title: Testosterone Replacement versus Placebo Bone and Bone Marrow in Castrated Mice

city: Seattle

first: Denise

last: Mauldin

address: 1100 Fairview Ave N D4-100

department: Human Biology

institute: Fred Hutchinson Cancer Research Center

summary:
Metastatic prostate cancer is commonly localized in bone and treated with androgen ablation, which is initially highly effective, but eventually results in resistant cancer cells. The decreased dependence on androgen is associated with activation of the androgen receptor (AR) via other growth factor receptor pathways. The current study analyzed
global gene expression in bone from castrated and sham operated C57BL/6 mice to identify factors that may be responsible for androgen signaling. Androgen suppression by castration increased the expression of 159 genes with insulin-like growth factor binding protein 5 (IGFBP5) being the most consistently increased. IGFBP5 protein was detected by immune histochemistry in osteoblasts, bone marrow stromal cells and
endothelial cells. In vitro treatment of marrow stromal cells with charcoal-stripped serum increased IGFBP5 mRNA expression approximately 25-fold and was reversed by androgen supplementation. Endogenous IGFBP5 was incorporated into extracellular matrix. In addition, treatment of extracellular matrices with exogenous IGFBP5 and IGF-1 or IGF-2 enhanced the growth of immortalized human prostate cells. These results suggest that androgen ablative therapy may increase IGFBP5 in the marrow microenvironment, thereby providing support for the development of therapy resistance prostate cancer cells.
Keywords: Bone Marrow Environment, Androgen Suppression, IGFBP5, prostate cancer

overall_design: Femural, tibial and humeral bones were collected from paired castrated  C57BL/6 mice received Testosterone or placebo pellets (12.5 mg 60 day slow release, Innovative Research of America, Sarasota, FL) for 4 weeks.  Each timepoint includes three replicates (mouse pairs) and cy dye labeling is alternated between experiment and control to account for dye effect.  Testosterone Replacement is the experimental channel and Placebo Control is the control channel.

first: Chang

last: Xu

first: Lynn

last: Graf

first: Ladan

last: Fazli

first: Ilsa

last: Coleman

first: Peter

middle: S

last: Nelson

first: Martin

last: Gleave

first: Stephen

middle: R

last: Plymate

first: Michael

last: Cox

first: Beverly

last: Torok-Storb

first: Beatrice

last: Knudsen


title: Experimental Selection for Drosophila Survival in Extremely Low O2 Environments

status: Public on Jun 01 2007

city: La Jolla

first: Dan

last: Zhou

state: CA

address: CMG 103, 9500 Gilman Drive, MC0735

department: Pediatrics

country: USA

institute: University of California, San Diego, School of Medicine

summary:
We have used long-term experimental selection over many generations to obtain Drosophila melanogaster strains that can live perpetually in extremely low, normally lethal O2 conditions (4-5% O2).  These strains show a dramatic phenotypic divergence from control animals, including a decreased recovery time after anoxia, a higher rate of O2 consumption in hypoxic conditions, and a decrease in body mass due to both decreased cell proliferation and cell size.  We used gene expression profiling to identify altered transcript levels in the adapted flies and we show that mutations in many of the corresponding genes confer the ability to survive under extremely low O2 conditions.
Keywords: genetic bases of hypoxia adaptation

overall_design: 27 isogenic D. melanogaster Lines were pooled and following long-term selection over generations with decreased oxygen level in the culture environment. The differences in gene expression were compared between adapted flies and generation matched naive controls by microarray. Pooled RNA samples from 27 parental lines were used as common reference.

first: Jin

last: Xue

first: Patrick

last: Morcillo

first: David

last: Lambert

first: Kevin

middle: P

last: White

first: Gabriel

middle: G

last: Haddad


title: Gene expression profiling of circulating plasma RNA from colorectal cancer patients

status: Public on Jun 06 2007

city: MADRID

first: Luis

last: LOMBARDIA

state: Madrid

address: Melchor Fernandez Almagro, 3

department: Molecualr Pathology Programme

country: Spain

institute: CNIO (Spanish National Cancer Center)

summary:
Gene expression profiling of circulating plasma RNA is a valuable tool to detect cancer and a potential clinical instrument to study tumor progression and therapy responsiveness. In this study, differential gene expression analysis was used to identify genes specifically increased in cancer patients.
Keywords: Disease state analysis

overall_design: We have measured the relative abundance of the different RNA species in plasma through cDNA microarray hybridization, by comparing RNA isolated and amplified from colorectal cancer patients (12) and from healthy donors (8). Each sample was competitively hybridized against a common reference formed by a pool of blood samples from 26 healthy donors.


title: Gap junction and purinergic P2 receptor proteins as a functional unit: insights from transcriptomics

status: Public on Jun 20 2007

city: Prairie View

first: Dumitru

middle: Andrei

last: Iacobas

state: TX

address: Ann Preston St

department: Undergraduate Medical Academy

institute: Prairie View A&M University

summary:
Gap junctions and purinergic P2 receptors (P2Rs) can be regarded as belonging to a common functional unit, given that they are involved in the transmission of calcium signals between cells. We have previously shown that deletion of the Gja1 gene alters expression levels of numerous genes encoding proteins with diverse functions, including purinergic, P2Rs, and have found that genes synergistically or antagonistically expressed in wildtype tissues are more prone to be similarly or oppositely regulated in Cx43-nulls. We have now explored the use of coordination analysis of gene expression as a strategy to identify interlinked genes encoding functionally related proteins and used pull-downs to evaluate their interlinkage. Our findings indicate that, in brain and in cultured astrocytes, several of these co-expressed genes encode proteins that are components of P2R signal transduction pathways and/or directly interact with these receptors, including the gap junction protein connexin43 (Cx43) and connexin45 (Cx45), as well as pannexins. It is proposed that coordination analysis of gene expression may provide a novel unbiased strategy for the identification of proteins belonging to supramolecular complexes.
Keywords: genetic modification

overall_design: The transcriptomes of cultured cortical astrocytes and whole brain of neonatal Cx43 null mice were compared to the transcriptomes of cortical astrocytes and whole brain of wildtype C57Bl/6 mice. 4 biological replicas were used for each sample type but wildtype cortical astrocytes were 6 replicas were used. Brain transcriptomes were compared using the sample reference method while for the astrocytes we have used the "multiple yellow" design described in Iacobas DA, Fan C, Iacobas S et al., Transcriptomic changes in developing kidney exposed to chronic hypoxia. Biochem Biophys Res Commun. 2006 349(1):329-38.

