title: CCRF-CEM Prednisolone treatment at 4h and 72h

status: Public on Mar 26 2011

summary:
It has been shown previously that glucocorticoids exert a dual mechanism of action, entailing cytotoxic, mitogenic as well as cell proliferative and anti-apoptotic responses, in a dose-dependent manner on CCRF-CEM cells at 72 h. Early gene expression response implies a dose-dependent dual mechanism of action of prednisolone too, something reflected on cell state upon 72 h of treatment.
In this work, a generic, computational microarray data analysis framework is proposed, in order to examine the hypothesis whether CCRF-CEM cells exhibit an intrinsic or acquired mechanism of resistance and to investigate the molecular imprint of this, upon prednisolone treatment. The experimental design enables the examination of both the dose (0 nM, 10 nM, 22 uΜ, 700 uΜ) effect of glucocorticoid exposure and the dynamics (early and late, namely 4 h, 72 h) of the molecular response of the cells at the transcriptomic layer.
In this work we demonstrated that CCRF-CEM cells may attain a mixed mechanism of response to glucocorticoids, however, there is clear evidence predicating towards an intrinsic mechanism of resistance. More specifically, at 4 h prednisolone appeared not to perform its expected function by down-regulating apoptotic genes, which is re-enforced by mechanisms, which down-regulate other sets of apoptotic genes. Also, low and high prednisolone concentrations up-regulates metabolic and signal-transduction related genes in both time points, thus grounding for a cell proliferation machinery. In addition, regulation of NF-κB-related genes implies an inherent mechanism of resistance through the established link of NF-κB inflammatory role and GC-induced resistance. The analysis framework applied here allows derivation of regulatory mechanisms activated by prednisolone through identification of early responding sets of genes. On the other hand, study of the prolonged exposure to glucocorticoids (72 h exposure) highlights the effect of homeostatic feedback mechanisms of the treated cells.
Overall, it appears that CCRF-CEM cells in this study exhibit a diversified, combined pattern of intrinsic and acquired resistance to prednisolone, yet with a tendency towards inherent resistant characteristics, through activation of different molecular courses of action.

overall_design:
For the assay of mRNA levels two sets of microarray chips were used (1,200 and 4,800 genes), obtained from TAKARA (Human Cancer Chip v.40 and IntelliGene™ II Human CHIP 1 respectively) (Chung, Park et al. 2004). The Human Cancer Chip v.40 platform is available in the GEO database under the accession number GPL13250 and the IntelliGene™ II Human CHIP 1 is available with the accession number GPL13251. Hybridization was performed with the CyScribe Post-Labeling kit (RPN5660, GE Healthcare, former Amersham Inc.) as described by the manufacturer. The fluorescent dyes used were Cy3 and Cy5. cDNAs were purified with PCR product clean-up kit (# 28104, QIAGEN Inc.). Slides were activated at 55o C for 30 min in 1% BSA. Samples were applied on the slides, and let to hybridize overnight at 55o C. The following day, slides were washed in 200 ml 0.1× SSC and 0,1% SDS for 3× 5 min, in 200 ml 0.1× SSC for 2× 5 min  and in 200 ml ddH2O for 30 sec. Slides were dried by centrifugation at 1500 rpm for 3 min and scanned with a microarray scanner. Slides were scanned with a microarray scanner (ScanArray 4000XL) (Perkin Elmer Inc. MA, former GSI Lumonics, USA). Images were generated with ScanArray microarray acquisition software (GSI Lumonics, USA). Image analysis and raw data acquisition were performed with the ImaGene® software from BioDiscovery (California, USA). Microarray experimental design consisted of a small loop-design at 4h as described previously (Churchill 2002; Altman and Hua 2006). Our design and experiments were described previously (Lambrou, Vlahopoulos et al. 2009). Briefly, there were three sets of experiments at 4h designated as follows: 0vs1: control vs. 10nM prednisolone, 1vs3: 10nM prednisolone vs. 700uM prednisolone and 0vs3: control vs. 700uM prednisolone. This loop-design had the following property. The sum of the ratios of the 0vs1 and 1vs3 experiments should theoretically equal the ratio in the 0vs3 experiment. In the following sections we describe how this was used in order to filter genes. Experiments at 72h were based on a simple reference design. In particular, the experimental setups consisted of the five following pairs: control vs. 10 nM prednisolone at 4 h (designated as ‘1’), 10 nM vs. 700 uM prednisolone at 4 h (designated as ‘2’), control vs. 700 uM prednisolone at 4 h (designated as ‘3’), 22 uM vs. 700 uM prednisolone at 72 h (designated as ‘4’), and control vs. 700 uM prednisolone at 72 h (designated as ‘5’).
The 4h experiments were performed in triplicates and the 72h experiments in single experiments.
Creation of a Common Gene Set: Due to the derivation of different genes in each of the five experimental setups and the different number of array features between the two microarray chips, the clustering included genes present in all setups, forming a common gene set. This resulted in strict filtering of huge numbers of genes, since only few are present simultaneously in all five experiments.  In total 586 genes were common in both platforms and these were used for further analyses.
Identification of Differentially Expressed Genes: In order to identify potentially differentially expressed (DE) genes between samples and among genes of the same experiment we have used a one-sample z-test and two-sample z-test (Yang, Chen et al. 2002). The DE genes per experiment were identified at a confidence level of 95%. The False Discovery Rate (FDR) has been calculated as described previously (Klipper-Aurbach, Wasserman et al. 1995; Storey and Tibshirani 2003; Storey and Tibshirani 2003).


