title: IRAK-4- and MyD88-dependent pathways are essential for the removal of developing autoreactive B cells in humans

status: Public on Oct 23 2008

first: Isabelle

last: Isnardi

address: 535E 70th Street

institute: Hospital for Special Surgery

summary: Most autoreactive B cells are normally counterselected during early B cell development.  To determine whether Toll-like receptors (TLRs) regulate the removal of autoreactive B lymphocytes, we tested the reactivity of recombinant antibodies from single B cells isolated from patients deficient for IL-1R-associated kinase (IRAK)-4, myeloid differentiation factor 88 (MyD88) and UNC-93B.  Indeed, all TLRs except TLR3 require IRAK-4 and MyD88 to signal and UNC-93B-deficient cells are unresponsive to TLR3, TLR7, TLR8 and TLR9.  All patients suffered from defective central and peripheral B cell tolerance checkpoints resulting in the accumulation of large numbers of autoreactive mature naïve B cells in their blood.  Hence, TLR7, TLR8, and TLR9 may prevent the recruitment of developing autoreactive B cells in healthy donors.  Paradoxically, IRAK-4-, MyD88- and UNC-93B-deficient patients did not display autoreactive antibodies in their serum nor developed autoimmune diseases, suggesting that IRAK-4, MyD88 and UNC-93B pathway blockade may thwart autoimmunity in humans.

overall_design: RNA was extracted from 105-3.105 batch sorted new emigrant and mature naïve B cells isolated from donors using the Absolutely RNA microprep kit (Stratagene).  100-200 ng of RNA was obtained per sample, and the quality of the purified RNA was assessed by the Bioanalyzer from Agilent.  Using the Ovation biotin system kit from Nugen, 30-50ng of RNA was amplified and labeled to produce cDNA.  Labeled cDNA was hybridized on chips containing the whole human genome (Human Genome U133 2.0 from Affymetrix). Raw data from new emigrant (1 healthy donor) and mature naive (4 healthy donors) B cells were analyzed in order to determine the expression of some molecules involved in the TLR pathway in these B cell population in humans.

first: Eric

last: Meffre


title: The skeletal muscle transcript profile reflects responses to inadequate protein intake in younger and older males

status: Public on Sep 22 2008

city: West Lafayette

first: Wayne

last: Campbell

state: IN

address: 700 West State Street

department: Foods and Nutrition

country: USA

institute: Purdue University

summary:
Inadequate protein intake initiates an accommodative response with adverse changes in skeletal muscle function and structure.  mRNA level changes due to short-term inadequate dietary protein might be an early indicator of accommodation.  The aims of this study were to assess the effects of dietary protein and the diet-by-age interaction on the skeletal muscle transcript profile.  Self-organizing maps were used to determine expression patterns across protein trials.
958 transcripts were differentially expressed (P<0.05) with diet and 853 had a diet-by-age interaction (P<0.05) using ANOVA.  The results for diet alone revealed that P63 was associated with up-regulation of transcripts related to ubiquitin-dependent protein catabolism and muscle contraction and P63 and P94 resulted in up-regulation of transcripts related to apoptosis and down-regulation of transcripts related to cell differentiation; muscle and organ development; extracellular space; and responses to stimuli and stress.  The diet-by-age expression patterns demonstrated that across the three protein trials transcripts related to protein metabolism were affected by age.  Changes in skeletal muscle mRNA levels in the younger and older males to protein intake near or below the RDA are indicative of an early accommodative response.  5052 transcripts were determined as differentially expressed (P<0.05) between the younger and older males, of which 2556 met the False Discovery Rate correction (P=0.0081).  The age-related changes in the transcript profile were consistent with aging skeletal muscle phenotypes including; mitochondrial dysfunction (UP- and DOWN-regulation), RNA splicing (UP), oxidative stress (UP), apoptosis (UP), and energy metabolism (DOWN).
Keywords: Age and dietary protein response

overall_design: 22 healthy free-living younger (21-43 y, n=12) and older (63-79 y, n=10) males completed three controlled feeding trials with protein intakes of 63% (P63: 0.50 g/kg), 94% (P94: 0.75 g/kg), and 125% (P125: 1.00 g/kg) of the recommended dietary allowance (RDA).  A fasting state vastus lateralis biopsy was taken from the dominant leg of each subject on day 12 of each trial following an overnight fast.  Total RNA was isolated from the muscle samples using Tri-Reagent and the manufacture's protocol.

middle: W

first: Anna

middle: E

last: Thalacker-Mercer

first: James

middle: C

last: Fleet

first: Bruce

middle: A

last: Craig


title: Expression profiles of human carotid plaques

status: Public on Sep 19 2008

city: Göteborg

first: Daniel

last: Hägg

address: Vita Stråket 15

department: Department of Molecular and Clinical Medicine

country: Sweden

institute: Göteborg University

summary: This analysis compares gene expression in human carotid plaques with gene expression in major tissues and cell types in the human body (GSE1133, Su et al. 2004).

overall_design: Human carotid endarterectomies were isolated from six subjects with symptomatic carotid artery disease.

first: Per-Arne

last: Svensson

first: Margareta

last: Jernås

first: Fredrik

last: Olson

first: Björn

last: Fagerberg


title: Overexpression of PKA regulatory subunits in ovarian cancer cells

status: Public on Sep 20 2008

city: baltimore

first: chris

last: cheadle

state: MD

address: 5200 Eastern Ave

department: SOM

institute: JHU

summary:
The regulatory subunit of cAMP-dependent protein kinase (PKA) exists in two isoforms, RI and RII, which distinguish the PKA isozymes, type I (PKA-I) and type II (PKA-II). Evidence obtained from a variety of different experimental approaches has shown that the relative levels of type I and type II PKA in cells can play a major role in determining the balance between cell growth and differentiation.  RIα transfected cells exhibit hyper-proliferative growth and RIIβ transfected cells revert to a relatively quiescent state. Profiling by microarray revealed equally profound changes in gene expression between RIα, RIIβ, and parental OVCAR cells.

Keywords: genetic modification

overall_design: In order to characterize the effect of PKA type I and type II regulatory subunits on gene transcription at a global level, the PKA regulatory subunit genes for RIα and RIIβ were stably transfected into cells of the ovarian cancer cell line (OVCAR8).   Three independent samples form each of RIα, RIIβ stably transfected cell lines as well as from parental cells were harvested for RNA isolation and subsequent microarray analysis.

first: Chris

last: Cheadle

first: Maria

last: Nesterova


title: RCV02 (cadA deficient) v wild-type enterohemorrhagic E. coli (EHEC)

status: Public on Sep 18 2008

city: Baltimore

first: David

last: Rasko

address: 801 W. Baltimore Street, Room 619

department: Istitute for Genome Sciences/Microbiology and Immunology

institute: University of Maryland School of Medicine

summary: Adherence of pathogenic Escherichia coli strains to intestinal epithelia is essential for infection. For enterohemorrhagic E. coli (EHEC) serotype O157:H7, we have previously demonstrated that multiple factors govern this pathogen’s adherence to HeLa cells (39). One of these factors is CadA, a lysine decarboxylase, and this protein has been proposed to negatively regulate virulence in several enteric pathogens. In the case of EHEC strains, CadA modulates expression of the intimin, an outer membrane adhesin involved in pathogenesis. Here, we experimentally inactivated cadA in O157:H7 strain 86-24 to investigate the role of this gene in EHEC adhesion to tissue culture monolayers, global gene expression patterns, and colonization of the infant rabbit intestine. As expected, the cadA mutant did not possess lysine decarboxylation activity and was hyper-adherent to tissue-culture cells. Adherence of the cadA mutant was nearly 2-fold greater than that of the wt and complementation of the cadA defect reduced adherence back to wt levels. Furthermore, the cadA mutant affected the expression of intimin protein. Disruption of the eae gene (encoding the intimin protein) in the cadA mutant significantly reduced its adherence to tissue-culture cells. However, adherence of the cadA eae double mutant was greater than that of an 86-24 eae mutant, suggesting that the enhanced adherence of the cadA mutant is not entirely attributable to enhanced expression of intimin in this  background. Gene array analysis revealed that the cadA mutation significantly altered EHEC gene expression patterns; expression of 1332 genes was down-regulated and 132 genes up-regulated in the mutant compared to the wild type strain. Interestingly, the gene expression variation shows an EHEC-biased gene alteration including intergenic regions. Two putative adhesins: flagella and F9 fimbriae were up-regulated in the cadA mutant, suggestive of their association with adherence in absence of the Cad regulatory mechanism. Remarkably, in the infant rabbit model, the cadA mutant out-competed the wild type strain in the ileum but not in the cecum or mid-colon, raising the possibility that CadA negatively regulates EHEC pathogenicity in a tissue-specific fashion.

overall_design: Arrays used as a starting point for further examination of the effects of the cadA deficiency. A limited number of biologically significant phenotypes and gene expression profiles were examined using qRT-PCR

first: Roberto

last: Vazquez-Juarez

first: Jeeba

last: Kuriakose

first: Jennifer

middle: M

last: Ritchie

first: Melissa

last: Kendall

first: Terry

last: Slater

first: Mala

last: Sinha

last: Luxon

first: Vsevolod

middle: L

last: Popov

first: Matthew

middle: K

last: Waldor

first: Vanessa

last: Sperandio

first: Alfredo

middle: G

last: Torres


title: Differential gene expression in omental adipose tissue from obese children

status: Public on Oct 01 2008

city: GRANADA

first: Concepcion

middle: María

last: Aguilera

state: Granada

address: Ramon y Cajal, 4

department: Intitute of Nutrition and Food Technology

country: Spain

institute: University of Granada

summary:
Characterization of genes associated with adipose tissue is key to understanding the pathogenesis of obesity and developing treatments for this disorder. Differential gene expression in the adipose tissue has been described in adulthood but none studies have been developed on childhood. The purpose of this study was to compare gene expression in omental adipose tissue from obese prepubertal and normal weight children. We selected 5 obese (BMI adjusted for age and sex z score >2) and 6 normal weight children. RNA was extracted from omental adipose tissue biopsies and cRNA was hybridizated on the human genome U133 Plus 2.0 Arrays (Affymetrix®). Microarray experiments were performed for each sample, and selected group of gene expression values were confirmed with real-time RT-PCR in 10 obese and 10 normal weigth prepubertal children. 1276 genes were found to be differentially expressed at P<0.05. Of those differential genes, 201 were upregulated (Fc>2) and 42 were downregulated (Fc<-2). Genes involved in metabolic and signalling pathways were altered in childhood obesity.
Keywords: disease state analysis

overall_design: Adipose tissue and blood samples were obtained from 27 children, 14 obese (BMI adjusted for age and sex z score > 2) and 13 non obese undergoing appendix surgery. About 400 mg of adipose tissue was taken and immediately immersed in RNAlater solution and stored at -80°C for gene expression analysis. Informed consent was obtained from all patients after the nature of the study was explained, and the experimental design was approved, from an ethical and scientific standpoint, by the Hospital’s Ethical Committee responsible for research.