middle: A

first: Sylvia

middle: O

last: Suadicani

first: Sanda

first: Christina

last: Chrisman

first: Michal

last: Cohen

middle: C

last: Spray

first: Eliana

last: Scemes


title: Cytotoxic activity of HTI-286 in prostate cancer

status: Public on Jun 29 2007

city: Vancouver

first: Susan

middle: L

last: Ettinger

state: BC

address: 2660 Oak Street

department: Urologic Sciences

country: Canada

institute: Faculty of Medicine

summary:
HTI-286, an analogue of hemiasterlin, interferes with microtubule dynamics and circumvents transport-based resistance to taxanes. In this study we evaluate the inhibitory effects of HTI-286 on human prostate cancer growth in vitro and in different in vivo models. The results show that HTI-286 is a potent inhibitor of proliferation and induced marked increases in apoptotic rates in all cell lines tested. Despite similar transcriptomic changes regarding cell death and cell cycle-regulating genes after exposure to HTI-286 or docetaxel, the gene array revealed distinct molecular signatures for both compounds.
Keywords: Drug comparison

overall_design:
LNCaP cells of the same passage were treated in 14 cm dishes for 72 h with 0.75 nM HTI-286 or 3 nM docetaxel and their corresponding vehicle controls (<0.0001% in solution). We chose a three day drug exposure because alterations of gene expression have been reported more evident after prolonged treatment. Total RNA of three biological replicates for 12 samples in total was isolated from cultured cell lines using the Trizol method (Invitrogen). The quality and quantity of RNA was assessed with an Agilent 2100 bioanalyzer (Agilent Technologies, Mississauga, ON, Canada).
Total RNA from each treatment sample (HTI-286 or docetaxel) was compared to its vehicle control sample on the same microarray. A dye swap for each pair was performed to account for dye bias. Microarrays of 21,000 (70-mer) human oligonucleotides representing 21,000 genes (Operon Technologies, Alameda, CA) printed in duplicate in 3xSSC onto aminosilane-coated slides (ERIE C28) were supplied by the Array Facility of The Prostate Centre at Vancouver General Hospital. Arrays were hybridized with 3DNA DNA Dendrimer Probes generated from 10 µg total RNA from LNCaP cells (n=3 for HTI-286 and vehicle control or n=3 for docetaxel and vehicle control) according to the manufacturer’s directions (Genisphere, Hatfield, PA). Briefly, reverse transcription incorporated a specific sequence present on the 5’ end of the RT primer supplied with the kit. The cDNA was hybridized to the array overnight at 42ºC. After stringent washes, the fluorescent 3DNA reagent which includes a ‘capture sequence’ complementary to the sequence at the 5’ end of the RT primer, was hybridized to the cDNA (47ºC for 2-3 h). Following further washes the arrays were immediately scanned on a ScanArrayExpress Microarray Scanner (PerkinElmer, Waltham, MA). Signal quality and quantity were assessed using Imagene 5.6 (BioDiscovery, San Diego, CA). Data from Imagene were analyzed in GeneSpring 6.1 (Silicon Genetics, Redwood City, CA) in order to profile changes in gene expression. Analyses performed in GeneSpring included background correction, LOWESS normalization and hierarchical clustering using standard correlation. Genes demonstrating normalized fold-changes >1.5 or <-1.5 in 3 of 3 experiments were then categorized by function using Ingenuity Pathways Analysis 5.0 (Ingenuity Systems, Redwood City, CA).

first: Boris

last: Hadaschik

first: Amina

last: Zoubeidi

first: Richard

middle: D

last: Sowery

first: Jenny

last: Baybik

first: Antonio

last: Hurtado-Coll

first: Michel

last: Roberge

first: Martin

middle: E

last: Gleave


title: Mouse Normal Variation

first: Denise

last: Mauldin

state: WA

summary:
 Cancer of the prostate is influenced both by genetic predisposition and environmental factors. The identification of genes capable of modulating cancer development has the potential to unravel disease heterogeneity and aid diagnostic and prevention strategies through improved understanding of gene-environment interactions. To this end, mouse models have been developed to isolate the influences of individual genetic lesions in the context of consistent genotypes and environmental exposures. However, the extent of normal prostatic phenotypic variability dictated by a genetic background potentially capable of influencing the process of carcinogenesis has not been established. In this study we used microarray analysis to quantitate transcript abundance levels in the prostates of five commonly studied inbred mouse strains. We applied a multiclass response t-test to identify genes whose expression in each strain significantly differed from the other four strains. Approximately 13% (932 genes) exhibited significant differential expression (range 1.3 to 190-fold) in one strain relative to other strains (FDR≤10%). The pattern of variability in transcript levels did not result from variations unique to a particular strain, but rather represented genetic variability across all five strains assessed. Expression differences were confirmed by qRT-PCR, or immunohistochemistry for several genes previously shown to influence cancer progression such as Psca, Mmp7, and Clusterin. Analyses of human prostate transcripts orthologous to variable murine prostate genes identified differences in gene expression in benign epithelium that correlated with the differentiation state of adjacent tumors. For example, the expression of apolipoprotein D, a gene known to enhance resistance to cell stress in Drosophila, was expressed at significantly greater levels in benign epithelium associated with high-grade versus low-grade cancers. These data support the concept that the cellular, tissue, and organismal context contribute to oncogenesis and suggest that a predisposition to a sequence of events leading to pathology may be determined prior to cancer initiation.
Keywords: mouse, prostate, transcript, variation, microarray, cancer, apolipoprotein D

overall_design: Prostates from twelve mice from each of five strains of Mus musculus: C57BL/6J, 129X1/SvJ, BALB/cAnCrl, FVB/NJ and DBA/2NCrl, were resected and individual lobes were dissected: DP: dorsal prostate; LP: lateral prostate; VP: ventral prostate; AP: anterior prostate. Each experimental sample represents a pool of equal amounts of RNA for each prostatic lobe from 3 animals. Four independent experimental samples were created per strain: 12 mice divided into 4 pools of 3 mice each for a total of 4 microarray experiments per strain. Amplified RNA from each experimental sample was hybridized against a reference sample (created by combining equal amounts of RNA from all the samples from all strains) onto custom mouse prostate cDNA microarrays using alternate dye-labeling to account for dye-specific effects.

