Integration of genomic and proteomic data to identify MYCN-regulated genes, proteins, and interaction networks in neuroblastoma cells.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 9584-9584
Author(s):  
Kolbrun Kristjansdottir ◽  
Kelly M. Bontempo ◽  
Kelly E. Regan ◽  
Saira Khan ◽  
Samuel Louis Volchenboum
Keyword(s):  
Proteomic Analysis ◽  
Global Analysis ◽  
Neuroblastoma Cell ◽  
Interaction Networks ◽  
Mycn Amplification ◽  
Western Blots ◽  
Protein Levels ◽  
Proteomic Data ◽  
Gene And Protein Expression ◽  
Mycn Expression

9584 Background: Neuroblastoma is the most common extracranial solid tumor found in children. The most common marker prognostic of poor outcome is amplification of the MYCN oncogene, yet the biological programs by which MYCN affects its aggressive phenotype are largely unknown. In order to identify biological pathways affected by MYCN amplification, we performed global analysis of genes and proteins in the Tet 21/N neuroblastoma cell line. Methods: MYCN expression in Tet21/N cells is regulated by tetracycline. Gene arrays were performed on MYCN-high and MYCN-low Tet21/N cells and SH-EP parental cells using Affymetrix GeneChip Human Exon 1.0 ST. For proteomic analysis, the cells were labeled with stable isotopes for relative quantitation, enriched for phosphoproteins to enhance detection of signaling proteins, and analyzed by GeLC-MS/MS analysis. Integrative pathway analysis was performed on the genomic and proteomic datasets using DAVE and GeneGo. Results: Integrating genomic and proteomic data in MYCN-high and MYCN-low expressing cells identified 88 proteins and 300 genes that change in abundance. We compare these results with previous studies and find both novel and previously identified genes and proteins. Integrating proteomic and genomic data identified over 50 gene products that are present in both analyses and are altered in abundance in response to modulating MYCN expression. The majority of these have discordant abundance changes, with protein levels more frequently altered with no change in gene expression. We have validated changes in protein levels using Western blots including NPM1 and MATR3. We present interaction networks and pathways that correlate with MYCN expression. Conclusions: We showed a significant discordance between gene and protein expression, underscoring the need for integrative genomic and proteomic analysis to describe complex systems. Our analysis identified previously reported and novel genes and proteins and networks regulated by MYCN expression that may provide new insights into the biology of MYCN-amplified tumors.

scholarly journals FOXR2 Stabilizes MYCN Protein and Identifies Non–MYCN-Amplified Neuroblastoma Patients With Unfavorable Outcome

2021 ◽  
pp. JCO.20.02540
Author(s):  
Felix Schmitt-Hoffner ◽  
Sjoerd van Rijn ◽  
Umut H. Toprak ◽  
Monika Mauermann ◽  
Felix Rosemann ◽  
...  
Keyword(s):  
Cell Lines ◽  
Tumor Regression ◽  
Neuroblastoma Cell ◽  
Unfavorable Outcome ◽  
Common Mechanism ◽  
Mycn Amplification ◽  
Data Sets ◽  
Alternative Mechanism ◽  
Protein Levels ◽  
Neuroblastoma Cell Lines