first: Ines

last: Tofe

first: Antonio

last: Suarez

first: Carolina

last: Gomez-Llorente

first: Ramon

last: Cañete

first: Angel

last: Gil


title: Oncogenomic analysis of mycosis fungoides reveals major differences with Sézary syndrome

status: Public on Sep 30 2008

city: Leiden

first: Marloes

last: van Kester

address: Einthovenweg 20

department: Dermatology

country: Netherlands

institute: Leiden University Medical Center

type: Genome variation profiling by genome tiling array

summary:
Mycosis fungoides (MF), the most common cutaneous T-cell lymphoma (CTCL), is a malignancy of mature, skin-homing T cells. Sézary syndrome (Sz) is often considered to represent a leukemic phase of MF. In this study the pattern of numerical chromosomal alterations in MF tumor samples was defined using array-based CGH; simultaneously gene expression was analyzed using microarrays. Highly recurrent chromosomal alterations in MF include copy number gain of 7q36, 7q21-7q22 and loss of 5q13 and 9p21. This pattern characteristic of MF differs markedly from chromosomal alterations observed in Sz. Integration of data from array-based CGH and gene expression analysis yielded several candidate genes with potential relevance in the pathogenesis of MF. We confirmed that the FASTK and SKAP1 genes, residing in loci with recurrent gain, demonstrated increased expression. The RB1 and DLEU1 tumor suppressor genes showed diminished expression associated with loss. In addition, it was found that presence of chromosomal alterations on 9p21, 8q24 and 1q21-1q22 was associated with poor prognosis in patients with MF. This study provides novel insight into genetic alterations underlying MF. Furthermore, our analysis uncovered genomic differences between MF and Sz, which suggest that the molecular pathogenesis and therefore therapeutic requirements of these CTCLs may be distinct.
To identify candidate oncogenes and tumor suppressor genes residing in chromosomal regions with recurrent copy number alteration in MF. To this end chromosomal alteration and gene expression patterns of 22 MF tumor samples were integrated to determine which genes located in minimal common regions (MCRs) with CNA demonstrated dysregulated expression associated with chromosomal alteration

Keywords: aCGH and gene expression integration

overall_design: 22 tumors for aCGH and gene expression

first: Remco

last: van Doorn

middle: S

last: Dijkman

first: Maarten

middle: H

last: Vermeer

first: Aat

last: Mulder

first: Karoly

last: Szuhai

first: Jeroen

last: Knijnenburg

first: Judith

last: Boer

first: Rein

last: Willemze

first: Cornelis

middle: P

last: Tensen


title: A comparative study of bone marrow and peripheral blood CD34+ myeloblasts in acute myeloid leukaemia

city: Hong Kong

first: Alice

last: Cheung

address: 21 Sassoon Road, Pokfulam

department: Medicine

country: China

institute: HKU

summary: To examine the differences between bone marrow (BM) and peripheral blood (PB) myeloblasts in acute myeloid leukaemia (AML), we compared CD34+ myeloblasts of paired BM and peripheral blood (PB) samples from AML patients using microarray.

overall_design: 5 paired BM and PB leukaemic samples from 5 AML patients

first: Howard

last: Chow

first: Raymond

last: Liang

first: Anskar

middle: Y

last: Leung


title: Effects of Leuzea carthamoides (maral root) in MCF-7 cells

status: Public on Sep 17 2008

city: Jena

first: Friedemann

last: Gaube

address: Semmelweisstr. 10

department: Pharmacy

country: Germany

institute: Friedrich-Schiller-University Jena

summary:
Products derived from roots of Leuzea carthamoides DC. (maral root) are being promoted as anti-aging and adaptogenic. The phytoecdysteroids are considered as active principles with numerous beneficial effects, but little is known about the pharmacological properties of Leuzea extracts. We, therefore, investigated the effects of a lipophilic Leuzea root extract on ER+ breast cancer MCF-7 cells at transcriptional level in comparison to 17beta-estradiol and the ER antagonist tamoxifen. With the extract 241 genes were regulated more than 1.5 fold. We observed gene regulation in an anti-proliferative and pro-apoptotic manner.
Additionally, expression of several enzymes with oxidoreductase activity was induced including a very strong increase of the phase I enzyme CYP1A1, a possible link to the AhR pathway, which might explain other expression results, e.g. the correlated regulation of about 20 genes by Leuzea compared to 17beta-estradiol.
Keywords: treatment with Leuzea carthamoides

overall_design: MCF-7 cells were treated for 24 h with a lipophilic (dichloromethane) Leuzea root extract, 17beta-estradiol, tamoxifen and the solvent control (0,1% DMSO) in duplicate

first: Stefan

last: Wolfl

first: Larissa

last: Pusch

first: Torsten

last: Kroll

first: Matthias

last: Hamburger


title: Human Initiator / Effector Gene Sets That Regulate Myometrial Contractility During Term and Preterm Labor

status: Public on Sep 27 2008

city: Kansas City

first: Carl

last: Weiner

state: KS

address: 3901 Rainbow Boulevard, MS 2028

institute: University of Kansas Medical Center

summary:
Distinct processes govern the transition from myometrial quiescence to activation during both term and preterm labor.  We sought the specific gene sets responsible for initiating term and preterm labor, along with a core set of effector genes necessary for labor independent of gestational age and the underlying trigger. The Effector Gene Set consisted of 49 genes present in both preterm and term labor but absent from non-labor samples.  122 genes were specific to preterm labor (Preterm Initiator Set) and 229 to term labor (Term Initiator Set).  The Term Initiator and the Effector Sets reflected predominantly inflammatory processes.  Surprisingly, the Preterm Initiator Gene Set reflected molecular and biological events almost exclusive of inflammation. Preterm and term labor differ dramatically in their unique, initiator gene profiles, suggesting alternative pathways underlie these events.  Inflammatory processes are ubiquitous to the Term Initiator and the Effector Gene Sets, supporting the idea term parturition is an inflammatory process.  The absence of inflammatory processes in the Preterm Initiator Set suggests inflammation is secondary to processes triggering spontaneous preterm birth, and could explain the lack of therapeutic efficacy associated with anti inflammatory/antibiotic regimens.
Keywords: myometrial gene expression, preterm versus term labor

overall_design: Myometrial gene expression was analyzed from samples obtained at term (n=6) or preterm (n=6) with and without labor using cDNA microarrays.  Patients in preterm labor all had intra amniotic inflammation.  Gene sets were generated using logical operations within a functional mapping tool (MetaCore™, GeneGo, St. Joseph, MI).  Relevant gene sets were validated with quantitative real-time polymerase chain reaction.

first: Clifford

last: Mason

first: Yafeng

last: Dong

first: Irina

last: Buhimschi

first: Peter

last: Swaan

first: Catalin


title: Discovery of somatic mutations in lung adenocarcinomas

status: Public on Sep 05 2008

city: St Louis

first: Rekha

last: Meyer

state: MO

address: 4320 Forest Park Av

institute: Washington University School of Medicine

summary: This was a collaborative study to discover somatic mutations in 188 lung adenocarcinomas. DNA sequencing of 623 genes with known or potential relationships to cancer revealed more than 1,000 somatic mutations across the samples. Our analysis identified 26 genes that are mutated at significantly high frequencies and thus are likely to play a role in carcinogenesis. The observed mutational profiles correlate with clinical features, smoking status, and DNA repair defects. These results are complemented by data integration including SNP array data and gene expression array data (deposited here). Our findings shed further light on several key signaling pathways involved in lung adenocarcinoma, and suggest new molecular targets for treatment.

overall_design: The overall design strategy was to sequence a large number of candidate cancer genes from patients diagnosed with adenocarcinoma with the objective of characterizing cancer genomes. For microarray analysis, a subset of 75 RNAs from a corresponding set of 188 lung adenocarcinomas DNAs were resequenced across 623 genes.

first: Mark

last: Watson

first: Richard

last: Wilson

first: Li

last: Ding


title: Effects of vasectomy on gene expression profiling along the human epididymis

city: Quebec

first: Ezequiel

last: Calvo

state: Quebec

address: 2705 Boul. Laurier

department: Molecular Endocrinilogy

country: Canada

institute: CRCHUL

summary:
Worldwide, almost 100 millions men rely on vasectomy for male contraceptive purposes. Due to changes in their personal life, an increasing number of these men request surgical vasectomy reversal. Unfortunately, a significant proportion of these men remain infertile, despite the reestablishment of patent ducts, possibly due to epididymal damages caused by vasectomy. In animal models, vasectomy affects different epididymal physiological and biochemical parameters. However, consequences of vasectomy on these biochemical parameters are poorly understood at the molecular level. Furthermore, results obtained with animal models cannot by extrapolated to human to understand the consequences of vasectomy on epididymal functions. Gene expression pattern of epididimydis is highly regulated. We previously showed that the human epididymal expression pattern of two genes is altered under vasectomy. To complete the list of epididymal genes affected by vasectomy, we analysed the epididymal gene expression profile of three vasectomised donors, using the affymetrix human GeneChip U133 Plus 2. These results were compared with gene expression pattern of three “normal” donors. The data generated allowed the identification of many human epididymal genes for which the expression is modified under vasectomy. Qt-PCR and western-blot analysis of six selected genes known to be expressed in specific epididymal segments were performed. The Qt-PCR results confirmed the selected transcripts expression pattern deduced from microarrays data, while the western-blot analysis revealed some differences in protein distribution along the epididymis when compared to the transcripts expression pattern. These results contribute to the understanding of the causes of the persistent of infertility even though spermogram values suggest surgical success of vasovasostomy.
Keywords: Tissue compartments comparison

overall_design: Vasectomised epididimus, corpus caput and cauda segments

first: Veronique

last: Thimon

first: Gilles

last: Frenette

first: Christine

last: Légaré

first: Omedine

last: Koukoui

first: Robert

last: Sullivan


title: Origin and pathogenesis of lymphocyte-predominant Hodgkin lymphoma as revealed by global gene expression analysis

city: Essen

first: Ralf

last: Küppers

address: Virchowstr. 173

department: Institute for Cell Biology (Tumor Research)

institute: University of Duisburg-Essen, Medical School

summary: The pathogenesis of nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) and its relationship to other lymphomas are largely unknown. This is partly due to the technical challenge of analyzing its rare neoplastic L&H cells, which are dispersed in an abundant non-neoplastic cellular microenvironment. We performed a genome-wide expression study of microdissected lymphocytic and histiocytic (L&H) lymphoma cells in comparison to normal and other malignant B cells, which indicates a relationship of L&H cells to and/or origin from germinal center B cells at transition to memory B cells. L&H cells show a surprisingly high similarity to the tumor cells of T cell-rich B cell lymphoma and classical Hodgkin lymphoma, a partial loss of their B cell phenotype and deregulation of many apoptosis-regulators and putative oncogenes. Importantly, L&H cells are characterized by constitutive NF-κB activity and aberrant ERK signaling. Thus, these findings shed new light on the nature of L&H cells, revealed several novel pathogenetic mechanisms in NLPHL, and may help in differential diagnosis and lead to novel therapeutic strategies.

overall_design: Analysis of differential gene expression in primary human lymphoma cells of nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) in comparison with primary lymphoma cells of classical Hodgkin lymphoma cells and other B-non-Hodgkin lymphoma (B-NHL) samples and subsets of non-neoplastic B lymphocytes isolated from blood or tonsils. 67 gene expression profiles were analysed.

first: Verena

last: Brune

first: Enrico

last: Tiacci

first: Ines

last: Pfeil

first: Claudia

last: Döring

first: Susan

last: Eckerle

first: Carel

last: van Noesel

first: Wolfram

last: Klapper

first: Brunangelo

last: Falini

first: Anja

last: von Heydebreck

first: Dirk

last: Metzler

first: Andreas

last: Bräuninger

first: Martin-Leo

last: Hansmann


title: Expression data from airway epithelial cell-conditioned monocyte-derive dendritic cells

status: Public on Sep 16 2008

city: Subiaco

first: Angela

last: Rate

state: Western Australia

address: 100 Roberts Rd

department: Cell Biology

country: Australia

institute: Telthon Institute for Child Health Research

summary:
Dendritic cells differentiate from their precursors in the airway mucosa under local environmental instruction.  Airway epithelial cells (AEC) are a potent source of both pro- and anti-inflammatory mediators and are in intimate contact with intraepithelial DC and their precursors. Thus, AEC are likely candidates for influencing this differentiation process in order to tailor the DC for optimal function in the airway mucosa.
We used Affymetirx microarrays to compare the gene expression profiles of monocyte derived DC (MDDC) populations differentiated with IL-4 and GM-CSF in the presence or absence of 16HBE 14o- epithelial cells.