first: Daniella

last: Bianchi-Frias

first: Colin

last: Pritchard

first: Brigham

middle: H

last: Mecham

first: Ilsa

last: Coleman

first: Peter

last: Nelson


title: Old versus Young Bone and Bone Marrow in Castrated Mice

summary:
Metastatic prostate cancer is commonly localized in bone and treated with androgen ablation, which is initially highly effective, but eventually results in resistant cancer cells. The decreased dependence on androgen is associated with activation of the androgen receptor (AR) via other growth factor receptor pathways. The current study analyzed
global gene expression in bone from castrated and sham operated  C57BL/6 mice to identify factors that may be responsible for androgen signaling. Androgen suppression by castration increased the expression of 159 genes with insulin-like growth factor binding protein 5 (IGFBP5) being the most consistently increased. IGFBP5 protein was detected by immune histochemistry in osteoblasts, bone marrow stromal cells and
endothelial cells. In vitro treatment of marrow stromal cells with charcoal-stripped serum increased IGFBP5 mRNA expression approximately 25-fold and was reversed by androgen supplementation. Endogenous IGFBP5 was incorporated into extracellular matrix. In addition, treatment of extracellular matrices with exogenous IGFBP5 and IGF-1 or IGF-2 enhanced the growth of immortalized human prostate cells. These results suggest that androgen ablative therapy may increase IGFBP5 in the marrow microenvironment, thereby providing support for the development of therapy resistance prostate cancer cells.
Keywords: Bone Marrow Environment, Androgen Suppression, IGFBP5, prostate cancer

overall_design: Femural, tibial and humeral bones were collected from paired castrated or sham-castrated C57BL/6 mice at 17 weeks (young) or 59 weeks (old).  Bone marrow was separated from bone for the young mice and arrayed separately.  Each timepoint includes three replicates (mouse pairs) and cy dye labeling is alternated between experiment and control to account for dye effect.

first: Chang

last: Xu

first: Lynn

middle: f

last: Graf

first: Ladan

last: Fazli

middle: M

first: Stephen

last: Plymate

first: Michael

last: Cox

first: Beverly

middle: J

last: Torok-Storb

first: Beatrice

last: Knudsen


title: S. coelicolor YSK4425 Time-course Study in R5- medium (Culture#2)

status: Public on Jun 23 2007

city: Minneapolis

first: Salim

last: Charaniya

state: MN

address: 421 Washington Ave SE., 151 Amundson Hall

department: Chemical Engineering and Materials Science

institute: University of Minnesota

summary:
Antibiotic biosynthesis in Streptomyces species is controlled by a complex genetic and biochemical network of global and pathway specific regulators.  Details of their precise interactions in mediating temporal and spatial expression of secondary metabolite genes remain poorly defined.  In this study, we employed whole-genome microarrays to investigate the temporal transcriptome profiles of S. coelicolor A3(2) afsS::apr mutant strain (YSK4425) and compare it to wild-type M145 strain. The regulatory protein encoded by afsS is known to affect antibiotic biosynthesis (Floriano, B., Bibb, M. 1996. afsR is a pleiotropic but conditionally required regulatory gene for antibiotic production in Streptomyces coelicolor A3(2). Mol Microbiol, 21, 385-96).
Keywords: Time course

overall_design: The time-course study involved analysis of 14 samples (15h, 17h, 19h, 21h, 23h, 25h, 28h, 31h, 32h, 35h, 37h, 38h, 41h, 45h). Of these, 15h, 17h, 19h, 21h, 23h, 25h, 32h, 41h, and 45h samples were analyzed on duplicate arrays (2 technical replicates). Genomic DNA of S. coelicolor wild type (M145) was used as a reference for all the arrays. cDNA was labeled with Alexa647 and genomic DNA was labeled with Cy3.

first: Wei

last: Lian

first: Karthik

last: Jayapal

first: Wei-Shou

last: Hu


title: Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells

status: Public on May 23 2007

city: Baltimore

first: Minoru

last: Ko

state: MD

address: 251 Bayview Blvd, Suite 100, 10C

department: National Institute on Aging

institute: NIH

summary:
To characterize the differentiation by Sox2 KO, we performed microarray analyses of mouse ES cell line 2TS22C during the time-course being induced of Sox2 KO
Keywords: development or differentiation design,time series design

overall_design: Inducible Sox2 KO cell line 2TS22C were assigned to eight experimental groups and cultured in GMEM medium (SIGMA) with LIF at 37°C in an atmosphere of 5% CO2 until the specified time point before the harvest: (1)for controls, cultured in the absence of Tc; (2) for 3h after the administration of Tc; (3) for 6h; (4) for 12h; (5) for 24h; (6) for 48h; (7) for 72h; (8) for 96h.

first: Shinji

last: Masui

first: Daisuke

last: Shimosato

first: Yayoi

last: Toyooka

first: Yuhki

last: Nakatake

first: Rika

last: Yagi

first: Kazue

last: Takahashi

first: Akihiko

last: Okuda

first: Ryo

last: Matoba

first: Alexei

last: Sharov

first: Hitoshi

last: Niwa


title: Effect of acute creatine monohydrate supplementation on skeletal muscle transcriptome

city: Hamilton

first: Adeel

last: Safdar

state: ON

address: 1200 Main Street West

department: Kinesiology

institute: McMaster University

summary:
Expression profiling of vastus lateralis muscle of young, healthy, non-obese men supplemented with creatine monohydrate vs. placebo for 10 days. Results identify transcriptional pathways activated in skeletal muscles as a result of acute creatine monohydrate supplementation.
Keywords: Supplementation response.

overall_design: In a randomized, placebo-controlled, crossover, double-blind design, 12 young, healthy, non-obese men were supplemented with either a placebo or creatine monohydrate (loading phase, 20 g/d x 3 d; maintenance phase, 5 g/d x 7 d) for 10 d.  Following a 28 day washout period, subjects were put on the alternate supplementation for 10 days.  Muscle biopsies of the vastus lateralis were obtained and were assessed for global mRNA expression using cDNA microarrays.

