

The formyl peptide receptor (FPR) is a key player in innate immunity and host defense mechanisms. In humans and other primates, a cluster of genes encodes two related receptors, FPR-like 1 and FPR-like 2 (FPRL1 and FPRL2). Despite their high sequence similarity, the three receptors respond to different sets of ligands and display a different expression pattern in leukocyte populations. Unlike FPR and FPRL1, FPRL2 is absent from neutrophils, and two endogenous peptide agonists, F2L and humanin, were recently described. In the present work, we investigated the detailed functional distribution of FPRL2 in leukocytes by quantitative PCR, flow cytometry, immunohistochemistry, and chemotaxis assays, with the aim of raising hypotheses regarding its potential functions in the human body. We describe that FPRL2 is highly expressed and functional in plasmacytoid dendritic cells and up-regulated upon their maturation. FPRL2 is also expressed in eosinophils, which are recruited but do not degranulate in response to F2L. FPRL2 is expressed and functional in macrophages differentiated from monocytes in vitro in different conditions. However, in vivo, only specific subsets of macrophages express the receptor, particularly in the lung, colon, and skin, three organs chronically exposed to pathogens and exogenous aggressions. This distribution and the demonstration of the production of the F2L peptide in mice underline the potential role of FPRL2 in innate immunity and possibly in immune regulation and allergic diseases.

A Systematic Screen for Transcriptional Regulators of the Yeast Cell Cycle

Transcription factors play a key role in the regulation of cell cycle progression, yet many of the specific regulatory interactions that control cell cycle transcription are still unknown. To systematically identify new yeast cell cycle transcription factors, we used a quantitative flow cytometry assay to screen 268 transcription factor deletion strains for defects in cell cycle progression. Our results reveal that 20% of nonessential transcription factors have an impact on cell cycle progression, including several recently identified cyclin-dependent kinase (Cdk) targets, which have not previously been linked to cell cycle transcription. This expanded catalog of cell-cycle-associated transcription factors will be a valuable resource for decoding the transcriptional regulatory interactions that govern progression through the cell cycle. We conducted follow-up studies on Sfg1, a transcription factor with no previously known role in cell cycle progression. Deletion of Sfg1 retards cells in G(1), and overexpression of Sfg1 delays cells in the G(2)/M phase. We find that Sfg1 represses early G(1), Swi5/Ace2-regulated genes involved in mother-daughter cell separation. We also show that Sfg1, a known in vitro cyclin-dependent kinase target, is phosphorylated in vivo on conserved Cdk phosphorylation sites and that phosphorylation of Sfg1 is necessary for its role in promoting cell cycle progression. Overall, our work increases the number of transcription factors associated with cell cycle progression, strongly indicates that there are many more unexplored connections between the Cdk-cyclin oscillator and cell cycle transcription, and suggests a new mechanism for the regulation of cell separation during the M/G(1) phase transition.

Reactivation of death receptor 4 (DR4) expression sensitizes medulloblastoma cell lines to TRAIL

Apoptosis, a key cellular response to therapeutic agents is often inactivated in tumor cells. In this study, we evaluated the expression of the tumor necrosis family of death receptors, DR4 and DR5, in medulloblastoma tumor samples and cell lines to determine if epigenetic modulation of gene expression could sensitize tumor cell lines to TRAIL-mediated apoptosis. Human medulloblastoma samples and cell lines were analyzed for DR4 and DR5 expression by quantitative PCR and immunofluorescence assays. Cell lines with downregulated expression of one or both genes were treated with the histone deacetylase inhibitor, MS-275, and the expression of DR4 and DR5 measured by quantitative PCR, Western blotting, flow cytometry and chromatin immunoprecipitation assays. Induction of apoptosis in the presence of MS-275 was evaluated by TUNEL assay and its ability to augment TRAIL-mediated cytotoxicity was determined by MTT assays, Western blotting and flow cytometry. Compared to normal cerebellum, DR4, but not DR5 expression was consistently downregulated in medulloblastoma tumor samples and in Daoy and D283 cell lines. Interestingly, MS-275 decreased cell growth and induced apoptosis in Daoy and D283 cells. In Daoy cells, this coincided with increased histone H3 and H4 acetylation at the DR4 promoter and enhanced DR4 gene and protein expression as well as elevated Caspase-8 activity. The involvement of DR4 in the cellular response to MS-275 was further confirmed by the observation that knockdown of DR4 and FADD abrogated apoptosis. Further, addition of TRAIL to MS-275 treated cells resulted in an enhancement of apoptosis, suggesting that the upregulated death receptors were functional. Our study provides an understanding of the role of DR4 in apoptosis of medulloblastoma cell lines and suggests a potential contribution of aberrant histone deacetylation to the resistance of medulloblastoma cells to therapeutic death.