PURPOSE Clinical outcomes of patients with neuroblastoma range from spontaneous tumor regression to fatality. Hence, understanding the mechanisms that cause tumor progression is crucial for the treatment of patients. In this study, we show that FOXR2 activation identifies a subset of neuroblastoma tumors with unfavorable outcome and we investigate the mechanism how FOXR2 relates to poor outcome in patients. MATERIALS AND METHODS We analyzed three independent transcriptional data sets of in total 1030 primary neuroblastomas with full clinical annotation. We performed immunoprecipitation for FOXR2 and MYCN and silenced FOXR2 expression in two neuroblastoma cell lines to examine the effect on cellular processes, transcriptome, and MYCN protein levels. Tumor samples were analyzed for protein levels of FOXR2 and MYCN. RESULTS In three combined neuroblastoma data sets, 9% of tumors show expression of FOXR2 but have low levels of MYCN mRNA. FOXR2 expression identifies a group of patients with unfavorable outcome, showing 10-year overall survival rates of 53%-59%, and proves to be an independent prognostic factor compared with established risk factors. Transcriptionally, FOXR2-expressing tumors are very similar to MYCN-amplified tumors, suggesting that they might share a common mechanism of tumor initiation. FOXR2 knockdown in FOXR2-expressing neuroblastoma cell lines resulted in cell cycle arrest, reduced cell growth, cell death, and reduced MYCN protein levels, all indicating that FOXR2 is essential for these tumors. Finally, we show that FOXR2 binds and stabilizes MYCN protein and MYCN protein levels are highly increased in FOXR2-expressing tumors, in several cases comparable with MYCN-amplified samples. CONCLUSION The stabilization of MYCN by FOXR2 represents an alternative mechanism to MYCN amplification to increase MYCN protein levels. As such, FOXR2 expression identifies another subset of neuroblastoma patients with unfavorable clinical outcome.

scholarly journals The Killing of Human Neuroblastoma Cells by the Small Molecule JQ1 Occurs in a p53-Dependent Manner

2020 ◽  
Vol 20 (13) ◽  
pp. 1613-1625
Author(s):  
Joseph Mazar ◽  
Caleb Gordon ◽  
Varun Naga ◽  
Tamarah J. Westmoreland
Keyword(s):  
Mechanism Of Action ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cells ◽  
Mycn Amplification ◽  
Dependent Manner ◽  
Human Neuroblastoma ◽  
Bet Inhibitor ◽  
Mycn Expression ◽  
Induced Apoptosis ◽  
Neuroblastoma Cell Lines

Background: MYCN amplification is a prognostic biomarker associated with poor prognosis of neuroblastoma in children. The overall survival of children with MYCN-amplified neuroblastoma has only marginally improved within the last 20 years. The Bromodomain and Extra-Terminal motif (BET) inhibitor, JQ1, has been shown to downregulate MYCN in neuroblastoma cells. Objective: To determine if JQ1 downregulation of MYCN in neuroblastomas can offer a target- specific therapy for this, difficult to treat, pediatric cancer. Methods: Since MYCN-amplified neuroblastoma accounts for as much as 40 to 50 percent of all high-risk cases, we compared the effect of JQ1 on both MYCN-amplified and non-MYCN-amplified neuroblastoma cell lines and investigated its mechanism of action. Results: In this study, we show that JQ1 can specifically target MYCN for downregulation, though this effect is not specific to only MYCN-amplified cells. And although we can confirm that the loss of MYCN alone can induce apoptosis, the exogenous rescue of MYCN expression can abrogate much of this cytotoxicity. More fascinating, however, was the discovery that the JQ1-induced knockdown of MYCN, which led to the loss of the human double minute 2 homolog (HDM2) protein, also led to the accumulation of tumor protein 53 (also known as TP53 or p53), which ultimately induced apoptosis. Likewise, the knockdown of p53 also blunted the cytotoxic effects of JQ1. Conclusion: These data suggest a mechanism of action for JQ1 cytotoxicity in neuroblastomas and offer a possible prognostic target for determining its efficacy as a therapeutic.

scholarly journals Placental expression of ACE2 and TMPRSS2 in maternal SARS-CoV-2 infection: are placental defenses mediated by fetal sex?

2021 ◽  
Author(s):  
Lydia L Shook ◽  
Evan A Bordt ◽  
Marie-Charlotte Meinsohn ◽  
David Pepin ◽  
Rose M De Guzman ◽  
...  
Keyword(s):  
Sex Differences ◽  
Protein Expression ◽  
Viral Entry ◽  
P Value ◽  
Western Blots ◽  
Fetal Sex ◽  
Placental Infection ◽  
Protein Levels ◽  
Gene And Protein Expression ◽  
The Impact