title: Gene expression in minor salivary gland of patients with Sjogren's syndrome (SS) and control

status: Public on May 01 2011

city: Bethesda

first: Niki

last: Moutsopoulos

address: 30 convent Dr

country: USA

institute: NIDCR

summary: To study the gene expression profile of salivary glands with varying degrees of inflammation in Sjogren's and non Sjogren's patients

overall_design: Total mRNA was extracted from whole minor salivary glands of patients with SS and control

first: Teresa

last: Wild

first: Zoila

last: Rangel

first: Peter

middle: J

last: Munson

first: Sharon

last: Wahl


title: Profiling of mesenchymal stem cells from adipose tissue of differentially aged mice

status: Public on Nov 30 2010

city: Verona

first: Mirco

last: Galiè

address: via Le Grazie, 8

department: Neuroscience, Biomedicine and Movement Sciences

country: Italy

institute: University of Verona

type:
Expression profiling by array
Non-coding RNA profiling by array

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: Recipient cell nuclear factors are required for reprogramming by nuclear transfer

status: Public on May 01 2010

city: New York

first: Dieter

middle: M

last: Egli

state: NY

address: 1150 St. Nicholas Avenue, room 607B, New York NY 10032

institute: Columbia University

summary:
Reprogramming occurs after nuclear transfer into zygotes whose genome was removed in mitosis, but not after nuclear transfer into zygotes enucleated in interphase
Egli et al. Development 2010 doi:10.1242/dev.046151

overall_design: Groups of 20 mouse embryos were used for the analysis. RNA amplification was done using Illumina total prep RNA amplification kit.  Total of 21 arrays.

first: Kevin

last: Eggan


title: Gene expression in tonsil and oral epithelia

status: Public on Dec 31 2007

summary:
To study characteristics of the orapharyngeal epithelia which may influence susceptibility or resistance to HIV, we performed microarray analysis of the tonsil and gingival epithelium.Tonsil epithelium has been implicated in HIV pathogenesis, but its role in oral transmission remains controversial. We performed microarray analysis of Laser Capture Microdissected tonsil and gingival epithelium. Our data revealed that genes related to immune functions such as antibody production and antigen processing were increasingly expressed in tonsil compared to the epithelium of another oro-pharyngeal site, gingival epithelium. Importantly, tonsil epithelium highly expressed genes associated with HIV entrapment and/or transmission, including the HIV co-receptor CXCR4 and the potential HIV binding molecules, FcRγIII, complement receptor 2, and various complement components. This increased expression of molecules involved in viral recognition, binding and entry may favor virus-epithelium interaction in an environment with reduced innate anti-viral mechanisms. Specifically, secretory leukocyte protease inhibitor, an innate molecule with anti-HIV activity, was minimal in the tonsil epithelium, in contrast to oral mucosa.  Collectively, our data suggest that increased expression of molecules associated with HIV binding and entry coupled with decreased innate anti-viral factors may render the tonsil a potential site for oral transmission.
Keywords: Cross sectional

overall_design:
To study characteristics of the orapharyngeal epithelia which may influence susceptibility or resistance to HIV, we performed microarray analysis of the tonsil and gingival epithelium.