Keywords: Comparison of two cell populations

overall_design: 16HBE 14o- epithelial cells were grown to semiconfluency in EMEM media supplemented with 10% FCS before CD14+ peripheral blood monocytes were added to the AEC with recombinant IL-4 and GM-CSF.  In parallel, monocytes were added to empty wells in media with IL-4/GM-CSF without the presence of the AEC.  Cells were cultured for 5 days at 37°C and 5%CO2 with media supplementation on day 3.  At the end of culture, cells were harvested and viable MDDC populations were isolated using flow cytometric sorting.  RNA was extracted from the purified MDDC populations and hybridised to Affymetrix microarrays.

first: Anthony

last: Bosco

first: Kathy

last: McKenna

first: John

last: Upham

first: Patrick

last: Holt


title: Genome-wide mapping cis-regulatory sequence elements in the human genome using Nuclease-Hypersensitive-CGH technique

city: Taipei

first: Ming-Ta

last: Hsu

address: No.155, Sec.2, Linong Street

country: Taiwan

institute: National Yang Ming University

type: Genome binding/occupancy profiling by genome tiling array

summary: Cis-regulatory sequences are the key elements for understanding global, context-dependent gene expression program in response to environmental challenges.  Because of uncertainty in their location relative to gene, nuclease hypersensitivity has been used as the robust experimental approach for identifying these regulatory elements.  We used oligonucleotide array chip and 454 sequencing platform to map the nuclease-hypersensitive sites (NHSS) in MCF-7 cell genome.  Small DNA fragments released after very mild micrococcal nuclease treatment were found to be highly associated with promoter, transcription factor binding sites, regulatory potential and gene expression.  In addition, sequencing data reveal three classes of novel NHSS not reported previously: 1) those containing regular repeat of NHSS (RNHSS), 2) those containing mitochondria sequence inserts (MNHSS), and 3) those containing highly clustered NHSS (CNHSS).  Blatting analysis shows that these novel NHSS are highly associated with genome rearrangements and creation of novel genes during primate genome evolution.  Repeated genes/ESTs in RNHSS are often widely expressed in human adult and fetal tissues.  The majority of NHSS is sensitive to alpha-amanitin treatment with the exception of RNHSS and some genes with amanitin-insensitive expression.  We suggest that study of these novel classes of NHSS may be crucial for understanding primate genome evolution.

overall_design:
Cells were washed three times with ice-cold 1X PBS. The cells were lysed with 700 μl of ice-cold MNase lysis buffer on ice for 15 min. The lysate was removed by suction, and then gently rinse the nuclei with 700 μl of MNase digestion buffer (without CaCl2). The reaction was performed by incubating cell nuclei in 650 μl of MNase digestion buffer (with CaCl2) and 0units (control) or 5units of MNase at 25 ℃ for 5 min. The reaction was terminated by adding 40 μl MNase stop buffer and 20 μl of 20 % SDS. DNA was then phenol/chloroform extracted and ethanol precipitate. Control total genomic DNA was labeled with Cy3, and 5U MNase treated nuclease-released small DNA fragments were labeled with Cy5. When small DNA fragments are hybridized with total genomic DNA, MNase sensitive DNA signal will be higher than control. The same strategy was applied with cells treated with 10μg/ml of alpha-amanitin for 48hrs.

Array-CGH protocols.
10 µg of the undigested and digested DNA were labeled with Cy3 or Cy5 respectively according to the manual of Agilent Oligo Microarray Kit. The log2 ratio (log2 Cy5/Cy3) were calculated and compared.

Affymetrix analysis.
Affymetrix microarray was performed using Human U133 2.0 (Affymetrix). Details of the methods for RNA quality, sample labeling, hybridization and expression analysis are according to the manual of Affymetrix Microarray Kit. Cells were either untreated (control) or treated with 10μg/ml of alpha-amanitin for 48hrs.


title: Genome-wide transcriptional response of an Avian Pathogenic Escherichia coli (APEC) Pst Mutant

status: Public on Sep 15 2008

city: St-Hyacinthe

first: Sébastien

last: Crépin

state: Québec

address: 3200 Sicotte

department: Microbiologie et pathologie

institute: Université de Montréal

summary:
Many reports show an association between the Pst system, the Pho regulon related genes and bacterial virulence. Our previous results showed that a functional Pst system is required for full virulence, resistance to serum, polymyxin B and acid shock. However, the interplay between the Pst system and virulence has an unknown molecular basis. To understand global APEC virulent strain responses to Pho regulon activation, we conducted transcriptome profiling experiments comparing the APEC chi7122 strain and its isogenic Pst mutant grown in rich phosphate medium using the Affymetrix GeneChip® E. coli Genome 2.0 Array. The Affymetrix GeneChip® E. coli Genome 2.0 Array contains the genome of the E. coli MG1655 and three pathogenic E. coli strain (EDL933, Sakai and CFT073) representing 20,366 genes. While comparing genes expression between Pst mutant and the wild type chi7122 strain, 471 genes are either up- (254) or down-regulated (217) of at least 1.5-fold, with a p-value inferior or equal to 0.05 and a false discovery rate of 2.71%.
Keywords: Escherichia coli, phosphate starvation response, Pho regulon, Pst system, Affymetrix, transcriptional analysis

overall_design: RNA extraction was performed onto the chi7122 and K3 strains on four biological replicates. An overnight culture grown at 37°C was diluted 100-fold into 5 ml of LB broth and was allowed to grow to mid-log phase (OD600 0,6). Cultures were centrifuged and RNAs were isolated by using the RiboPure™-Bacteria Kit (Ambion, Austin, TX), according to the manufacturer’s recommendations, with the exception that the DNAse 1 treatment was performed twice. RNA extractions were performed on four different days (the chi7122 and K3 RNA were extracted at the same time) and a total of eight hybridizations were performed. Hybridization was carried out at McGill University & Genome Quebec Innovation Centre according to the Affymetrix recommendation (Affymetrix Expression Manual Section 3 701029 rev. 4). GeneChip scan was carried out at McGill University & Genome Quebec Innovation Centre according to the Affymetrix recommendation (Affymetrix Expression Manual Section 3 701029 rev. 4), and data were processed using the FlexArray software (Michal Blazejczyk, Mathieu Miron, Robert Nadon (2007). FlexArray: A statistical data analysis software for gene expression microarrays. Genome Quebec, Montreal, Canada). Raw data were normalized using the RMA algorithm and log2 were generated. The expression value was generated by subtracting the mean value of each replicate of the mutant strain by wild-type strain.

first: Martin

last: Lamarche

first: Philippe

last: Garneau

first: Julie

last: Proulx

last: Séguin

first: Charles

last: Dozois

first: Josée

last: Harel


title: Identifying Novel Predictors of Resistance to DNA Damaging Agents

status: Public on Sep 23 2008

city: Chapel Hill

first: Rebecca

middle: Catherine

last: Fry

address: 1213 MHRC

department: Environmental Sciences and Engineering

institute: UNC-Chapel Hill

summary:
We have investigated whether gene expression signatures can be used to predict inter-individual responses to DNA damaging agents
We used microarrays to detail inter-individual variation in gene expression across a healthy population under basal and damage induced conditions
Keywords: dose (MNNG)

overall_design: lymphoblastoid cell lines that are part of the NIHPDR were examine for gene expression differences as well as killing upon exposure to MNNG

first: J

last: Svensson

first: Chandni

last: Valiathan

first: Emma

last: Wang

first: Bradley

middle: J

last: Hogan

first: Sanchita

last: Bhattacharya

first: James

last: Bugni

last: Whittaker

first: Leona

middle: D

last: Samson


title: Involvement of hCMV in the ontogeny of CD4+ T-LGL

status: Public on Sep 01 2008

city: Salamanca

first: Arancha

last: Rodríguez-Caballero

address: Avda. Universidad de Coimbra. Campus Unamuno

institute: Cancer Research Center. University of Salamanca.

summary: Recent studies suggest the potential involvement of common antigenic stimuli on the ontogeny of monoclonal TCRalphabeta+/CD4+/NKa+/CD8-/+dim T-large granular lymphocyte (LGL) lymphocytosis. Since healthy individuals show (oligo)clonal expansions of hCMV-specific TCRVbeta+/CD4+/cytotoxic/memory T-cells, we investigate the potential involvement of hCMV in the origin and/or expansion of monoclonal CD4+ T-LGL. A detailed characterization of those genes that underwent changes in T-LGL cells responding to hCMV was performed by microarray gene expression profile (GEP) analysis.

overall_design:
Total RNA was isolated from magnetic-activated cell sorter (MACS)-freshly purified hCMV-stimulated CD69+, hCMV-stimulated CD69- and unstimulated monoclonal CD4+ T-LGL lymphocytes from PB samples from four TCRVbeta+/CD4+ T-LGL lymphocytosis patients (purity of ≥98%). Briefly, 100 ng of total RNA from each of the 12 purified cell fractions was amplified and labeled using the GeneChip two cycle cDNA synthesis kit and the GeneChip IVT labeling kit (Affymetrix Inc., Santa Clara, CA), respectively. Then it was hybridized to the Human Genome U133 Plus 2.0 Array (Affymetrix).
In parallel, total RNA was also isolated from highly purified (≥ 98% purity) hCMV-stimulated (specific) CD69+ CD4+ T-lymphocytes isolated from PB samples from hCMV-seropositive healthy donors (n=5, mean age of 36 years) using a FACSAria flow cytometer (BDB). To get pure and highly concentrated RNA, the silica membrane technology NucleoSpin® RNA XS (Macherey-Nagel, Düren, Germany) was used. Total RNA was then amplified, labeled and hybridized to the  Human Genome U133 Plus 2.0 Array (Affymetrix) as described above.

last: Rodriguez-Caballero

first: Andrés

last: Garcia-Montero

first: Paloma

last: Barcena

first: Julia

last: Almeida

first: Francisco

last: Ruiz-Cabello

first: Maria

last: Tabernero

first: Pilar

last: Garrido

first: Santiago

last: Muñoz-Criado

first: Yorick

last: Sandberg

first: Anton

last: Langerak

first: Marcos

last: Gonzalez

first: Ana

last: Balanzategui

first: Alberto

last: Orfao


title: HLA ligand profiles of primary renal cell carcinoma maintained in metastases.sue

status: Public on Sep 03 2008

city: Tuebingen

first: Andreas

middle: Oliver

last: Weinzierl

address: Auf der Morgenstelle 15

department: Department of Immunology

institute: University of Tuebingen

summary: In recent years, several approaches have been taken in the peptide-based immunotherapy of metastatic renal cell carcinoma (RCC), although little is known about HLA presentation on metastases compared to primary tumor and normal tissue of RCC. In this study we compared primary tumor, normal tissue and metastases with the aim of identifying similarities and differences between these tissues. We performed this comparison for two RCC patients on the level of the HLA ligandome using mass spectrometry and for three patients on the level of the transcriptome using oligonucleotide microarrays. The quantitative results show that primary tumor is more similar to metastasis than to normal tissue, both on the level of HLA ligand presentation and mRNA. We were able to characterize a total of 142 peptides in the qualitative analysis of HLA-presented peptides. Six of them were significantly overpresented on metastasis, among them a peptide derived from CD151; fourteen were overpresented on both primary tumor and metastasis compared to normal tissue, among them an HLA ligand derived from tumor protein p53. Thus, we could demonstrate that peptide-based immunotherapy might affect tumor as well as metastasis of RCC, but not healthy kidney tissue. Furthermore we were able to identify several peptides derived from tumor-associated antigens that are suitable for vaccination of metastatic RCC.