first: A

first: N

last: Yardley

first: R

last: Snow

first: S

last: Melov

first: M

last: Tarnopolsky


title: Expression data for hindlimb and tail samples in a metamorphosis assay for thyroid axis disruption - inhibitors

status: Public on May 12 2007

city: Victoria

first: Caren

last: Helbing

state: British Columbia

address: PO Box 3055 Stn CSC

department: Biochemistry and Microbiology

institute: University of Victoria

summary:
Thyroid hormones (TH), thyroxine (T4) and 3, 5, 3’-triiodothyronine (T3), play crucial roles in regulation of growth, development and metabolism in vertebrates and are targets for endocrine disruptive agents. Perturbations in TH action can contribute to the development of disease states and the U.S. Environmental Protection Agency is developing a high throughput screen using TH-dependent metamorphosis of the Xenopus laevis tadpole as an assay platform. Currently this methodology relies on external morphological endpoints and changes in central thyroid axis parameters. However, exposure-related changes in gene expression in TH-sensitive tissue types that occur over shorter time frames have the potential to augment this screen. Using a combination of cDNA array and real time quantitative polymerase chain reaction (QPCR) analyses, this study identifies molecular markers in tissues peripheral to the central thyroid axis. We examine the hindlimb and tail of tadpoles up to 96 hours of continuous exposure to T3, T4, methimazole, propylthiouracil, or perchlorate. Several novel biomarker candidates are indicated that include transcripts encoding importin, RNA helicase II/Gu, and defender against death protein, DAD1. In combination with previously-identified biomarker candidates, these transcripts will greatly augment the predictive and diagnostic power of the Xenopus metamorphosis assay for perturbation of TH action.
Keywords: time course

platform_organism:
Xenopus laevis
Aquarana catesbeiana

overall_design:
Twenty-eight early prometamorphic (NF stage 54;(Nieuwkoop and Faber, 1956; Shi, 2000)) tadpoles were continuously exposed to three separate T4 concentrations (10, 20.1, and 40.3 nM), and a LSW control in one exposure set, or five different T3 concentrations (0.48, 0.97, 1.92, 3.84, and 7.68 nM), and a LSW control in the second exposure set as described in detail in Zhang et al (2006). Each chemical exposure concentration was replicated twice along with the associated LSW control. At 24 h, 48 h and 96 h two animals per exposure replicate (four animals total per each individual treatment) were randomly selected, euthanized in MS-222, and preserved in RNAlater (Ambion Inc., Austin, Texas, USA) for analysis of gene expression. On exposure day 14, all remaining organisms were euthanized in MS-222, weighed, and developmentally staged in a blind evaluation. Animals exposed to either chemical showed an acceleration of metamorphosis which was published previously (Zhang et al., 2006).

NF stage 54 tadpoles were continuously exposed to a single concentration of PTU (20 mg/L), MMI (100 mg/L) or PER (4 mg/L).  We have previously shown that exposure to these concentrations resulted in an increase in thyroid gland size at day 8 and significantly delayed metamorphosis at 14 days (Degitz et al., 2005; Tietge et al., 2005). The exposure regimen details are recorded elsewhere (Zhang et al., 2006). Briefly, tadpoles were randomly placed into 24 tanks (20 tadpoles/tank) and exposed (six tanks/chemical) to a single concentration of each chemical. At 24 h, 48 h and 96 h 5 tadpoles from two of the 6 tanks (10 tadpoles per each individual treatment) were randomly selected, euthanized in MS-222, and preserved in RNAlater (Ambion Inc., Austin, Texas, USA) for analysis of gene expression.


title: Expression data for brain samples in a metamorphosis assay for thyroid axis disruption - inhibitors

summary:
Thyroid hormones (TH), thyroxine (T4) and 3, 5, 3’-triiodothyronine (T3), play crucial roles in regulation of growth, development and metabolism in vertebrates and their action are targets for endocrine disruptive agents. Perturbations in TH action can contribute to the development of disease states and the U.S. Environmental Protection Agency is developing a high throughput screen using TH-dependent amphibian metamorphosis as an assay platform. Currently this methodology relies on external morphological endpoints and changes in central thyroid axis parameters. However, exposure-related changes in gene expression in TH-sensitive tissue types that occur over shorter time frames have the potential to augment this screen. This study aims to characterize and identify molecular markers in the tadpole brain. Using a combination of cDNA array analysis and real time quantitative polymerase chain reaction (QPCR), we examine the brain of tadpoles following 96 hours of continuous exposure to T3, T4, methimazole, propylthiouracil, or perchlorate. This tissue was more sensitive to T4 rather than T3, even when differences in biological activity were taken into account. This implies that a simple conversion of T4 to T3 cannot fully account for T4 effects on the brain and suggests distinctive mechanisms of action for the two THs. While the brain shows gene expression alterations for methimazole and propylthiouracil, the environmental contaminant, perchlorate, had the greatest effect on the levels of mRNAs encoding proteins important in neural development and function. Our data identify gene expression profiles that can serve as exposure indicators of these chemicals.
Keywords: time course

overall_design: NF stage 54 tadpoles were continuously exposed to a single concentration of PTU (20 mg/L), MMI (100 mg/L) or PER (4 mg/L).  We have previously shown that exposure to these concentrations resulted in an increase in thyroid gland size at day 8 and significantly delayed metamorphosis at 14 days (Degitz et al., 2005; Tietge et al., 2005). The exposure regimen details are recorded elsewhere (Zhang et al., 2006). Briefly, tadpoles were randomly placed into 24 tanks (20 tadpoles/tank) and exposed (six tanks/chemical) to a single concentration of each chemical. At 24 h, 48 h and 96 h 5 tadpoles from two of the 6 tanks (10 tadpoles per each individual treatment) were randomly selected, euthanized in MS-222, and preserved in RNAlater (Ambion Inc., Austin, Texas, USA) for analysis of gene expression.


title: Chromosome 8 BAC Array CGH and Expression Studies Identify Amplification and Overexpression of TRMT12 in Breast Cancer

status: Public on May 07 2007

city: Bethesda

first: Christopher

last: Pan

address: Rm 5228, Bldg 50, South Dr.