Background: Sex differences in vulnerability to and severity of SARS-CoV-2 infection have been described in non-pregnant populations. ACE2 and TMPRSS2, host molecules required for viral entry, are regulated by sex steroids and expressed in the placenta. We sought to investigate whether placental ACE2 and TMPRSS2 expression vary by fetal sex and in the presence of maternal SARS-CoV-2 infection. Methods: Placental ACE2 and TMPRSS2 were quantified in 68 pregnant individuals (38 SARS-CoV-2 positive, 30 SARS-CoV-2 negative) delivering at Mass General Brigham from April to June 2020. Maternal SARS-CoV-2 status was determined by nasopharyngeal RT-PCR. Placental SARS-CoV-2 viral load was quantified. RTqPCR was performed to quantify expression of ACE2 and TMPRSS2 relative to the reference gene YWHAZ. Western blots were performed on placental homogenates to quantify protein levels. The impact of fetal sex and SARS-CoV-2 exposure on ACE2 and TMPRSS2 expression was analyzed by 2-way ANOVA. Results: SARS-CoV-2 virus was undetectable in all placentas. Maternal SARS-CoV-2 infection impacted TMPRSS2 placental gene and protein expression in a sexually dimorphic fashion (2-way ANOVA interaction p-value: 0.002). We observed no impact of fetal sex or maternal SARS-CoV-2 status on placental ACE2 gene or protein expression. Placental TMPRSS2 expression was significantly correlated with ACE2 expression in males (Spearman's rho=0.54, p=0.02) but not females (rho=0.23, p=0.34) exposed to maternal SARS-CoV-2. Conclusions: Sex differences in placental TMPRSS2 but not ACE2 were observed in the setting of maternal SARS-CoV-2 infection. These findings may have implications for offspring vulnerability to placental infection and vertical transmission.These findings may have implications for offspring vulnerability to placental infection and vertical transmission.

scholarly journals Direct current stimulation enhances neuronal alpha-synuclein degradation in vitro

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Gessica Sala ◽  
Tommaso Bocci ◽  
Valentina Borzì ◽  
Marta Parazzini ◽  
Alberto Priori ◽  
...  
Keyword(s):  
Direct Current ◽  
Molecular Mechanisms ◽  
Neuroblastoma Cell ◽  
Alpha Synuclein ◽  
Human Neuroblastoma Cell ◽  
Human Neuroblastoma ◽  
Direct Current Stimulation ◽  
Gene And Protein Expression ◽  
Autophagic Degradation ◽  
On Line

AbstractDespite transcranial Direct Current Stimulation (DCS) is currently proposed as a symptomatic treatment in Parkinson’s disease, the intracellular and molecular mechanisms elicited by this technique are still unknown, and its disease-modifying potential unexplored. Aim of this study was to elucidate the on-line and off-line effects of DCS on the expression, aggregation and degradation of alpha-synuclein (asyn) in a human neuroblastoma cell line under basal conditions and in presence of pharmachologically-induced increased asyn levels. Following DCS, gene and protein expression of asyn and its main autophagic catabolic pathways were assessed by real-time PCR and Western blot, extracellular asyn levels by Dot blot. We found that, under standard conditions, DCS increased monomeric and reduced oligomeric asyn forms, with a concomitant down-regulation of both macroautophagy and chaperone-mediated autophagy. Differently, in presence of rotenone-induced increased asyn, DCS efficiently counteracted asyn accumulation, not acting on its gene transcription, but potentiating its degradation. DCS also reduced intracellular and extracellular asyn levels, increased following lysosomal inhibition, independently from autophagic degradation, suggesting that other mechanisms are also involved. Collectively, these findings suggest that DCS exerts on-line and off-line effects on the expression, aggregation and autophagic degradation of asyn, indicating a till unknown neuroprotective role of tDCS.

scholarly journals Effect of Alpha-1 Antitrypsin on CFTR Levels in Primary Human Airway Epithelial Cells Grown at the Air-Liquid-Interface

Molecules ◽  
2021 ◽  
Vol 26 (9) ◽  
pp. 2639
Author(s):  
Frauke Stanke ◽  
Sabina Janciauskiene ◽  
Stephanie Tamm ◽  
Sabine Wrenger ◽  
Ellen Luise Raddatz ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Human Lung ◽  
Liquid Interface ◽  
Lung Epithelial Cells ◽  
Western Blots ◽  
Mesenchymal Transition ◽  
Protein Levels ◽  
Lung Epithelial ◽  
Cftr Protein ◽  
Air Liquid Interface