overall_design: Three clear cell renal cell carcinomas including autologous normal tissue and autologous metastasis were analyzed.

first: Juliane

last: Stickel

middle: O

first: Nina

last: Hillen

first: Oliver

last: Drews

first: Mathias

last: Schuler

first: Jörg

last: Hennenlotter

first: Dorothee

last: Wernet

first: Arnulf

last: Stenzl

first: Stefan

last: Stevanovic


title: Differentiation associated changes in gene expression profiles for interstitial cystitis and control urothelial cells

city: Lexington

first: Deborah

last: Erickson

state: KY

address: 800 Rose Street, MS275

department: Surgery

institute: University of Kentucky

type: Expression profiling by high throughput sequencing

type: Methylation profiling by high throughput sequencing

summary: Purpose:  Evaluate gene expression profiles after inducing differentiation in cultured interstitial cystitis (IC) and control urothelial cells. Materials and Methods:  Bladder biopsies were taken from IC patients and controls (women having surgery for stress incontinence).  Primary cultures were grown in Keratinocyte Growth Medium with supplements.  To induce differentiation, in some plates the medium was changed to DMEM-F12 with supplements.  RNA was analyzed with Affymetrix chips.  Three nonulcer IC patients were compared with three controls. Results:  After inducing differentiation, 302 genes with a described function were altered at least 3-fold with p <0.01 in both IC and control cells.  Functions of the162 upregulated genes included cell adhesion (e.g. claudins, occludin, cingulin); urothelial differentiation, retinoic acid pathway and keratinocyte differentiation (e.g. skin cornified envelope components).  The 140 downregulated genes included genes associated with basal urothelium (e.g.  p63, integrins ?4, ?5 and ?6, basonuclin 1 and extracellular matrix components), vimentin, metallothioneins and members of the Wnt and Notch pathways.  Comparing IC vs. control cells after differentiation, only seven genes with a described function were altered at least 3-fold with p <0.01.  PI3, SERPINB4, CYP2C8, EFEMP2 and SEPP1 were decreased in IC; AKR1C2 and MKNK1 were increased in IC. Conclusions:  Differentiation-associated changes occurred in both IC and control cells. Comparing IC vs. control revealed very few differences.  This study may have included IC patients with minimal urothelial deficiency and/or selected the cells that were most robust in culture.  Also, the abnormal urothelium in IC may be due to post-translational changes and/or the bladder environment.

overall_design: Human female urothelial cell cultures, differentiated vs. non-differentiated,interstitial cystitis vs. control

middle: R

first: Steven

last: Schwarze

first: Justin

last: Dixon

first: Curtis

last: Clark

first: Matt

last: Hersh


title: A high efficiency system for the generation and study of human induced pluripotent stem cells

status: Public on Sep 12 2008

city: NYC

first: Tim

last: Ahfeldt

state: NY

address: 1425 Madison Ave

department: Neuroscience

institute: Icahn School of Medicine at Mount Sinai

summary: Direct reprogramming of human fibroblasts to a pluripotent state has been achieved through ectopic expression of the transcription factors OCT4, SOX2, and either cMYC and KLF4 or NANOG and LIN28. Little is known, however, about the mechanisms by which reprogramming occurs, which is in part limited by the low efficiency of conversion. To this end, we sought to create a doxycycline-inducible lentiviral system to convert primary human fibroblasts and keratinocytes into human induced pluripotent stem (hiPS) cells. hiPS cells generated with this system were molecularly and functionally similar to human embryonic stem (hES) cells, demonstrated by gene expression profiles, DNA methylation status, and differentiation potential. While expression of the viral transgenes was required for several weeks in fibroblasts, we found that 10 days was sufficient for the reprogramming of keratinocytes, suggesting that the kinetics of reprogramming are cell-type dependent. Using our inducible system, we developed a strategy to induce hiPS cell formation at high frequency by generating differentiated cells that contain the viral transgenes in a pattern that enables successful induction of pluripotency. Upon addition of doxycycline to differentiated hiPS-derived cells, we obtained “secondary” hiPS cells at a frequency at least 100-fold greater than the initial conversion. The ability to reprogram cells with high efficiency provides a unique platform to dissect the underlying molecular and biochemical processes that accompany nuclear reprogramming.

overall_design: Total RNA was isolated from control HUES lines, hBJ (donor line) 4 primary HIPS lines and 1 secondary HIPS line using an RNeasy kit (Qiagen). Samples were processed as independent triplicates. RNA probes for microarray hybridization were prepared and hybridized to Affymetrix HGU133 plus 2 oligonucleotide microarrays. Data was extracted and analyzed using Rosetta Resolver system. During importation, the data was subjected to background correction, intrachip normalization, and the Rosetta Resolver Affymetrix GeneChip error model (Weng et al, 2006). For the generation of intensity plots, genes that showed greater than a two-fold difference in expression value (p<0.01) in HUES8 hES cells and BJ fibroblasts were noted (19,663 probes) and their expression analyzed. A hierarchical clustering was performed.

first: Nimet

last: Maherali

first: Alessandra

last: Rigamonti

first: Jochen

last: Utikal

first: Chad

last: Cowan

first: Konrad

last: Hochedlinger


title: BMPR2 mutation carriers with and without evidence of PAH

city: Nashville

first: James

last: West

state: TN

address: 4200 E 9TH Ave, Box B133

institute: Vanderbilt University

summary:
Background: While BMPR2 mutation strongly predisposes to pulmonary arterial hypertension (PAH), only 20% of mutation carriers develop clinical disease. This finding suggests that modifier genes contribute to FPAH clinical expression. Since modifiers are likely to be common alleles, this problem is not tractable by traditional genetic approaches. Further, examination of gene expression is complicated by confounding effects attributable to drugs and the disease process itself. Methods: To resolve these problems, B-cells were isolated, EBV-immortalized, and cultured from familial PAH patients with BMPR2 mutations, mutation positive but disease-free family members, and family members without mutation. This allows examination of differences in gene expression without drug or disease-related effects. These differences were assayed by Affymetrix array, with follow-up by quantitative RT-PCR and additional statistical analyses. Results: By gene array, we found consistent alterations in multiple pathways with known relationship to PAH, including actin organization, immune function, calcium balance, growth, and apoptosis. Selected genes were verified by quantitative RT-PCR using a larger sample set. Analysis of overrepresented gene ontology groups suggests that it is pathway-specific, not gene-specific changes that carry increased risk of disease. Conclusions: B-cell lines are a valuable and accessible tool for assaying alterations in gene expression free from drug and disease effects. Predisposition to disease within BMPR2 mutation carriers was linked to several pathways, including proliferation, GTP signaling, and stress response.
Keywords: Search for modifier genes

overall_design: Immortalized B-cells from BMPR2 mutation carriers with and without disease are compared to search for modifier genes

first: Joy

last: Cogan

first: Mark

last: Geraci

first: Linda

last: Robinson

first: John

last: Newman

last: Phillips

first: Kirk

last: Lane

first: Barbara

last: Meyrick

first: Jim

last: Loyd


title: Molecular subtypes of DLBCL have distinct chromosomal aberrations

status: Public on Sep 04 2008

city: Bethesda

first: Louis

middle: M.

last: Staudt

address: 9000 Rockville Pike, Bldg 10, Rm 4N114

department: Lymphoid Malignancies Branch

institute: National Cancer Institute

summary:
We performed array comparative genomic hybridization (aCGH) and gene expression profiling in 203 samples of diffuse large B cell lymphoma (DLBCL). By gene expression, at least three molecular subtypes of DLBCL termed as germinal center B cell-like (GCB) DLBCL, activated B cell-like (ABC) DLBCL, and primary mediastinal B cell lymphoma (PMBL) can be distinguished. Combining gene expression profiling and aCGH, revealed copy number abnormalities that had strikingly different frequencies in the three molecular DLBCL subtypes. These data provide genetic evidence that the DLBCL subtypes are distinct diseases that utilize different oncogenic pathways.
Keywords: clinical history design

overall_design: The retrospective study included RNA and DNA extracted from 203 clinical samples.


title: Expression analysis of growing and senescent activated hepatic stellate cells

status: Public on Aug 22 2008

city: Cold Spring Harbor

first: Valery

last: Krizhanovsky

address: 1 Bungtown Rd

institute: Cold Spring harbor Laboratory

summary:
Cellular senescence is an irreversible proliferative arrest and can be triggered in many cell types in response to diverse forms of cellular damage or stress.
We used microarrays to compare gene expression profile between growing and senescent human activated hepatic stellate cells.

Keywords: cell type comparison

overall_design:
Two separate preparation of activated hepatic stellate cells were treated with DNA damaging agent to induce senescence or vechicle to remain growing.
RNA was extracted from both replications and used for hybridization on Affymetrix arrays to determine expression differences.

first: Scott

middle: W

last: Lowe


title: Role of Caveolin 1, E-Cadherin, Enolase 2 and PKCa on resistance to methotrexate in human HT29 colon cancer cells

status: Public on Sep 08 2008

city: Barcelona

first: Carlos

last: Ciudad

state: Barcelona

address: Av. Juan XXIII-27

department: Biochemistry and Molecular Biology

institute: University of Barcelona

summary:
A summary of the work associated to these microarrays is the following:

Methotrexate (MTX) is one of the earliest cytotoxic drugs used in cancer therapy, and despite the isolation of multiple other folate antagonists, methotrexate maintains its significant role as a treatment for different types of cancer and other disorders. The usefulness of treatment with methotrexate is limited by the development of drug resistance, which may be acquired through different ways. To get insights into the mechanisms associated with drug resistance and sensitization we have performed a functional analysis of genes deregulated in methotrexate resistant cells, either due to its co-amplification with the DHFR gene or as a result of a transcriptome screening using microarrays. Genes adjacent to dhfr locus and included in the 5q14 amplicon were overexpressed in HT29 MTX-resistant cells. Treatment with siRNAs against those genes caused a slight reduction in cell viability in both HT29 sensitive and resistant cells. On the other hand, microarray analysis of HT29 and HT29 MTX resistant cells unveiled overexpression of caveolin 1, enolase 2 and PKCa genes in treated cells without concomitant copy number gain. siRNAs against these three genes effectively reduced cell viability and caused a decreased MTX resistance capacity. Moreover, overexpression of E-cadherin, which was found underexpressed in MTX-resistant cells, also sensitized the cells toward the chemotherapeutic agent. We provide functional evidences indicating that caveolin 1 and E-cadherin may play a critical role in cell survival and may constitute potential targets for coadjuvant therapy.
Keywords: DHFR, Methotrexate, drug resistance

overall_design: Two cell lines are compared in the study, which are HT29 colon cancer cells sensitive to methotrexate and HT29 cells resistant to 10e-5M MTX. Six samples are provided which correspond to triplicated of each cell line. The samples provided were subsequently normalyzed and analyzed using the specific software GeneSpring GX v7.3.1.