department: NHGRI

type: Genome variation profiling by genome tiling array

summary:
Genomic changes in chromosome 8 are commonly observed in breast cancer cell lines and tumors.  Fine mapping of such genomic changes, and evaluation of associated expression changes, are expected to provide reagents for diagnosis, insights into understanding the disease, and open up avenues for novel therapeutic intervention.  We made an effort to search for genes on chromosome 8 with altered copy number and expression.  A high resolution (0.1Mb) bacterial artificial chromosome (BAC) array of chromosome 8 that can detect single copy changes was developed using Phi29 DNA polymerase amplified BAC DNA.  Hybridization of DNA from a breast cancer cell line (SKBR3) with two known amplified regions (8q21 and 8q24) resulted in resolving this region into 6 distinct amplicons, in addition identified 3 deleted regions.  The boundaries and the peak of each region were verified by FISH, and the extent of amplification/deletion for each region was validated by qPCR.  Using these BAC arrays, CGH was performed with a total of eight breast cancer cell lines, and common regions of genomic amplification/deletion were identified by segmentation analysis.  Two consensus regions in 8q24 included a 1.2 Mb region (125.3-126.5 Mb) and a 1.0 Mb region (128.1-129.1 Mb) that were amplified in 7/8 cell lines, and in the 8q21 region there was a smaller 0.88 Mb region (86.62-87.50 Mb) amplified in 4/8 cell lines.  A global expression analysis was performed for all these cell lines using a high-density oligonucleotide array to identify the genes whose expression correlated with amplification/deletion.  Representative genes from 5 commonly amplified regions (REXO1L1, TMEM55A, TRMT12, MTSS1, EIF2C2, MYC) and 2 deleted regions (DPYSL2, NRG1 and NDRG1) were verified by qPCR and RT-qPCR for genomic and expression changes.  Validation by RT-qPCR using RNA from 30 breast tumors showed that the TRMT12 (125.5 Mb) gene were overexpressed  in many of the tumors.  TRMT12 is  homolog of a yeast gene encoding tRNA methyltransferase involved in biosythesis of a modified base in a tRNA.  It would be interesting to explore the role of TRMT12 and the RNA processing pathway in tumorigenesis.
Keywords: Comparative Genomic Hybridization (CGH), Breast cancer cell lines, Expression, BAC array, Oligo expression array

overall_design: A high-density BAC arrays for chromosome 8 was generated by printing on glass slides BAC DNAs amplified using Phi29 DNA polymerases. These arrays were used to evaluate copy number changes in chromosome 8 for 8 breast cancer cell lines. Segmentation analysis identified common regions of copy number gain/loss. RNA from these 8 cell lines was analyzed for global expression changes using an oligonucleotide (HEEBO) expression array. The consistent expression changes in genes for chromosome were identified, and a fraction of these genes validated by RT-qPCR. Overexpression in tumor RNA was validated for two genes that showed genomic amplification.

first: Virginia

middle: W

last: Rodriguez

first: Yidong

last: Chen

first: Abdel

last: Elkahloun

first: Settara

last: Chandrasekharappa


title: Brain samples in a metamorphosis assay for thyroid axis disruption

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

overall_design: Refer to individual Series


title: Gene expression profiling classifies lung squamous cell carcinomas with N1 positive lymph nodes

status: Public on May 01 2007

city: Brisbane

first: Jill

middle: Everland

last: Larsen

state: Queensland

address: Rode Road, Chermside

department: Thoracic Medicine

country: Australia

institute: The Prince Charles Hospital

summary:
Using gene expression microarrays, we tested whether lung SCCs that have metastasised to loco-regional lymph nodes (N1/2m) are different to those that directly invade and involve local nodes (N1d). Using 22,323 element microarrays, a non-parametric test identified 126 genes/transcripts that discriminated between N0 (n=35) and N1/2m (n=16) tumours (Wilcoxon-Mann-Whitney U test, P<0.01). Hiearchical clustering of all 59 tumours (including 8 N1d tumours) demonstrated the N1d tumours clustered with the N0 tumours rather than the N1/2m tumours. Next, we built class prediction models from the 35 N0 tumours and 16 N1/2m tumours to predict the class of N1d tumours. All models consistently classified N1d tumours as similar to N0 tumours. This could explain some of the variable response of some N1 tumours to adjuvant chemotherapy, and suggest refinement of the TNM "N" stage nomenclature to adjust for this biological observation to ensure appropriate patients benefit from treatment.
Keywords: non-small cell lung carcinoma, squamous cell, nodal metastasis, TNM staging, expression profiling

overall_design: Expression profiling using 22K element microarrays of 59 primary lung squamous cell carcinomas.

first: Sandra

last: Pavey

first: Belinda

last: Clarke

first: Maree

last: Colosimo

first: Rayleen

middle: V

last: Bowman

first: Ian

last: Yang

first: Nicholas

middle: K

last: Hayward

first: Kwun

last: Fong


title: Nm23-H1 suppresses tumor cell motility by downregulating the LPA receptor

status: Public on Apr 30 2007

summary:
Exogenous overexpression of the metastasis suppressor gene Nm23-H1 reduces the metastatic potential of multiple types of cancer cells. In addition, Nm23-H1 expression suppresses in vitro tumor cell motility and invasion, important correlates of metastasis. The molecular mechanism by which Nm23-H1 suppresses metastasis, motility and invasion has yet to be defined. However, mutational analysis of Nm23-H1 has revealed that substitution mutant P96S and S120G are unable to inhibit cell motility. To elucidate the molecular mechanism of Nm23-H1 motility suppression, we performed expression microarray analysis of an MDA-MB- 435 carcinoma cell line overexpressing wild-type Nm23-H1 and cross-compared the expression profile to lines expressing P96S and S120G mutants. Nine genes, MET, PTN, SMO, FZD1, L1CAM, MMP2, NETO2, CTGF and EDG2, were down regulated by wild-type but not by mutant Nm23-H1 expression (p<0.05). Reduced expression of these genes coincident with elevated Nm23-H1 expression was observed in human breast tumor cohorts, a panel of breast carcinoma cell lines and hepatocellular carcinomas from control and Nm23-M1 knockout mice.
The functional significance of down regulated genes was assessed by transfection and in vitro motility assays. Only EDG2 overexpression significantly restored motility to Nm23-H1- suppressed cancer cells and it enhanced motility by 60-fold in these cells. In addition, silencing EDG2 expression with siRNA, reduced the motile phenotype of metastatic breast cancer cells. These data suggest that Nm23-H1 suppresses metastasis, at least in part, through downregulation of EDG2 expression and that compounds directed toward EDG2 may be useful for treating aggressive, low Nm23-H1-expressing breast tumors.
Keywords: Human MDA-MB-435 Breast cancer derived cell lines

overall_design:
Total RNA was extracted from confluent MDA-MB-
435-carcinoma cell lines H1-177 overexpressing wild type Nm23-H1, S-22 (P96S mutant) and I-205 (S120G mutant).  The RNA was purified using the Trizol reagent from Invitrogen, Inc., following the manufacturer’s suggested protocol. Triplicate sample hybridizations were done for comparing H1-177 to C-100 (including one dye swap), Four replicates hybridizations were done for comparing H1-177 to S-22 (including two dye swap) and four replicates hybridizations were done for comparing H1-177 to I-205 (including two dye swap). The raw data for the dye swap experiment was reversed so that the data match the other two replicates.