The cystic fibrosis transmembrane conductance regulator (CFTR) gene is influenced by the fundamental cellular processes like epithelial differentiation/polarization, regeneration and epithelial–mesenchymal transition. Defects in CFTR protein levels and/or function lead to decreased airway surface liquid layer facilitating microbial colonization and inflammation. The SERPINA1 gene, encoding alpha1-antitrypsin (AAT) protein, is one of the genes implicated in CF, however it remains unknown whether AAT has any influence on CFTR levels. In this study we assessed CFTR protein levels in primary human lung epithelial cells grown at the air-liquid-interface (ALI) alone or pre-incubated with AAT by Western blots and immunohistochemistry. Histological analysis of ALI inserts revealed CFTR- and AAT-positive cells but no AAT-CFTR co-localization. When 0.5 mg/mL of AAT was added to apical or basolateral compartments of pro-inflammatory activated ALI cultures, CFTR levels increased relative to activated ALIs. This finding suggests that AAT is CFTR-modulating protein, albeit its effects may depend on the concentration and the route of administration. Human lung epithelial ALI cultures provide a useful tool for studies in detail how AAT or other pharmaceuticals affect the levels and activity of CFTR.

scholarly journals Evaluation of a Genome-ScaleIn SilicoMetabolic Model for Geobacter metallireducens by Using Proteomic Data from a Field Biostimulation Experiment

2012 ◽  
Vol 78 (24) ◽  
pp. 8735-8742 ◽  
Author(s):  
Yilin Fang ◽  
Michael J. Wilkins ◽  
Steven B. Yabusaki ◽  
Mary S. Lipton ◽  
Philip E. Long
Keyword(s):  
Proteomic Analysis ◽  
In Silico ◽  
Field Experiments ◽  
Metabolic Model ◽  
Geobacter Metallireducens ◽  
Content Type ◽  
Proteomic Data ◽  
Subsurface Environment ◽  
A Genome ◽  
Genome Scale

ABSTRACTAccurately predicting the interactions between microbial metabolism and the physical subsurface environment is necessary to enhance subsurface energy development, soil and groundwater cleanup, and carbon management. This study was an initial attempt to confirm the metabolic functional roles within anin silicomodel using environmental proteomic data collected during field experiments. Shotgun global proteomics data collected during a subsurface biostimulation experiment were used to validate a genome-scale metabolic model ofGeobacter metallireducens—specifically, the ability of the metabolic model to predict metal reduction, biomass yield, and growth rate under dynamic field conditions. The constraint-basedin silicomodelof G. metallireducensrelates an annotated genome sequence to the physiological functions with 697 reactions controlled by 747 enzyme-coding genes. Proteomic analysis showed that 180 of the 637G. metallireducensproteins detected during the 2008 experiment were associated with specific metabolic reactions in thein silicomodel. When the field-calibrated Fe(III) terminal electron acceptor process reaction in a reactive transport model for the field experiments was replaced with the genome-scale model, the model predicted that the largest metabolic fluxes through thein silicomodel reactions generally correspond to the highest abundances of proteins that catalyze those reactions. Central metabolism predicted by the model agrees well with protein abundance profiles inferred from proteomic analysis. Model discrepancies with the proteomic data, such as the relatively low abundances of proteins associated with amino acid transport and metabolism, revealed pathways or flux constraints in thein silicomodel that could be updated to more accurately predict metabolic processes that occur in the subsurface environment.

scholarly journals Altered V-ATPase expression in renal intercalated cells isolated from B1 subunit-deficient mice by fluorescence-activated cell sorting

2013 ◽  
Vol 304 (5) ◽  
pp. F522-F532 ◽  
Author(s):  
Luca Vedovelli ◽  
John T. Rothermel ◽  
Karin E. Finberg ◽  
Carsten A. Wagner ◽  
Anie Azroyan ◽  
...  
Keyword(s):  
Cell Sorting ◽  
Collecting Duct ◽  
Egfp Expression ◽  
Intercalated Cells ◽  
Wild Type ◽  
Western Blots ◽  
Atpase Subunit ◽  
Protein Levels ◽  
Baseline Conditions ◽  
Deficient Mice