Human palatine tonsils were obtained from routine therapeutic tonsillectomies (sleep apnea and non-tonsillitis) performed on otherwise healthy adults at the George Washington University Hospital with informed consent (IRB #099920). Gingival tissues were collected from healthy sites with probing depths < 3mm, with no clinical evidence of inflammation during routine therapeutic periodontal surgery at the University of Maryland, Department of Periodontics, with informed consent (IRB#1201211). Tissues were immediately snap frozen for the microarray studies. CM performed immediately at 10x magnification, using a Leica AS LMD system (Leica Microsystems). Areas unequivocally identified as epithelium were outlined , laser-dissected and captured and RNA from the tissues isolated. Preparation of biotin-labeled cRNA, hybridization, and scanning were performed according to manufacturer’s two cycle protocol (Affymetrix, Santa Clara, CA). Fluorescence intensity was measured using the Affymetrix GeneChip scanner and GeneChip Operating Software (GCOS, Affymetrix).

Samples GSM173679, GSM173680,GSM173681, GSM173682, GSM173683, GSM173684, GSM173685, GSM173686, GSM173688, GSM173690  were processed togehter as "batch 1", while samples GSM173687 and GSM173689 were processed as "batch 2" together with sample GSM173691 which is a repeat of GSM173690, which was processed for comparison between the two batches.

first: Salvador

last: Nares

first: Nikolaos

last: Nikitakis

first: Jie

last: Wen

first: John

last: Sauk


title: Transcription-driven Chromatin Repression of Intragenic Promoters

status: Public on Apr 26 2018

city: Frederiksberg C

first: Maxim

last: Ivanov

address: Thorvaldsensvej 40

department: Dept. of Plant and Environmental Sciences

country: Denmark

institute: University of Copenhagen

type: Expression profiling by high throughput sequencing

summary: The goal of this study is to investigate a chromatin-based mechanism that suppresses intragenic initiation of RNAPII transcription in A.thaliana. We demonstrate that RNAPII transcription across gene promoters represses their function in plants. The FACT histone chaperone complex is required for this repression mechanism by co-transcriptionally enforcing elevated levels of H3K4me1 at repressed intragenic promoters;

overall_design: TSS-Seq was used to map the transcription initiation sites with single base resolution in A.thaliana seedlings (Col-0 and FACT-deficient genotypes). We also used RNA-Seq to quantify gene expression changes in the same genotypes;

first: Mathias

last: Nielsen

first: Ryan

last: Ard

first: Xueyuan

last: Leng

last: Kindgren

first: Vicente

last: Pelechano

first: Sebastian

last: Marquardt


title: Gene expression profiles of Ras mutants.

status: Public on Feb 21 2009

city: Houston

first: Jeffrey

last: Chang

address: 6431 Fannin St

institute: University of Texas

summary:
Gene expression profiles were collected from HEK-HT cells expressing H-Ras with Ras-activating (G12V), Raf-activating (G12V,T35S), RalGEF-activating (G12V,E37G), or PI3K-activating (G12V,Y40C) mutations.

Keywords: Comparison of Ras pathway components.

overall_design: THX=control; 12V=G12V mutation; 35S=G12V,T35S mutation; 37G=G12V,E37G mutation; 40C=G12V,Y40C mutation.  5 replicates of each.  Total of 25 samples.

middle: T

first: Joseph

middle: R

last: Nevins


title: The effects of kiwifruit extracts on colonic gene and protein expression levels in interleukin 10 gene-deficient mice.

status: Public on Mar 05 2011

city: Palmerston North

first: Helge

last: Dzierzon

address: Bachelar Road

department: Bioinformatics

country: New Zealand

institute: Plant and Food Research Ltd.

summary:
Inflammatory Bowel Disease (IBD) is a term describing a collection of conditions characterised by chronic inflammatory disorder of the gastrointestinal tract involving an inappropriate immune response to commensal microorganisms in a genetically susceptible host. Four kiwifruit extracts, aqueous and ethyl acetate extracts of gold kiwifruit (Actinidia chinensis) or green kiwifruit (A. deliciosa), have previously demonstrated anti-inflammatory activity using in vitro models of IBD. This study examined whether these kiwifruit extracts had immune modulating effects in vivo against inflammatory processes known to be increased in patients with IBD.
KFEs were used as a dietary intervention in Il10-/- mice (an in vivo model of IBD) and the C57BL/6J background strain in a 3 x 2 factorial design. While all Il10-/- mice developed significant colonic inflammation compared to the C57BL/6J mice, this was not affected by the inclusion of KFE in the diet. Whole genome gene and protein expression level profiling indicated that KFEs influenced immune signalling pathways and metabolic processes within the colonic tissue; however, the effects were subtle. In particular, adaptive immune pathways were reduced by three out of four kiwifruit extracts, with greater reduction seen in the C57BL/6J mice. This suggests that while immune-modulating activity was present in vivo, KFEs did not reduce inflammatory processes relevant to IBD.