first: Christine

last: Horak

first: Patricia

middle: S

last: Steeg


title: TGFβ Signaling in Prostate Stroma Supports Carcinoma Growth by Upregulating Stromal Genes Related to Tissue Remodeling

status: Public on Apr 28 2007

summary:
Increasing evidence points to an active stromal involvement in cancer initiation and progression. Cytokines derived from tumor cells are believed to modulate stromal cells to produce growth and angiogenic factors which in turn provide the tumor with the necessary microenvironment for expansion and invasion. Transforming growth factor beta (TGFβ) has been implicated as a candidate cytokine to mediate this communication. However, how its signaling in stromal cells regulates tumorigenesis and tumor progression remains unresolved. We show that normal, pre-senescent fibroblasts or prostate stromal cells cotransplanted with prostate carcinoma cells subcutaneously into nude mice reduced tumor latency and accelerated tumor growth. When their TGFβ signaling was blocked, the fibroblasts and stromal cells still stimulated tumor initiation, but no longer supported tumor growth as control cells did. The loss of the tumor growth-promoting activity of the stromal cells with attenuated TGFβ signaling was not associated with altered cellular senescence or tumor angiogenicity. TGFβ and the medium conditioned by the prostate carcinoma cells stimulated myofibroblast differentiation of the intact stromal cells, but not the stromal cells with attenuated TGFβ signaling. Gene microarray and quantitative RTPCR analyses showed that TGFβ upregulated a host of genes in stromal cells that are involved in tissue remodeling and wound healing. Thus, our study provides evidence for TGFβ as a supporting agent in tumor progression through the induction of a perpetual wound healing process in the tumor microenvironment.
Keywords: Human prostate stromal cells (PrSC)

overall_design: Total RNA was extracted from confluent PrSC cells and DNRII transfected PrSC cells with and without 24-hour treatment with 5.0 ng/mL TGFb3.  The RNA was purified using a RiboPure™Kit from Ambion, Inc., following the manufacturer’s suggested protocol. Triplicate sample hybridizations were done for each comparison. TGFbeta treated PrSC cell versus non treated PrSC cells and TGFbeta treated DNRII PrSC cell versus non treated PrSC cells. For each comparison, one dye swap experiment was performed. The raw data for the dye swap experiment was reversed so that the data match the other two replicates.

first: Erik

last: Verona

first: Lu-Zhe

last: Sun


title: Expression data for brain samples in a metamorphosis assay for thyroid axis disruption

first: Caren

last: Helbing

summary:
Thyroid hormones (TH), thyroxine (T4) and 3, 5, 3’-triiodothyronine (T3), play crucial roles in regulation of growth, development and metabolism in vertebrates and their action are targets for endocrine disruptive agents. Perturbations in TH action can contribute to the development of disease states and the U.S. Environmental Protection Agency is developing a high throughput screen using TH-dependent amphibian metamorphosis as an assay platform. Currently this methodology relies on external morphological endpoints and changes in central thyroid axis parameters. However, exposure-related changes in gene expression in TH-sensitive tissue types that occur over shorter time frames have the potential to augment this screen. This study aims to characterize and identify molecular markers in the tadpole brain. Using a combination of cDNA array analysis and real time quantitative polymerase chain reaction (QPCR), we examine the brain of tadpoles following 96 hours of continuous exposure to T3, T4, methimazole, propylthiouracil, or perchlorate. This tissue was more sensitive to T4 rather than T3, even when differences in biological activity were taken into account. This implies that a simple conversion of T4 to T3 cannot fully account for T4 effects on the brain and suggests distinctive mechanisms of action for the two THs. While the brain shows gene expression alterations for methimazole and propylthiouracil, the environmental contaminant, perchlorate, had the greatest effect on the levels of mRNAs encoding proteins important in neural development and function. Our data identify gene expression profiles that can serve as exposure indicators of these chemicals.
Keywords: dose response

overall_design: Twenty-eight early prometamorphic (NF stage 54;(Nieuwkoop and Faber, 1956; Shi, 2000)) tadpoles were continuously exposed to three separate T4 concentrations (10, 20.1, and 40.3 nM), and a LSW control in one exposure set, or five different T3 concentrations (0.48, 0.97, 1.92, 3.84, and 7.68 nM), and a LSW control in the second exposure set as described in detail in Zhang et al (2006). Each chemical exposure concentration was replicated twice along with the associated LSW control. At 24 h, 48 h and 96 h two animals per exposure replicate (four animals total per each individual treatment) were randomly selected, euthanized in MS-222, and preserved in RNAlater (Ambion Inc., Austin, Texas, USA) for analysis of gene expression.  On exposure day 14, all remaining organisms were euthanized in MS-222, weighed, and developmentally staged in a blind evaluation. Animals exposed to either chemical showed an acceleration of metamorphosis which was published previously (Zhang et al., 2006).


title: Genomic analysis of COP9 signalosome function in Drosophila melanogaster

status: Public on Apr 12 2007

city: Tel Aviv

first: Danny

last: Chamovitz

address: Ramat aviv

country: Israel

institute: Tel Aviv University

summary:
The COP9 signalosome (CSN), an eight-subunit protein complex, is conserved in all higher eukaryotes. CSN intersects the ubiquitin-proteasome pathway, modulating signaling pathways controlling various aspects of development. We are using Drosophila as a model system to elucidate the function of this important complex. Transcriptome data was generated for four csn mutants, sampled at three developmental time points. Our results are highly reproducible, being confirmed using two different experimental setups that entail different microarrays and different controls. Our results indicate that the CSN acts as a transcriptional repressor during Drosophila development, resulting in achronic gene expression in the csn mutants. "Time shift" analysis with the publicly-available Drosophila transcriptome data indicates that genes repressed by the CSN are normally induced primarily during late embyogenesis, or during metamorphosis. These temporal shifts are likely due to the roles of the CSN in regulating transcription factors. A null mutation in CSN subunit 4, and hypomorphic mutations in csn5 lead to more severe defects than seen in the csn5null mutants strain, suggesting that CSN5 carries only some of the CSN function.
Keywords: time course csn mutants

overall_design: four mutants (csn4null, csn5null, csn51, csn53) in two CSN subunits (CSN4 and CSN5) sampled at three time points of larval development 60, 72, and 96 hours after egg deposition (AED)Experiments were conducted such that mRNA populations isolated from mutants were compared with mRNA populations from age-matched WT larvae i.e. each array was hybridized with mRNA extractions from age matched mutant and WT larvae. Four biological repeats which included dye reversals were conducted for each experiment