Unlike human patients with mutations in the 56-kDa B1 subunit isoform of the vacuolar proton-pumping ATPase (V-ATPase), B1-deficient mice (Atp6v1b1−/−) do not develop metabolic acidosis under baseline conditions. This is due to the insertion of V-ATPases containing the alternative B2 subunit isoform into the apical membrane of renal medullary collecting duct intercalated cells (ICs). We previously reported that quantitative Western blots (WBs) from whole kidneys showed similar B2 protein levels in Atp6v1b1−/− and wild-type mice (Păunescu TG, Russo LM, Da Silva N, Kovacikova J, Mohebbi N, Van Hoek AN, McKee M, Wagner CA, Breton S, Brown D. Am J Physiol Renal Physiol 293: F1915–F1926, 2007). However, WBs from renal medulla (including outer and inner medulla) membrane and cytosol fractions reveal a decrease in the levels of the ubiquitous V-ATPase E1 subunit. To compare V-ATPase expression specifically in ICs from wild-type and Atp6v1b1−/− mice, we crossed mice in which EGFP expression is driven by the B1 subunit promoter (EGFP-B1+/+ mice) with Atp6v1b1−/− mice to generate novel EGFP-B1−/− mice. We isolated pure IC populations by fluorescence-assisted cell sorting from EGFP-B1+/+ and EGFP-B1−/− mice to compare their V-ATPase subunit protein levels. We report that V-ATPase A, E1, and H subunits are all significantly downregulated in EGFP-B1−/− mice, while the B2 protein level is considerably increased in these animals. We conclude that under baseline conditions B2 upregulation compensates for the lack of B1 and is sufficient to maintain basal acid-base homeostasis, even when other V-ATPase subunits are downregulated.

scholarly journals Comparison of age‐dependent alterations in thioredoxin 2 and thioredoxin reductase 2 expressions in hippocampi between mice and rats

2021 ◽  
Vol 37 (1) ◽  
Author(s):  
Yeon Ho Yoo ◽  
Dae Won Kim ◽  
Bai Hui Chen ◽  
Hyejin Sim ◽  
Bora Kim ◽  
...  
Keyword(s):  
Thioredoxin Reductase ◽  
Pyramidal Cells ◽  
Brain Regions ◽  
Young Age ◽  
Cresyl Violet ◽  
Western Blots ◽  
Protein Levels ◽  
Cresyl Violet Staining ◽  
Age Dependent ◽  
The Brain

Abstract Background Aging is one of major causes triggering neurophysiological changes in many brain substructures, including the hippocampus, which has a major role in learning and memory. Thioredoxin (Trx) is a class of small redox proteins. Among the Trx family, Trx2 plays an important role in the regulation of mitochondrial membrane potential and is controlled by TrxR2. Hitherto, age-dependent alterations in Trx2 and TrxR2 in aged hippocampi have been poorly investigated. Therefore, the aim of this study was to examine changes in Trx2 and TrxR2 in mouse and rat hippocampi by age and to compare their differences between mice and rats. Results Trx2 and TrxR2 levels using Western blots in mice were the highest at young age and gradually reduced with time, showing that no significant differences in the levels were found between the two subfields. In rats, however, their expression levels were the lowest at young age and gradually increased with time. Nevertheless, there were no differences in cellular distribution and morphology in their hippocampi when it was observed by cresyl violet staining. In addition, both Trx2 and TrxR2 immunoreactivities in the CA1-3 fields were mainly shown in pyramidal cells (principal cells), showing that their immunoreactivities were altered like changes in their protein levels. Conclusions Our current findings suggest that Trx2 and TrxR2 expressions in the brain may be different according to brain regions, age and species. Therefore, further studies are needed to examine the reasons of the differences of Trx2 and TrxR2 expressions in the hippocampus between mice and rats.