first: Elisabet

last: Selga

first: Cristina

last: Morales

first: Verónica

last: Noé

first: Miguel

middle: A

last: Peinado


title: Transcriptonal profile of cord lining epithelial cells after phage integrase mediated integration

status: Public on Aug 23 2008

city: Singapore

first: Jaichandran

last: Sivalingam

address: 11 Hospital Drive

department: Division of Medical Sciences

country: Singapore

institute: National Cancer Center

summary:
The two obstacles that impede a wider application of genetically modified cells expressing therapeutic transgenes for ex vivo gene therapy are the immune mediated rejection of the transplanted cells, combined with their potential to cause iatrogenic oncogenesis. In this study we describe a new cellular vehicle for this form of therapy,
termed the cord lining epithelial cell (CLEC). CLECs are derived from the human amnion and incorporate many of the immunoregulatory functions associated with the fetal/maternal interface. We show that CLECs can be safely transfected by phage φC31 integrase to accomplish site-specific integration of a therapeutic human transgene. We also show that transplanted CLECs are not oncogenic in vivo and can be maintained in immunocompetent mice where acute xeno-rejection rapidly destroys other human cell types. Finally, we demonstrate the utility of CLECs for ex vivo gene therapy by delivering human coagulation factor 8 to mice with Hemophilia A.

overall_design: The transcriptome datasets of human umbilical cord lining epithelial cells were compared before(CLEC) and 1 month after(CLEC-GFP) phage integrase mediated integration of EGFP cDNA into the genome. Transcriptome datasets were generated in singles and genes differentiallty expressed in cells before and after phage integrase treatment were analysed. Genes differentially expressed by at least 2 fold as compared to untreated CLEC were considered to be significantly dysregulated. List of genes with significant dysregulation were used for further analysis and used to determine if genomic integration events had resulted in any potential geno-toxicity.


title: Estrogen- and Myc-regulated genes in MCF-7 breast cancer cells

status: Public on Aug 21 2008

city: Sydney

first: Liz

last: Musgrove

state: NSW

address: 384 Victoria Street, Darlinghurst

department: Cancer Research Program

institute: Garvan Institute of Medical Research

summary:
Estrogen-responsive genes were identified by transcript profiling of estrogen-treated MCF-7 breast cancer cells.
The gene expression profile generated after estrogen treatment was compared with that following inducible expression of c-Myc or c-Zip (a deletion mutant of c-Myc that lacks the N-terminal transactivation domains) in clonal MCF-7 cell lines.

Keywords: Single time point

overall_design: RNA was collected in three independent experiments, each including parental MCF-7 cells treated with 17b-estradiol (E2) or ethanol (EtOH), zinc-treated p∆MT-c-Myc cells, zinc-treated p∆MT-c-Zip cells and zinc-treated empty vector (p∆MT) cells. Cells were arrested for 48 h with 10 nM ICI 182780 and then treated for 6 h with either 100 nM E2 or ethanol vehicle, or 75 mM zinc for the stably transfected cell lines.

first: Elizabeth


title: Time course gene expression of HUVEC after TNF-alpha treatment

status: Public on Sep 13 2008

city: Komaba

first: Shuichi

last: Tsutsumi

country: Japan

institute: The University of Tokyo

summary:
The many steps involved in the production of a mature mammalian mRNA are extensively coupled, and levels of both precursors and products can be measured using expression and genomic tiling microarrays.  Different probes in these arrays targeting the same transcript often give different signals; then, precursor (nascent) RNA – which is present transiently at low concentrations – is difficult to detect.
Keywords: TNFa stimulation time course of HUVEC

overall_design: Using tumor necrosis factor a (TNF-alpha) to switch on transcription of selected human genes rapidly and synchronously, we apply samples collected every 15 min to arrays

first: Tatsuhiko

last: Kodama

first: Meng

last: Xu

first: Yoshihiro

last: Ohta

first: Takashi

last: Minami

first: Shu-ichi

first: Daisuke

last: Komura

first: Kenji

last: Inoue

first: Mika

last: Kobayashi

first: Akashi

last: Izumi

first: Mai

last: Miura

first: Hiroko

last: Meguro

first: Miyako

last: Fujii

first: Nobuhiko

last: Oshida

first: Jun'ichi

last: Kitakami

first: Hiroshi

last: Kimura

first: Shumpei

last: Ishikawa

first: Takao

last: Hamakubo

first: Hiroyuki

last: Aburatani

first: Peter

last: Cook

first: Sigeo

last: Ihara


title: Primary human peripheral Blood B cell Acute Stimulation - anti_IgM

first: Louis

last: Staudt

summary:
To identify genes in human peripheral blood B cells whose expression changes with acute stimulation through the antigen receptor using anti_IgM.
Keywords: time series design

overall_design: Peripheral human blood B cells were purified and gene expression from unstimulated cells (resting) was compared to cells stimulated with anti_IgM for 1hr and 3hrs using Affymetrix U133 arrays. Two unistimulated cell microarrays were biological replicates.


title: The undifferentiated phenotype in neuroblastoma depends on Cyclin D1 and CDK4 activity

status: Public on Aug 20 2008

city: Amsterdam

first: Richard

last: Volckmann

state: Noord-Holland

address: van der Boechorststraat 6a

department: Laboratory of Experimental Oncology and Radiobiology

institute: msterdam UMC and University of Amsterdam

summary:
Genomic aberrations of Cyclin D1 (CCND1) and CDK4 in neuroblastoma indicate that dysregulation of the G1 entry checkpoint is an important cell cycle aberration in this pediatric tumor. Here we report that analysis of Affymetrix expression data of primary neuroblastic tumors shows an extensive over-expression of Cyclin D1 and CDK4 which correlates with histological subgroups and prognosis respectively. Immunohistochemical analysis demonstrated an over-expression of Cyclin D1 in neuroblasts and a low Cyclin D1 expression in all cell types in ganglioneuroma. This suggests an involvement of G1 regulating genes in neuronal differentiation processes which we further evaluated using RNA interference against Cyclin D1 and its kinase partner CDK4 in several neuroblastoma cell lines. This resulted in pRb pathway inhibition as shown by an almost complete disappearance of CDK4 specific pRb phosphorylation; reduction of E2F transcriptional activity and a decrease of Cyclin A protein levels. The Cyclin D1 and CDK4  knock-down resulted in a significant reduction in cell proliferation, a G1 specific cell cycle arrest and moreover an extensive neuronal differentiation. Affymetrix microarray profiling of siRNA treated cells revealed a shift in expression profile towards a neuronal phenotype. Several new potential downstream players are identified. We conclude that neuroblastoma functionally depend on over-expression of G1 regulating genes to maintain their undifferentiated phenotype.
Keywords: Neuroblastoma, CCND1, Cyclin D1, CDK4

overall_design: The Cell line IMR-32 at time point 0 and transiently transfected with siRNA against GFP, Cyclin D1 and CDK4 at time point 48 hours. All experiments are biological triplicates.

first: Jan

last: Molenaar

first: Marli

middle: E

last: Ebus

last: Koster

middle: v

last: Sluis

first: Carel

last: van Noesel

first: Rogier

last: Versteeg

first: Huib

middle: N

last: Caron


title: Gene expression analysis of T-ALL cell lines treated with gamma-secretase inhibitor

city: New York

first: Adolfo

last: Ferrando

address: 1130 St Nicholas Ave Irving Cancer Research Center 5-505A

department: Institute for Cancer Genetics

institute: Columbia University

summary:
Gain-of-function mutations in NOTCH1 are common in T-cell lymphoblastic leukemias making this receptor a promising target for drugs such as gamma-secretase inhibitors (GSI), which block a proteolytic cleavage required for NOTCH1 activation. However, the enthusiasm for these therapies has been tempered by tumor resistance and the paucity of information on the oncogenic programs regulated by oncogenic NOTCH1. Analysis of gene expression in GSI-responsive and GSI-resistant cell lines treated with Compound E identifies differential resopnses to GSI.
Keywords: Drug response

overall_design: Samples for microarray analysis were prepared and hybridized in Affymetrix Human U133 Plus 2.0 arrays according to the manufacturer’s instructions and as previously described. RNA was extracted from duplicate cultures of GSI-sensitive (ALL-SIL, CUTLL1, DND41, HPB-ALL, KOPTK1) and GSI-resistant (CCRF-CEM, MOLT3, P12 ICHIKAWA, PF382 and RPMI8402) T-ALL cell lines treated for 24 h with vehicle (DMSO) or 500 nM CompE.  Interarray intensity differences were normalized with Dchip.

first: Teresa

last: Palomero

first: MariaLuisa

last: Sulis

first: Maria

last: Cortina

first: Pedro

last: Real

first: Kelly

last: Barnes

last: Ciofani

first: Esther

last: Caparros

first: Jean

last: Buteau

first: Kristy

last: Brown

first: Sherrie

middle: L

last: Perkins

first: Govind

last: Bhagat

first: Archana

last: Mishra

first: Giuseppe

last: Basso

first: Ramon

last: Parsons

first: JuanCarlos

last: Zúñiga-Pflücker

last: Dominguez


title: Impaired T-cell function in patients with novel ICOS

status: Public on Sep 26 2008

city: Bunkyo-ku

first: naomi

last: takahashi

state: Tokyo

address: 1-5-45 Yushima

department: Department of Pediatrics and Developmental Biology

institute: Tokyo Medical and Dental University

summary: Interaction of ICOS - ICOS ligand is required for the germinal center formation, T-cell immune responses, and development of autoimmune diseases.  Human ICOS deficiency with the identical ICOS mutation has been identified in nine patients worldwide.  In vitro studies showed T-cell defect of the patients was mild, and in vivo autoimmunity was uncommon and mild.  Here we report in-depth analysis of T-cell function in two siblings with novel ICOS deficiency.  While the brother displayed mild skin infections, psoriasis-like skin region, and defective immunoglobulin class switching, the sister had more severe symptoms, which included immunodeficiency, rheumatoid arthritis, inflammatory bowel disease, interstitial pneumonitis, and psoriasis.  Despite of normal CD3/CD28-induced proliferation and IL-2 production in vitro, peripheral blood T-cells from both patients demonstrated decreased percentage of CD4 central and effector memory T-cells and impaired production of Th1, Th2, and Th17 cytokines upon CD3/CD28 costimulation or upon PMA/ionophore stimulation.  The defective polarization into effector cells were associated with impaired induction of T-bet, GATA3 and MAF and RORC.  Reduced CTLA-4+CD45RO+ FoxP3+ regulatory T-cells and diminished induction of inhibitory cell surface molecules including CTLA-4 were also observed in the patients.  Further analysis of the gene expression and immune functions of the patients demonstrated increased induction of RANKL, lack of IFN-g response, and loss of Itch expression upon activation in the female case with autoimmunity.  Our study suggests extensive T-cell dysfunction and loss of balance between effector cells and regulatory cells in the ICOS-deficient patients may account for their immunodeficiency and/or autoimmune disorder.

overall_design: One control and two patients samples. Negatively-selected CD4 T-cells were incubated with or without stimulants (plate-bound anti-CD3mAb and anti-CD28mAb).  The cells were collected at 24h time point.