title: Global gene expression profiling reveals similarities and differences among mouse pluripotent stem cells

status: Public on May 11 2007

first: Minoru

last: Ko

summary:
We present a compendium DNA microarray analysis of multiple mouse ESCs and EGCs from different genetic backgrounds (strains 129 and C57BL/6) cultured under standard conditions and in differentiation-promoting conditions by the withdrawal of Leukemia Inhibitory Factor (LIF) or treatment with retinoic acid (RA). All pluripotent cell lines showed similar gene expression patterns, which separated them clearly from other tissue stem cells with lower developmental potency. Differences between pluripotent lines derived from different sources (ESC vs. EGC) were smaller than differences between lines derived from different mouse strains (129 vs. C57BL/6).
Keywords: cell type comparison design

overall_design: Multiple ESC and EGC cell lines were cultured without feeders on gelatin coated, 6-well plates, 100,000 cells/well (10,000 cells/cm2) in 3 conditions: 1) in complete ES medium: DMEM, 15% FBS; LIF (ESGRO) 1000 u/ml; 1mM sodium pyruvate; 0.1 mM NEAA, 2 mM glutamate, 0.1 mM beta-mercapto ethanol, and penicillin/streptomycin (50U/50ug per ml); 2) in ES medium without LIF; 3) in complete ES medium plus 1 uM RA. Cells were incubated at 37 0C; 5% CO2. Medium was changed daily. On the day 3 Trizol was added, RNA extracted using Phase lock gelTM (Eppendorf) columns according to manufacturer protocol. RNA was precipitated with isopropanol, washed with 70% ethanol and dissolved in DEPC dH2O.

first: Lioudmila

last: Sharova

first: Alexei

last: Sharov

first: Yulan

last: Piao

first: Nabeebi

last: Shaik

first: Terry

last: Sullivan

first: Colin

last: Stewart

first: Brigid

last: Hogan


title: Microarray analysis in the zebrafish liver and telencephalon after exposure to low concentration of 17a-EE2

status: Public on Mar 31 2007

city: Ottawa

first: Emily

middle: R

last: Gerrie

address: 20 Marie Curie

department: Biology

institute: University of Ottawa

summary:
Experiment exposed adult male zebrafish to 10ng/L 17-alpha ethinylestradiol for three weeks.
Gene expression profiling of liver and telencephalon following exposure.
Keywords: estrogenic exposure

overall_design:
4 arrays per group:
Liver = slides 1, 2, 3, 4 (slides 3 and 4 are dye swaps)

Telencephalon = slides 5, 6, 7, 8 (slides 7 and 8 are dye swaps)

first: Christopher

last: Martyniuk

first: Jason

middle: T

last: Popesku

first: Marc

last: Ekker

first: Vance

last: Trudeau


title: Transcriptional response of S.cerevisiae to aeration after anaerobic growth

status: Public on Mar 06 2007

city: Randwick

first: Anthony

middle: G.

last: Beckhouse

state: NSW

address: High St

department: BABS

institute: Ramaciotti Centre for Gene Function Analysis

summary:
To determine the transcriptional changes that occur when yeast is shifted from anaerobic growth to an aerobic environment over a period of 120 minutes.
Keywords: time course, stress response, environmental response, aerobic, anaerobic

overall_design: This experiment consists of 11 arrays; however, the normalization strategy involves condensing the dye-swap technical replicate data for each time-point (except for the 0min vs 0min control) into a single Sample, for a total of 6 Sample records. Biological replicates are considered to be included as the time-point samples originated from separate flasks and were compared to a pooled sample of RNA from the anaerobic samples (0min).

middle: G

first: Chris

last: Grant

first: Peter

last: Rogers

first: Ian

last: Dawes

first: Vincent

last: Higgins


title: Piperonyl butoxide induces the expression of P450 and GST genes in Drosophila melanogaster

status: Public on Mar 18 2007

city: Parkville

first: Lee

last: Willoughby

state: Victoria

address: 30 Flemington Road

department: Genetics

institute: University of Melbourne

summary:
Piperonyl butoxide (PBO) is an insecticide synergist known to inhibit the activity of cytochrome P450 enzymes.  PBO is currently used in many insecticide formulations, and has also been suggested as a pre-treatment in some pesticide applications. Little is known about how insects respond to PBO exposure at the gene transcription level. We have characterised the transcriptional response of the Drosophila melanogaster genome after PBO treatment, using both a custom designed “detox” microarray containing cytochrome P450 (P450), glutathione S-transferase (GST) and esterase genes, and a full genome microarray. We identify a subset of P450 and GST genes, along with additional metabolic genes, that are highly induced by PBO.  The gene set is an extremely similar gene set to that induced by phenobarbital, a compound for which pre-treatment is known to confer tolerance to a range of insecticide compounds.  The implications of the induction of gene families known to metabolise insecticides and the use of PBO in pest management programs are discussed.
Keywords: Induction response after treatment by PBO

overall_design:
Drosophila melanogaster adult males were exposed to PBO. 8 replicates of 50 individuals were exposed to PBO and an additional 8 replicates of 50 individuals were exposed to the appropriate control (that is the same exposure conditions, without the addition of the compound).
For each compound, RNA was extracted from all of the 8 samples and then pooled, before labelling. For each experiment exposed/unexposed labelling dye swaps were used to account for potential differences in labelling efficiency.

first: Philip

last: Batterham

first: Phillip

last: Daborn


title: Gene expression signatures that predict radiation exposure (mouse)

status: Public on Mar 01 2007

city: Durham

first: Holly

middle: Kloos

last: Dressman

state: NC

address: 101 Science Drive

department: Institute for Genome Sciences and Policy

institute: Duke University Medical Center

summary:
Gene expression analysis of peripheral blood leukocytes (PB MNCs) to develop expression profiles that accurately reflect prior radiation exposure.
Keywords: Comparative, exposure dosage, C57BI6 Murine Irradiation Studies

overall_design:
We have made use of gene expression analysis of peripheral blood mononuclear cells (PB MNCs) to develop expression profiles that accurately reflect prior radiation exposure. Importantly, we demonstrate that expression profiles can be developed that not only predict radiation exposure in mice but also distinguish the level of radiation exposure, ranging from 50 cGy to 1000 cGy. Likewise, a molecular signature of radiation response developed solely from irradiated human patient samples can predict and distinguish irradiated human PB samples from non-irradiated samples with an accuracy of 90%, sensitivity of 85% and specificity of 94%. We further demonstrate that a radiation profile developed in the mouse can correctly distinguish PB samples from irradiated and non-irradiated human patients with an accuracy of 77%, sensitivity of 82% and specificity of 75%. Taken together, these data demonstrate that molecular profiles can be generated which are highly predictive of different levels of radiation exposure in mice and humans.
Mouse Dataset only