Tissue transglutaminase and apoptosis: sense and antisense transfection studies with human neuroblastoma cells

1994 ◽  
Vol 14 (10) ◽  
pp. 6584-6596
Author(s):  
G Melino ◽  
M Annicchiarico-Petruzzelli ◽  
L Piredda ◽  
E Candi ◽  
V Gentile ◽  
...  
Keyword(s):  
Cell Death ◽  
Structural Changes ◽  
Tissue Transglutaminase ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cells ◽  
Neuroblastoma Cell Line ◽  
Human Neuroblastoma Cell ◽  
Human Neuroblastoma ◽  
Transfected Cells ◽  
Protein Levels

In this report, we show that the overexpression of tissue transglutaminase (tTG) in the human neuroblastoma cell line SK-N-BE(2) renders these neural crest-derived cells highly susceptible to death by apoptosis. Cells transfected with a full-length tTG cDNA, under the control of a constitutive promoter, show a drastic reduction in proliferative capacity paralleled by a large increase in cell death rate. The dying tTG-transfected cells exhibit both cytoplasmic and nuclear changes characteristic of cells undergoing apoptosis. The tTG-transfected cells express high Bcl-2 protein levels as well as phenotypic neural cell adhesion molecule markers (NCAM and neurofilaments) of cells differentiating along the neuronal pathway. In keeping with these findings, transfection of neuroblastoma cells with an expression vector containing segments of the human tTG cDNA in antisense orientation resulted in a pronounced decrease of both spontaneous and retinoic acid (RA)-induced apoptosis. We also present evidence that (i) the apoptotic program of these neuroectodermal cells is strictly regulated by RA and (ii) cell death by apoptosis in the human neuroblastoma SK-N-BE(2) cells preferentially occurs in the substrate-adherent phenotype. For the first time, we report here a direct effect of tTG in the phenotypic maturation toward apoptosis. These results indicate that the tTG-dependent irreversible cross-linking of intracellular protein represents an important biochemical event in the induction of the structural changes featuring cells dying by apoptosis.

Constitutive activation of STAT-3 and downregulation of SOCS-3 expression induced by adrenalectomy

2001 ◽  
Vol 281 (6) ◽  
pp. R2048-R2058 ◽  
Author(s):  
Abram M. Madiehe ◽  
Ling Lin ◽  
Christy White ◽  
H. Doug Braymer ◽  
George A. Bray ◽  
...  
Keyword(s):  
Dna Binding ◽  
Signaling Pathway ◽  
Leptin Receptor ◽  
Binding Activity ◽  
Janus Kinase ◽  
Adrenal Steroids ◽  
Western Blots ◽  
Protein Levels ◽  
Dna Binding Activity ◽  
Stat Signaling

Removal of adrenal steroids by adrenalectomy (ADX) slows or reverses the development of many forms of obesity in rodents, including those that are leptin or leptin receptor deficient. Obesity is associated with hyperleptinemia and leptin resistance. We hypothesized that glucocorticoids impair leptin receptor signaling and that removal thereof would activate the Janus kinase (JAK)-signal transducers and activators of transcription (STAT) signaling pathway. The inhibitory effect of leptin (2.5 μg icv) on food intake was enhanced in ADX rats. A combination of ribonuclease protection assays, RT-PCR, Western blots, and mobility shift assays was used to evaluate the leptin signaling pathway in whole hypothalami from sham-operated, ADX and corticosterone-replaced ADX (ADX-R) Sprague-Dawley rats that were treated acutely with either saline vehicle or leptin intracerebroventricularly. ADX increased the expression of leptin receptor mRNA, increased STAT-3 mRNA and protein levels, induced constitutive STAT-3 phosphorylation and DNA binding activity, and also reduced suppressor of cytokine signaling-3 (SOCS-3) mRNA and protein levels. ADX and leptin treatment increased STAT-3 phosphorylation, but with no concomitant increase in DNA binding activity. Leptin and ADX decreased NPY mRNA expression, but their combination did not further decrease NPY mRNA. Corticosterone supplementation of ADX rats partially reversed many of these effects. In conclusion, ADX through activation of STAT-3 and inhibition of SOCS-3 activates the JAK-STAT signaling pathway. These effects most probably explain the ability to prevent the development of obesity by removal of adrenal steroids.

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