first: Naomi

last: Takahashi

first: Kenji

last: Matsumoto

first: Hirohisa

last: Saito

first: Toshihiro

last: Nanki

first: Nobuyuki

last: Miyasaka

first: Tetsuji

last: Kobata

first: Miyuki

last: Azuma

first: Lee

last: Sang-Kyou

first: Shuki

last: Mizutani

first: Morio

last: Tomohiro


title: Endometrium of hormone-treated postmenopausal women

city: Rotterdam

first: Leen

last: Blok

address: Dr Molewaterplein 40

department: Obstetrics & Gynecology

institute: Erasmus University Medical Center

summary:
Title: Transcriptome analysis of human endometrial tissues from healthy post-menoupausal women reflecting the endometrial response to 3-weeks treatment with tibolone, E2 and E2+MPA.
In an observational, open, non-randomized, controlled study uterine tissues were collected in order to generate endometrial gene expression profiles. healthy postmenopausal women were enrolled into the following treatment groups: Control-group; Tibolone-group, 2,5 mg of tibolone (administered orally) every day, starting 21 days prior to surgery; Estradiol-group, 2 mg of estradiol (administered orally) every day, starting 21 days prior to surgery; Estradiol+progestagen-group, 2 mg of estradiol (administered orally) and 5 mg of MPA (Medroxy Progesteroneacetate, administered orally) every day, starting 21 days prior to surgery. Pure (100%) endometrium was isolated from the snap-frozen uterine tissues and used for RNA isolation. RNA was labeled and hybridized to whole genome Affymetrix U133plus2 GeneChips containing 54,614 probe sets, representing approximately 47,000 transcripts.
Relative to the control group, 940 genes are regulated in the endometrium of E2 treated patients, whereas only 198 genes are significantly regulated in endometria from tibolone or E2+MPA treated patients. Furthermore, only 9% of E2 regulated genes are also regulated by tibolone (85 out of 940), only 5% are also regulated by E2+MPA treatment and the overlap between tibolone and E2+MPA treatment is about 10%. This indicated that tibolone-treatment results in a weak endometrial profile similarity to E2 treatment and no profile similarity to E2+MPA treatment. A more detailed analysis showed that down stream processes, such as regulation of the cell cycle, angiogenesis and cell proliferation are almost not affected by tibolone treatment but, in contrast, are significantly affected by E2. For example, upon staining with Ki67, a marker for mitotic activity, significantly increased stromal as well as glandular cell proliferation was observed in the endometria from the E2-only treated group, while tibolone treatment resulted only in a slight increase in stromal cell proliferation (and no increase in glandular cell proliferation). These results indicate that in contrast to long-term tibolone use, short-term (21-days) use results in some estrogenic stimulation of the endometrium, which is clearly far less and rather different from what is observed during E2 treatment.
References:
Klaassens et al., 2006
Hanifi-Moghaddam et al., 2007
Verheul et al., 2007

overall_design:
This study was designed as a controlled clinical trial. Patients who visited our clinics to undergo vaginal hysterectomy for treatment of prolapse, were eligible to participate in this study. The trial was performed in the period before the scheduled surgery. After informed consent, the patients were sequentially assigned to one of the following treatment groups:

-	  Control-group (no hormonal treatment);
-	  Tibolone-group (2.5 mg tibolone (Livial, N.V. Organon, Oss, The Netherlands) administered orally every day, starting 21 days prior to surgery);
-	  E2 group (2 mg of estradiol administered orally every day, starting 21 days prior to surgery);

-	  E2+MPA-group (2 mg estradiol + 5 mg MPA administered orally every day, starting 21 days prior to surgery).

Pure endometrium was isolated and used for profiling. 31 samples, 3 of which were duplicates, were analyzed.

first: Anet

last: Klaassens

first: Heidy

last: Van Wijk

first: Payman

last: Hanifi-Moghaddam

first: Bianca

last: Sijmons

first: Patricia

middle: C

last: Ewing

first: Marjan

last: Ten Kate-Booij

first: Sjarlot

last: Kooi

first: Wilfred

middle: F

last: Van Ijcken

first: Peter

last: Van der Spek

first: M

last: Den Bakker

first: Herman

last: Verheul

first: Helenus

last: Kloosterboer

first: Curt

last: Burger


title: mRNA expression profiling of nasopharyngeal carcinoma

status: Public on Aug 16 2008

city: Madison

first: Paul

last: Ahlquist

state: WI

address: 1525 Linden Dr.

department: Oncology

institute: University of Wisconsin-Madison

summary:
Nasopharyngeal carcinoma is an Epstein-Barr virus-associated epithelial cancer with high prevalence in Southeast Asia. mRNA expression levels were measured for essentially all human genes and all latent Epstein-Barr virus (EBV) genes in nasopharyngeal carcinoma tissue samples and normal nasopharyngeal tissues. Data were analyzed for differential gene expression between tumor and normal tissue and  for correlations with levels of viral gene expression. Primary publications: Sengupta et al, 2006, Cancer Research 66(16): 7999-8006. Dodd et al, 2006, Cancer Epidemiology, Biomarkers & Prevention 15(11): 2216-2225.
In subsequent studies using the same set of tissue samples, microRNA levels were measured in tumors and normal tissues and analyzed for correlations with differential target gene expression (Sengupta et al, 2008, Proc. Nat. Acad. Sci. USA 105: 5874-5878.)

Keywords: mRNA expression profiling

overall_design: Total RNA extracted from laser-captured epithelium from 31 nasopharyngeal carcinomas and 10 normal healthy nasopharyngeal tissue specimens.


title: Myeloma versus Lymphoma cell lines

first: Louis

last: Staudt

summary:
To identify genes more highly expressed in Myeloma celllines v.s lymphoma cell lines.
Keywords: cell type comparison design

overall_design: Cell line cDNA was hybridized to Affymetrix U133 arrays for comparison.


title: Microarray analysis of human monocytes infected with Francisella tularensis

status: Public on Aug 13 2008

city: Columbus

first: Jonathan

middle: P

last: Butchar

state: OH

address: 400 W. 12th Avenue

department: Internal Medicine

institute: The Ohio State University

summary:
Differential expression in human peripheral blood monocytes between F. novicida-infected and uninfected, and between Francisella tularensis tularensis isolate Schu S4 and uninfected.
The goal was to examine genomewide transcriptional reponses to these two strains, and identify differentially-regulated genes that may help explain the virulence of Schu S4.

Keywords: Immune Response, Human Monocytes, Bacteria, Francisella

overall_design: Human monocytes were infected with the Schu S4 isolate of Francisella tularensis tularensis (n=4), with F. tularensis subspecies novicida isolate U112 (n=4) or were left uninfected (n=6).  Gene expression values were calculated using the gcrma package in R and BioConductor, and limma to identify differentially expressed genes.  Submitted here are expression values calculated using R 2.7.1 and BioConductor 2.2 (FreeBSD/amd64) but the original were done using R 2.6.1 and BioConductor 2.1 (FreeBSD/amd64).  Twelve other chips were pooled with these 14 for preprocessing.

first: Susheela

last: Tridandapani


title: Gene expression profiling of HhAntag-treated pancreatic xenografts

city: South San Francisco

last: Haverty

state: CA

address: 1 DNA Way

department: Bioinformatics

institute: Genentech, Inc.

summary:
Four vehicle-treated and four HhAntag-treated pancreatic xenograft tumors were profiled for gene expression changes using Affymetrix U133 Plus 2.0 and Affymetrix Mouse Genome 430 2.0 arrays.

Keywords: comparative gene expression, hedgehog, hh

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: A primary human pancreatic tumor xenograft (1051178-A) was established by direct implantation of surgical material into female CD1 nu/nu mice of 6-8 weeks of age.  Tumors were serially passaged into larger cohorts of mice for efficacy testing and subsequently distributed into tumor volume-matched cohorts upon tumors reaching between 200 to 350 mm3.  HhAntag was resuspended in 0.5% methyl-cellulose, 0.2% Tween-80 (MCT) and administered orally twice daily at 75 mg/kg from a 10 mg/ml suspension.  MCT alone served as vehicle control.  Tumor xenografts (4/group) were excised following 21 days of dosing and RNA was extracted.  Preparation of complementary RNA, Human Genome U133 Plus 2.0 array and Mouse Genome 430 2.0 array hybridizations, and subsequent data analysis were carried out using Affymetrix protocols, with signal intensities being determined by the MAS5.0 algorithm.

first: Bob

last: Yauch

first: Fred

last: DeSauvage


title: Transcriptional regulation by the novel Rho GTPase RhoBTB2.

city: Bristol

first: Harry

last: Mellor

state: Avon

address: School of Medical Sciences, University Walk

department: Department of Biochemistry

country: United Kingdom

institute: University of Bristol

summary:
RhoBTB2 is a novel Rho GTPase that undergoes loss, underexpression and mutation in breast and lung cancer. We have shown that we can mimic loss of RhoBTB2 through siRNA treatment of primary cells. We propose to perform comparative microarray analysis of primary lung cells to establish the identification of the gene targets of RhoBTb2 regulation.
Keywords: Effects of siRNA expression

overall_design: Primary lung cells (NHBE) transfected with siRNA oligonucleotides against cyclophilin or Lamin A/C (controls) were compared to cells transfected with siRNA oligonucleotides against RhoBTB2. We used two siRNA oligonucleotides against RhoBTB2 (1 and 2) to determine any non-specific effects. Cells expressed the siRNA oligonucleotides for 72 hours before total RNA extraction. 10 samples have been analysed.

first: Caroline

middle: M

last: McKinnon

first: Kate

last: Lygoe

first: Lara

last: Skelton

first: Richard

last: Mitter


title: Global gene expression from SOX7 and SOX17 over-expressing human embryonic stem cells (CA1 and CA2 lines)

status: Public on Aug 11 2008

city: Toronto

first: Cheryle

last: Seguin

state: Ontario

address: 101 College St

institute: The Hospital for Sick Children

summary:
This study aimed to understand the transcriptional networks regulating endoderm specification from HESC and therefore explored the phenotype of CA1 and CA2 HESC constitutively over-expressing SOX7 or SOX17. Cell lines were created using an inducible construct whereby clonal populations containing transgene integration are selected by Neomycin resistance without expressing of the gene of interest (NoCre controls). Transgene expression is induced via Cre-mediated recombination and selected for puromycin resistance (SOX O/E). The phenotype of the resulting cells suggests that SOX7 expressing HESC represent stable extraembryonic endoderm progenitors, while SOX17 expressing HESC represent early definitive endoderm progenitors. Both in vitro and in vivo SOX7 expressing HESC are restricted to the extraembryonic endoderm lineage, while SOX17 expressing HESC demonstrate mesendodermal specificity.  In vitro, SOX17 expressing HESC efficiently produce mature definitive endoderm derivatives.
The molecular phenotype of the resulting SOX7 and SOX17 expressing HESC was characterized by microarray analysis
Keywords: cell line comparison

overall_design: Total RNA was extracted from confluent monolayer cultures of SOX7 over-expressing HESC, SOX17 over-expressing HESC, and their respective control parental HESC lines (designated NoCre Sox7 and NoCre Sox17).