SUBMITTER_CITATION: Edmunds, Shelley Jane. The effects of kiwifruit extracts on gene and protein expression in in vitro and in vivo mouse models of inflammatory bowel disease. Diss. University of Auckland, New Zealand, 2010. http://hdl.handle.net/2292/6623

overall_design: Experimental design. Two experiments were conducted, one using extracts from gold kiwifruit and one using extracts from green kiwifruit. Within each experiment, both Il10-/- and C57 mice were randomly divided into three diet treatment groups, to ive the following 12 treatments:  1) Gold Kiwifruit Experiment, C57BL/6J mice, Control diet (AIN-76A),  2) Gold Kiwifruit Experiment, C57BL/6J mice, Aqueous Kiwifruit Extract Supplemented Diet (AIN-76A + 5% Aqueous KFE)  3) Gold Kiwifruit Experiment, C57BL/6J mice, Ethyl Acetate Kiwifruit Extract Supplemented Diet (AIN-76A + 0.11% Ethyl Acetate KFE)  4) Gold Kiwifruit Experiment, Il10-/- mice, Control diet (AIN-76A),  5) Gold Kiwifruit Experiment, Il10-/- mice, Aqueous Kiwifruit Extract Supplemented Diet (AIN-76A + 5% Aqueous KFE)  6) Gold Kiwifruit Experiment, Il10-/- mice, Ethyl Acetate Kiwifruit Extract Supplemented Diet (AIN-76A + 0.11% Ethyl Acetate KFE)   7) Green Kiwifruit Experiment, C57BL/6J mice, Control diet (AIN-76A),  8) Green Kiwifruit Experiment, C57BL/6J mice, Aqueous Kiwifruit Extract Supplemented Diet (AIN-76A + 5% Aqueous KFE)  9) Green Kiwifruit Experiment, C57BL/6J mice, Ethyl Acetate Kiwifruit Extract Supplemented Diet (AIN-76A + 0.11% Ethyl Acetate KFE)  10) Green Kiwifruit Experiment, Il10-/- mice, Control diet (AIN-76A),  11) Green Kiwifruit Experiment, Il10-/- mice, Aqueous Kiwifruit Extract Supplemented Diet (AIN-76A + 5% Aqueous KFE)  12) Green Kiwifruit Experiment, Il10-/- mice, Ethyl Acetate Kiwifruit Extract Supplemented Diet (AIN-76A + 0.11% Ethyl Acetate KFE)   Six biological replicates were analysed from each treatment group, except for groups 3 and 7 where five replicates were analysed due to array quality issues with the sixth replicate. Each replicate contained mRNA from one mouse. A reference design was used, where each slide was hybridised with mRNA from one sample (green channel) and mRNA from a common reference pool (red channel).

first: Shelley

last: Edmunds

first: Marcus

last: Davy

first: Nicole

middle: C

last: Roy

first: Matthew

middle: P

last: Barnett

first: Shuotun

last: Zhu

first: Donald

last: Love

first: William

middle: A

last: Laing


title: The CrebA/Creb3-like transcription factors are major and direct regulators of secretory capacity

status: Public on Oct 18 2010

city: Baltimore

first: Deborah

last: Andrew

state: MD

address: 725 N. Wolfe St., Hunterian G-1

department: Cell Biology

institute: Johns Hopkins University School of Medicine

summary:
Abstract: Secretion occurs in all cells, with relatively low levels in most cells and extremely high levels in specialized secretory cells, such as those of the pancreas, salivary and mammary glands.  Here, we report that the CrebA/Creb3-like family of bZip transcription factors functions to upregulate expression of both the general protein machinery required in all cells for secretion and of cell-type specific secreted proteins.  Drosophila CrebA directly binds the enhancers of secretory pathway genes and is both necessary and sufficient to activate expression of every secretory pathway component gene examined thus far.  Microarray profiling reveals that CrebA also upregulates expression of genes encoding cell type specific secreted components.  Finally, we find that the human CrebA orthologues, Creb3L1 and Creb3L2, have the ability to upregulate the secretory pathway in non-secretory cell types.

This SuperSeries is composed of the SubSeries listed below.

sample_organism:
Drosophila melanogaster
Homo sapiens

platform_organism:
Drosophila melanogaster
Homo sapiens