IGF Signaling Is Critical for Growth and Survival of T-Cell Acute Lymphoblastic Leukemia Cells and Is Potentiated by Notch Upregulation of IGF1R

T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy of immature T cell progenitors in which we described activating mutations of Notch1 to occur in over 50% of cases. As well, others have identified loss-of-function mutations in Sel10/Fbw7 to occur in 8–16% of cases, which also enhance Notch signaling. Notably, inhibition of Notch signaling in these cells induces growth arrest and in some cases apoptosis as well. Subsequent studies have characterized c-myc as a critical downstream target of Notch signaling in this context. More recently, mutations in PTEN (occurring in 17% of cases) were shown to potentiate PI3K/Akt signaling and proposed to confer resistance to Notch signaling inhibition, but then impose “addiction” to PI3K/Akt. Most leukemias likely derive cooperative growth/survival signals from Notch and PI3K/Akt pathways, as evidenced by synergistic effects of gamma-secretase inhibitors (GSI) which block Notch signaling and rapamycin which blocks mTOR downstream of PI3K/Akt. In mouse models, combined activation of c-myc and b-catenin with inactivation of PTEN elicited T-ALL which was devoid of Notch mutations, suggesting Notch signaling coordinately activates c-myc, Wnt, and PI3K/Akt signaling pathways. In studying the mechanism for PI3K/Akt activation in T-ALL with activated Notch signaling, we examined insulin-like growth factor receptor-1 (IGF1R) as a candidate upstream initiator of PI3K/Akt activation. We observed IGF1R expression consistently in T-ALL cells from primary human leukemias and cell lines, as well as in primary mouse leukemias derived experimentally by retroviral transduction of marrow with activated forms of Notch1. In all cases, inhibition of IGF1R signaling either with blocking antibody or small molecule kinase inhibitors resulted in growth suppression and apoptosis of leukemia cells. We further observed that inhibition of Notch signaling either by small molecule GSI or transduction with a dominant negative coactivator, Mastermind, resulted in a 2–3 fold decrease in IGF1R expression. Given that the PI3K/Akt/mTOR pathway is known to be important for growth/survival of T-ALL leukemia cells, we hypothesized that Notch might be potentiating activation of this pathway by upregulating IGF1R expression. In fact, we found leukemia cells with active Notch and higher IGF1R levels showed 20-fold greater sensitivity to IGF-1 ligand induced Akt activation as compared to leukemia cells with inactive Notch and lower IGF1R levels. This enhanced signaling output cannot be explained by Notch suppression of PTEN as this effect was noted in both PTEN wild-type and PTEN null leukemia cells. Additionally, cells with wild type PTEN demonstrated only minimal if any changes in PTEN protein levels after Notch inhibition as measured by a highly quantitative flow cytometry assay. In sum, we have identified IGF-1 signaling as being critical to growth and survival of T-ALL leukemia cells, and provide evidence that Notch may potentiate PI3K/Akt signaling by upregulating expression of IGF1R. These data are of immediate clinical relevance as several IGF1R inhibitors are currently in Phase 3 clinical trial and hopefully will provide additional therapeutic options to refractory/relapsed patients and/or in combination with front-line therapy in newly diagnosed patients.

Effects of Azithromycin on the Expression of ATP Binding Cassette Transporters in Epithelial Cells from the Airways of Cystic Fibrosis Patients

Induction of ATP Binding Cassette (ABC) proteins involved in chloride transport has been proposed as a possible mechanism of the beneficial effects of azithromycin (AZM) in cystic fibrosis (CF) patients. This study focused on the effects of AZM on mRNA and protein expression of Multidrug Resistance-associated Protein 1 (MRP1) and Multidrug Resistance Protein 1 (MDR1) by real-time quantitative PCR, flow cytometry and gene reporter assays in two CF and two isogenic non-CF airway epithelial cell lines. We detected higher levels of MRP1 and lower levels of MDR1 mRNA in CF versus non-CF cells while both proteins were not differentially expressed. After AZM treatment we found modest differences in MRP1 and MDR1 mRNA expression while protein levels were unaffected. The ability of AZM to regulate MRP1 promoter transcriptional activity was excluded by gene reporter assays. Our data do not support the hypothesis of induction of ABC transporters by AZM.

Suppression of Microglial Inflammatory Activity by Myelin Phagocytosis: Role of p47-PHOX-Mediated Generation of Reactive Oxygen Species

Multiple sclerosis (MS) is pathologically characterized by inflammatory demyelination and neuronal injury. Although phagocytosis of myelin debris by microglia and macrophages in acute MS lesions is well documented, its pathophysiological significance is unclear. Using real-time quantitative PCR, flow cytometry, ELISA, and reactive oxygen species (ROS) measurement assays, we demonstrated that phagocytosis of myelin modulates activation of microglial cells prestimulated by interferon-gamma (IFN-gamma) or a combination of IFN-gamma and lipopolysaccharide with a biphasic temporal pattern, i.e., enhanced production of proinflammatory mediators during the first phase (< or = 6 h), followed by suppression during the second (6-24 h) phase. In this second phase, myelin phagocytosis leads to an enhanced release of prostaglandin E2 and ROS in microglia, whereas the production of anti-inflammatory cytokines (particularly interleukin-10) remains unchanged. Suppression of inflammatory microglial activation by myelin phagocytosis was reversed by treatment with superoxide dismutase and catalase, by inhibition of the NADPH-oxidase complex, or by specific knockdown of the NADPH-oxidase-required adaptor p47-phagocyte oxidase (PHOX). Furthermore, we observed that myelin phagocytosis destabilized tumor necrosis factor-alpha and interferon-induced protein-10 mRNA through an adenine-uridine-rich elements-involved mechanism, which was reversed by blocking the function of NADPH-oxidase complex. We conclude that phagocytosis of myelin suppresses microglial inflammatory activities via enhancement of p47-PHOX-mediated ROS generation. These results suggest that intervention in ROS generation could represent a novel therapeutic strategy to reduce neuroinflammation in MS.