first: Janet

last: Rossant


title: Expression Profile of CREB knockdown in Myeloid Leukemia Cells

status: Public on Aug 30 2008

city: los angeles

first: matteo

last: pellegrini

address: 610 charles young drive east

institute: UCLA

summary:
Background.  The cAMP Response Element Binding Protein, CREB, is a transcription factor that regulates cell proliferation, differentiation, and survival in several model systems, including neuronal and hematopoietic cells.  We demonstrated that CREB is overexpressed in acute myeloid and leukemia cells compared to normal hematopoietic stem cells.  CREB knockdown inhibits leukemic cell proliferation in vitro and in vivo, but does not affect long-term hematopoietic reconstitution.  Therefore, we propose CREB to be a potential target for therapy.  To understand downstream pathways regulating CREB, we performed expression profiling with RNA from the K562 myeloid leukemia cell line.
Results.
By combining our expression data from CREB knockdown cells with prior ChIP data on CREB binding we were able to identify a list of putative CREB regulated genes.  We performed extensive analyses on the top genes in this list as high confidence CREB targets.  We found that this list is enriched for genes involved in cancer, and unexpectedly, highly enriched for histone genes.  Furthermore, histone genes regulated by CREB were more likely to be specifically expressed in hematopoietic lineages.  The transcription factor Elk-1 was upregulated in response to CREB deletion.
Conclusions.
We have identified a high confidence list of CREB targets in K562 cells.  These genes allow us to begin to understand the mechanisms by which CREB contributes to acute leukemia.  In particular, we speculate that the regulation of histone genes may play an important role in this process, by possibly altering the regulation of DNA replication during the cell cycle.

overall_design:
Cell lines. The following human leukemia cell lines were transduced with shRNAs: K562 (Iscoves + 10% FCS) and TF-1 (RPMI + 10%FCS + rhGM-CSF.  Cells were cultured at 37oC, 5% CO2 and split every 3 to 4 days.  Primary AML bone marrow samples were processed as previously described [12].  All human samples were obtained with approval from the Institutional Review Board and consents were signed, according to the Helsinki protocol.

shRNA sequence design and constructs. The CREB specific shRNA sequences were selected and validated based on accepted parameters established by Tuschl et al.  [25-27]; CREB shRNA-1, CREB shRNA-2, CREB shRNA-3.  Controls included empty vector, luciferase shRNA, and scrambled shRNA. shRNA sequences are: CREB shRNA-1(5’GCAAATGACAGTTCAAGCCC3’), shRNA-2 (5’GTACAGCTGGCTAACAATGG3’), shRNA-3 (5’GAGAGAGGTCCGTCTAATG3’), Luciferase shRNA (5’GCCATTCTATCCTCTAGAGGA3’), Scramble shRNA (5’GGACGAACCTGCTGAGATAT3’). Short-hairpin sequences were synthesized as oligonucleotides and annealed according to standard protocol.  Annealed shRNAs were then subcloned into pSICO-R shRNA vectors from the Jacks laboratory at MIT [28]. The second generation SIN vector HIV-CSCG was used to produce human shRNA vectors [29].

Microarray analysis. Total RNA (10 μg) was extracted from K562 cells transduced with vector alone or CREB shRNA was submitted to the UCLA DNA Microarray Facility. RNA samples were labeled and hybridized by standard protocol to Affymetrix GeneChip Human Genome  U133+ Array Set HG-U133A array.  Gene expression values were calculated using the MAS5 software. The expression values are quantile normalized across all arrays.  We obtained the expression profiles for a control set and CREB downregulated K562 cells.  These are first quantile normalized, and then a t-test is performed between the two groups to identify significantly differentially regulated genes.  The analysis was performed using Matlab (Mathworks, Inc.).  A normal quantile plot of T is shown in figure qqplot.tiff.  Data points with t-scores < 1/(2N) display with red circles, were N is the total number of genes.  We therefore see that there are a significant number of differentially expressed genes, which are either direct or indirect targets of CREB.

first: Matteo

last: Pellegrini

first: Jerry

last: Cheng

first: Jon

last: Voutila

first: Dejah

last: Judelson

first: Stanley

last: Nelson

first: Kathleen

last: Sakamoto


title: A six-gene signature predicting breast cancer lung metastasis

status: Public on Aug 15 2008

city: SAINT-CLOUD

first: clairac

last: geraldine

address: 35 rue Daily

department: Oncogenetique

country: France

institute: Centre Rene Huguenin

summary:
The lungs are a frequent target of metastatic breast cancer cells, but the underlying molecular mechanisms are unclear. All existing data were obtained either using statistical association between gene expression measurements found in primary tumors and clinical outcome, or using experimentally derived signatures from mouse tumor models. Here, we describe a distinct approach that consists to utilize tissue surgically resected from lung metastatic lesions and compare their gene expression profiles with those from non-pulmonary sites, all coming from breast cancer patients.
We demonstrate that the gene expression profiles of organ-specific metastatic lesions can be used to predict lung metastasis in breast cancer. We identified a set of 21 lung metastasis-associated genes. Using a cohort of 72 lymph node-negative breast cancer patients, we developed a six-gene prognostic classifier that discriminated breast primary cancers with a significantly higher risk of lung metastasis. We then validated the predictive ability of the six-gene signature in 3 independent cohorts of breast cancers consisting of a total of 721 patients. Finally, we demonstrated that the signature improves risk stratification independently of known standard clinical parameters and a previously established lung metastasis signature based on an experimental breast cancer metastasis model.
Keywords: Disease state analysis

overall_design: We used microarrays to identify lung metastasis-related genes in a series of 23 patients with breast cancer metastases. No replicate, no reference sample.

first: Thomas

last: Landemaine

first: Amanda

last: Jackson

first: Akeila

last: Bellahcène

first: Nadia

last: Rucci

first: Soraya

last: Sin

first: Berta

last: Martin Abad

first: Angels

last: Sierra

first: Alain

last: Boudinet

first: Jean-Marc

last: Guinebretière

first: Enrico

last: Ricevuto

first: Catherine

last: Noguès

first: Marianne

last: Briffod

first: Ivan

last: Bièche

first: Pascal

last: Cherel

first: Teresa

last: Garcia

first: Vincent

last: Castronovo

first: Anna

last: Teti

first: Rosette

last: Lidereau

first: Keltouma

last: Driouch


title: KINK-1, a novel small-molecule inhibitor of IKKbeta, enhances susceptibility of melanoma cells to antitumoral treatment

status: Public on Aug 09 2008

city: Wuerzburg

first: Margarete

last: Schoen

institute: RVZ

summary:
Interference with chemoresistance to enhance the efficacy of chemotherapeutics may be of great utility for cancer therapy. We have identified KINK-1 (Kinase Inhibitor of NF-kappaB-1), a highly selective small-molecule IKKkappa inhibitor, as a potent suppressor of both constitutive and induced NF-kappaB activity in melanoma cells. While KINK-1 profoundly diminished various NF-kappaB-dependent gene products regulating proliferation, cytokine production or anti-apoptotic responses, the compound by itself showed little antiproliferative or pro-apoptotic activity on the cellular level. However, its combination with some cytostatics markedly enhanced their antitumoral activities in vitro, and doxorubicin-induced NF-kappaB activation, a mechanism implicated in chemoresistance, was abrogated by KINK-1. In addition, when KINK-1 was combined with doxorubicin in an in vivo melanoma model, experimental metastasis was significantly diminished as compared to either treatment alone. Induction of chemoresistance by KINK-1 in vivo was not observed. Thus, KINK-1 or related substances might increase the susceptibility of tumors to chemotherapy.
Keywords: time-course

overall_design: one control and two time point (12hrs and 24hrs) are analyzed, with one replicate each (6 arrays total)

last: Schön

first: Gregor

last: Wienrich

first: Helga

last: Sennefelder

first: Susanne

last: Kneitz

first: Verena

last: Vöhringer

first: Katharina

last: Amschler

first: Karl

last: Ziegelbauer

first: Michael


title: Anticancer metabolites discovered by Computational Metabolomics

status: Public on Aug 04 2008

city: Atlanta

first: Nathan

middle: J.

last: Bowen

state: GA

address: 223 James P Brawley Drive

department: Center for Cancer Research and Therapeutic Development

institute: Clark Atlanta University

summary:
CoMet, a fully automated Computational Metabolomics method to predict changes in metabolite levels in cancer cells compared to normal references has been developed and applied to Jurkat T leukemia cells with the goal of testing the following hypothesis: up or down regulation in cancer cells of the expression of genes encoding for metabolic enzymes leads to changes in intracellular metabolite concentrations that contribute to disease progression. Nine metabolites predicted to be lowered in Jurkat cells with respect to normal lymphoblasts were examined: riboflavin, tryptamine, 3-sulfino-L-alanine, menaquinone, dehydroepiandrosterone, α-hydroxystearic acid, hydroxyacetone, seleno-L-methionine and 5,6-dimethylbenzimidazole. All, alone or in combination, exhibited antiproliferative activity. Of eleven metabolites predicted to be increased or unchanged in Jurkat cells, only two (bilirubin and androsterone) exhibited significant antiproliferative activity. These results suggest that cancer cell metabolism may be regulated to reduce the intracellular concentration of certain antiproliferative metabolites, resulting in uninhibited cellular growth and have the implication that many other endogenous metabolites with important roles in carcinogenesis are awaiting discovery.
Keywords: cell type

overall_design:
The first step of the CoMet approach consists of the classification of each enzyme-coding human gene into four possible groups: G1) upregulated in cancer cells, G2) downregulated in cancer cells, G3) expressed in both, normal and cancer cells, at levels that are statistically indistinguishable, and G4) not expressed in both, normal and cancer cells. We used two types of data for the classification: the log base 2 signal intensities and the presence calls of the corresponding probe sets, as reported by the Affymetrix Microarray Suite Software 5.0 (MAS 5.0). First, an “off” status is provisionally assigned to each gene in each of the two studied conditions (normal and cancer) if the mean fraction of presence calls labeled as “marginal” or “absent” in the corresponding probe sets is at least 80%; otherwise, an “on” status is assigned. Then, each gene is temporarily classified into the G1, G2, G3 or G4 group, according to its on/off status in normal and cancer conditions. Finally, genes in the temporary G3 or G4 groups are transferred to the G1 or G2 groups if they fulfill the following criterion for differential expression: the signal intensities in normal and cancer samples exhibit a statistically significant difference in at least 40% of the corresponding probe sets, as evaluated by an ANOVA two-tailed test with P < 0.005.

RNA extraction, amplification and microarray data processing. Total RNA was extracted from cell lines using Trizol (Invitrogen) and processed using the RiboAmp OA or HS kit (Arcturus) in conjunction with the IVT Labeling Kit from Affymetrix, to produce an amplified, biotin-labeled mRNA suitable for hybridizing to GeneChip Probe Arrays (Affymetrix). Labeled mRNA was hybridized to GeneChip Human Genome U133 Plus 2.0 Arrays in the GeneChip Hybridization oven 640, further processed with the GeneChip Fluidics Station 450 and scanned with the GeneChip Scanner. Affymetrix .CEL files were processed using the Affymetrix Expression Console (EC) Software Version 1.1. Files were processed using the default MAS5 3’ expression workflow which includes scaling all probes to a target intensity (TGT) of 500. Spiked in report controls used were AFFX-BioB, AFFX-BioC, AFFX-BioDn, and AFFX-CreX. Affymetrix .CEL files for three normal lymphoblast samples used as a normal reference to compare Jurkat cells expression data were directly retrieved from the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/, samples GSM113678, GSM113802 and GSM113803 of untreated GM15851 cells from the Series GSE5040).

first: Adrian

middle: K

last: Arakaki

first: Roman

last: Mezencev

first: Ying

last: Huang

first: John

last: McDonald

first: Jeffrey

last: Skolnick


title: Expression data from p73 over-expression in H1299 cells

status: Public on Aug 06 2008

city: Nashville

first: Jennifer

last: Pietenpol

address: 23rd Ave South at Pierce

department: Biochemistry

institute: Vanderbilt University Medical Center

summary: The integral role of p53 in tumor suppression has promted many laboratories to perform extensive analyses of signaling pathways downstream of the p53 family of sequence-specific DNA binding transcription factors (p53 and its homologs p63 and p73).  Despite the ability of p73 to regulate many p53 family target genes, little is known about the specific pathways that modulate p73 during development, tumorigenesis and tumor therapy.  In this study we present a gene signature-based approach for connecting signaling pathways to transcription factors, as exemplified by p73.  We generated a p73 gene signature by integrating whole-genome chromatin immunoprecipitation and expression profiling.

overall_design: H1299 lung carcinoma cells were infected with p73 expressing or control adenovirus for 5 h and then harvested.

last: Rosenbluth


title: Identification of candidate p73 target genes

type: Genome binding/occupancy profiling by high throughput sequencing

summary:
The integral role of p53 in tumor suppression has promted many laboratories to perform extensive analyses of signaling pathways downstream of the p53 family of sequence-specific DNA binding transcription factors (p53 and its homologs p63 and p73).  Despite the ability of p73 to regulate many p53 family target genes, little is known about the specific pathways that modulate p73 during development, tumorigenesis and tumor therapy.  In this study we present a gene signature-based approach for connecting signaling pathways to transcription factors, as exemplified by p73.  We generated a p73 gene signature by integrating whole-genome chromatin immunoprecipitation and expression profiling.

This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: Anti-IGF-IR antibody h10H5 induces a unique transcriptional profile in SK-N-AS human neuroblastoma xenograft tumor

status: Public on Sep 01 2008

city: south San Francisco

first: Jerry

last: Tang

address: 1 DNA Way MS 93

institute: Genentech Inc

summary:
The insulin-like growth factor (IGF) system consists of two ligands (IGF-I and IGF-II), which both signal through type I IGF receptor (IGF-IR) to stimulate proliferation and inhibit apoptosis, with activity contributing to malignant growth of many types of human cancers. We have developed a humanized, affinity-matured anti-human IGF-IR monoclonal antibody (h10H5), which binds with high affinity and specificity to the extracellular domain. h10H5 inhibits IGF-IR-mediated signaling by blocking IGF-I and IGF-IIbinding and by inducing cell surface receptor down-regulation via internalization and degradation. In vitro, h10H5 exhibits anti-proliferative effects on cancer cell lines. In vivo, h10H5 demonstrates single-agent anti-tumor efficacy in human SK-N-AS neuroblastoma and SW527 breast cancer xenograft models, and even greater efficacy in combination with the chemotherapeutic agent Docetaxel or an anti-VEGF antibody. Anti-tumor activity of h10H5 is associated with decreased AKT activation and glucose uptake, and a 316-gene transcription profile with significant changes involving DNA metabolic and cell cycle machineries. These data support the clinical testing of h10H5 as a biotherapeutic for IGF-IR-dependent human tumors.

Keywords: Gene expression changes as markers for drug activity

overall_design: Two treatment groups with four tumor samples per group are collected and analyzed,

first: Yonglei

last: Shang

first: Yifan

last: Mao

last: Batson

first: Suzie

middle: J

last: Scales

first: Gail

last: Phillips

first: Mark

last: Lackner

first: Klara

last: Totpal

first: Simon

last: Williams

first: Jihong

last: Yang

first: Zhijun

first: Zora

last: Modrusan

first: Christine

last: Tan

first: Wei

last: Liang

first: Siao

last: Tsai

first: Alexander

last: Vanderbilt

first: Kenji

last: Kozuka

first: Klaus

last: Hoeflich

first: Janet

last: Tien

first: Sarajane

last: Ross

first: Congfen

last: Li

first: Sang

middle: H

last: Lee

first: An

last: Song

first: Yan

last: Wu

first: Jean

last: Stephan

first: Avi

last: Ashkenazi

first: Jiping

last: Zha


title: Characterization of the gene expression changes associated with melanoma-endothelial cell communication

status: Public on Aug 02 2008

city: Baltimore

first: Megan

middle: Jean

last: Stine

state: MD

address: 1650 Orleans Street, CRB1 336

institute: The Johns Hopkins University School of Medicine

summary:
Average life expectancy for patients with metastatic melanoma is less than 6 months, and only a handful of treatment options are available. If the disease can be stopped before it spreads to other organs, life expectancy is greatly increased. The goal of this project is to identify possible regulators of melanoma metastasis by determining genes whose expression is modulated when the cells are grown in contact with endothelial cells. Identification of genes involved in this cell-cell communication could have therapeutic implications.
Keywords: co-culture

overall_design: Using a lentiviral system, melanoma cell line 1205Lu was transduced with GFP, and HUVEC (human umbillical vein endothelial cells) cells at passage 2 were transduced with RFP. These labeled cells were plated at near confluency either alone (controls) or mixed at a 1:1 ratio (co-cultures) for 48 hours in endothelial growth medium (EGM-2, Cambrex). The labeled cells were then sorted by FACS and RNA was collected from all 4 cultures. There are a total of four samples: GFP-1205Lu cells grown alone, GFP-1205Lu cells separated from co-culture with RFP-HUVEC cells, RFP-HUVEC cells grown alone, and RFP-HUVEC cells separated from co-culture with GFP-1205Lu cells. The JHMI Microarray Core was responsible for sample processing and initial data processing using Affymetrix-recommended protocols (Affymetrix GeneChip Expression Analysis Technical Manual, 2004).

first: Rhoda

last: Alani


title: Effects of tobacco smoke on gene expression and cellular pathways in a cellular model of oral leukoplakia

status: Public on Aug 01 2008

city: NEW YORK

first: Piali

last: Mukherjee

state: New York

department: Medicine

institute: Weill Cornell Medicine

summary:
In addition to being causally linked to the formation of multiple tumor types, tobacco use has been associated with decreased anticancer treatment efficacy and reduced survival time. A detailed understanding of the cellular mechanisms that are affected by tobacco smoke should facilitate the development of improved preventive and therapeutic strategies.  We have investigated the effects of a tobacco smoke (TS) extract on the transcriptome of MSK-Leuk1 cells, a cellular model of oral leukoplakia. Using Affymetrix HGU133 Plus 2 arrays, 411 differentially expressed probesets were identified. The observed transcriptome changes were grouped according to functional information, and translated into molecular interaction network maps and signaling pathways. Pathways related to cellular proliferation, inflammation, apoptosis and tissue injury appeared to be perturbed.  Analysis of networks connecting the affected genes identified specific molecular interactions, hubs and key transcription regulators affected by TS. Thus TS was found to induce several EGFR ligands forming an EGFR-centered molecular interaction network, as well as several AhR-dependent genes, including the xenobiotic metabolizing enzymes CYP1A1 and CYP1B1.  Notably, the latter findings in vitro are consistent with our parallel finding that levels of CYP1A1 and CYP1B1 were increased in oral mucosa of smokers.  Collectively, these results offer insights into the mechanisms underlying the procarcinogenic effects of TS and raise the possibility that inhibitors of EGFR or AhR signaling will prevent or delay the development of tobacco smoke-related tumors. Moreover, the inductive effects of TS on xenobiotic metabolizing enzymes may help explain reduced efficacy of chemotherapy, and suggest targets for chemopreventive agents in smokers.
Keywords: microarray, tobacco smoke, gene expression, oral, aryl hydrocarbon receptor

overall_design: In this study MSK-Leuk1 cells established from a dysplastic leukoplakia lesion adjacent to a squamous cell carcinoma of the tongue were exposed to tobacco condensate for 0.5 hr, 3 hr, 6 hr, 12 hr and 24 hr, respectively and compared to untreated controls. Six biological replicates were used at each timepoint for each condition (Control or Tobacco Smoke treated).

first: Zeynep

last: Gumus

first: Baoheng

last: Du

first: Ashutosh

last: Kacker

first: Jay

middle: O

last: Boyle

first: Jennifer

last: Bocker

first: Kotha

last: Subbaramaiah

first: Andrew

last: Dannenberg

first: Harel

last: Weinstein


title: Overexpression of the Apoptotic Cell Removal Receptor, MERTK, in Alveolar Macrophages of Cigarette Smokers

first: Yael

last: Strulovici-Barel

address: 1300 York Avenue

department: Department of Genetic Medicine

institute: Weill Cornell Medical College

summary:
Mononuclear phagocytes play an important role in the removal of apoptotic cells by expressing cell surface receptors that recognize and remove apoptotic cells. Based on the knowledge that cigarette smoking is associated with increased lung cell turnover, we hypothesized that alveolar macrophages (AM) of normal cigarette smokers may exhibit enhanced expression of apoptotic cell removal receptor genes. AM obtained by bronchoalveolar lavage of normal non-smokers (n=11) and phenotypic normal smokers (n=13, 36 ± 6 pack-yr) were screened for mRNA expression of all known apoptotic cell removal receptors using Affymetrix HG-U133 Plus 2.0 chips with TaqMan RT-PCR confirmation. Of the 14 known apoptotic receptors expressed, only MER Tyrosine Kinase (MERTK), a transmembrane tyrosine kinase receptor, was significantly up-regulated in smokers. MERTK expression was then assessed in AM of smokers vs nonsmokers by TaqMan RT-PCR, immunohistochemistry, Western and flow analysis. Smoker AM had up-regulation of MERTK mRNA levels (smoker vs non-smoker, 3.6-fold by microarray, p<0.003; 9.5-fold by TaqMan RT-PCR, p<0.02). Immunohistochemistry demonstrated a qualitative increase in MERTK protein expression on AM of smokers. Increased protein expression of MERTK on AM of smokers was confirmed by Western and flow analyses (p< 0.007 and p< 0.0002, respectively). MERTK, a cell surface receptor that recognizes apoptotic cells, is expressed on human AM, and its expression is up-regulated in AM of cigarette smokers. This may reflect an increased demand for removal of apoptotic cells in smokers, an observation with implications for the development of chronic obstructive pulmonary disease (COPD), a disorder associated with dysregulated apoptosis of lung parenchymal cells.
Keywords: Comparison of gene expression in alveolar macrophages of normal non-smokers and normal smokers.

overall_design: Alveolar macrophages were obtained from bronchoalveolar lavage.

first: Angeliki

last: Kazeros

first: Ben-Gary

last: Harvey

first: Brendan

last: Carolan

first: Holly

last: Vanni

first: Anja

last: Krause

first: Ronald

middle: G

last: Crystal


title: Expression in HepG2 cells with overexpression of TMPRSS6 or its mutant version.

city: La Jolla

first: Yu

last: Xia

address: 10550, N. Torrey Pines Rd.

department: Genetics

institute: The Scripps Research Institute

summary:
TMPRSS6 is a type II transmembrane serine protease and is revealed by our work to be part of a low-iron sensing pathway. When animal gets iron deficient, TMPRSS6 is required to shut off hepcidin gene, so as to allow iron to be uptaken from GI tract. The mutant mouse, which was generated by ENU mutagenesis, has developed microcytic anemia. The phenotype is caused by a splicing error in Tmprss6 gene. However, the mechanism of TMPRSS6 effect remains elusive. To gain further insight into the molecular components of the TMPRSS6 signaling pathway, we overexpressed either TMPRSS6 or its mutant version of protein in human liver carcinoma cell line HepG2 cells, and compared the transcription status betweem these two treatments.
Keywords: genetic modification

overall_design: Triplicate per transfection with wildtype Tmprss6 cDNA, mutant version of Tmprss6 cDNA and empty vector as control, respectively. 36 hours post transfection, cells were harvested and subjected to RNA extraction.

first: Xin

first: Bruce

last: Beutler