first: Garrett

last: Muramoto

first: Nelson

last: Chao

first: Sarah

last: Meadows

first: Dawn

last: Marshall

first: Geoffrey

last: Ginsburg

first: Joseph

last: Nevins

first: John

middle: P

last: Chute


title: Gene expression signatures that predict radiation exposure

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: Evaluating the effects of activating mutations of human LH receptor in MA-10 cell

status: Public on Mar 08 2007

first: TIN-LAP

last: LEE

address: 2C08 Building 49

department: NICHD

institute: National Institutes of Health

summary:
To study the potential tumorigenic effect of two activating mutations of hLHR identified in patients, we generated in vitro cell model using mouse Leydig MA-10 cells. The two mutations, Asp578His and Asp578Gly, were stably transfected to MA-10 cell to create MA-10-Asp578His and MA-10-Asp578Gly line.
The profile of expressed genes in cells expressing the mutated hLHR was compared with that of control cells using cDNA microarrays with NIA mouse clone sets.
Keywords: Genetic modification analysis

overall_design: Both stably transfected cells were compared to empty vector control. MA-10 cell was used as reference for all the samples. Indirect labelling of Cy3 and Cy5 was applied to subject and control. Each sample contains three biological replicates.

first: Micheal

last: Leung

first: Owen

last: Rennert

first: Wai-Yee

last: Chan


title: Analysis of the transcriptional response of Candida parapsilosis following exposure to farnesol

city: Dublin

first: Geraldine

last: Butler

address: Belfield

department: School of Biomolecular and Biomedical Science

country: Ireland

institute: University Colege Dublin Conway Institute

summary:
 We used the results of a genome sequence survey to design and manufacture partial genomic microarrays that were applied to determining the transcriptional response of C. parapsilosis to the presence of exogenous farnesol.
Keywords: drug response, farnesol,

overall_design:
Transcriptional profiling was performed following exposure to 50 microM
 of farnesol compare to the control experiment (no treatment). Six independent experiment comparisons (including 3 dye swaps) were performed.

first: Tristan

last: Rossignol

first: Mary

last: Logue


title: Genes involved in doxorubicin drug resistance in 143b osteosarcoma cell line

city: Chennai

first: Rajkumar

last: T

state: Tamilnadu

address: Adyar

department: Molecular Oncology

country: India

institute: Cancer Institute (WIA)

summary:
The study was done to identify the differentially expressed genes in response to Doxorubicin drug resistance in the 143B human osteosarcoma cell line, using the Microarray technology.

Keywords: drug resistance; dual channel; cell line based; doxorubicin resistance; Human osteosarcoma cell line

overall_design: A two-dye experiment model, using the wild type 143B osteosarcoma cell line (Control) and the 143B Doxorubicin drug resistant cell line (Test), was done. Using the GPL2869 Platform (70-mer oligo based array), 4 experiments were done, two were regular (GSM76017 and GSM76020) (Wild type = Cy3; drug resistant = Cy5) and two were dye swap (GSM76018 and GSM76019).

first: Thangarajan

last: Rajkumar

first: Manoharan

last: Yamuna

first: Nirmala

last: Nancy


title: Differential gene expression in mice liver following primaquine administration

status: Public on Feb 27 2007

city: Lucknow

first: Sanjeev

last: Noel

state: Uttar Pradesh

address: M G Marg

department: Toxicology

institute: Central Drug Research Institute

summary:
Global gene expression in livers of primaquine treated mice was compared with control mice at different time points.
Keywords: Time course

overall_design: Mice (Mus musculus) belonging to control and primaquine-administered groups (n=4) were sacrificed. The livers were snap frozen in liquid nitrogen, and subsequently stored at -80ºC till further use. Liver samples were homogenized and total RNA was extracted with TRI reagent. 25 ug of total RNA was converted into labeled cDNA using CyScribe first strand cDNA labeling kit. The Cy5 and Cy3 labeled cDNAs were resuspended in Cyscribe Hyb buffer containing 10ug/ml sheared Salmon sperm DNA and 10ug/ml Yeast tRNA. The labeled samples were hybridized to the arrays and incubated for 16-18hrs at 42ºC. Fluorescent array images were collected for both Cy3 and Cy5 with molecular dynamics III scanner supported with ImageQuant v5.0. Image intensity data were extracted and analyzed with ArrayVision v8.0 analysis software. Background corrected data was LOWESS normalized and log ratios were calculated using Avadis v4. Further, mean log ratios were calculated for duplicate spots. Replicate experiments were carried out in dye swap manner for each time point.

first: Srikanta

last: Rath


title: Carbon tetrachloride and Acetaminophen hepatotoxicity study

summary:
Differential gene expression in mice liver after carbon tetrachloride and acetaminophen administration. Livers from control mice were compared with drug treated mice livers at different time points.
Keywords: Time-course

overall_design: Mice (Mus musculus) belonging to control and carbon tetrachloride and acetaminophen-administered groups (n=4) were sacrificed. The livers were snap frozen in liquid nitrogen, and subsequently stored at -80ºC till further use. Liver samples were homogenized and total RNA was extracted with TRI reagent. 25 ug of total RNA was converted into labeled cDNA using CyScribe first strand cDNA labeling kit. The Cy5 and Cy3 labeled cDNAs were resuspended in Cyscribe Hyb buffer containing 10ug/ml sheared Salmon sperm DNA and 10ug/ml Yeast tRNA. The labeled samples were hybridized to the arrays and incubated for 16-18hrs at 42ºC. Fluorescent array images were collected for both Cy3 and Cy5 with molecular dynamics III scanner supported with ImageQuant v5.0. Image intensity data were extracted and analyzed with ArrayVision v8.0 analysis software. Background corrected data was LOWESS normalized and log ratios were calculated using Avadis v4. Further, mean log ratios were calculated for duplicate spots. Replicate experiments were carried out in dye swap manner for each time point.


title: Rosiglitazone Signature in Mouse White Adipose Tissue

status: Public on Feb 17 2007

city: Seattle

first: eric

middle: e

last: schadt

address: 401 Terry Ave N

institute: Merck

summary:
The purpose of this experiment was to determine the murine white adipose expression traits that were changed in response to treatment with Rosiglitazone.
Keywords: drug treatment response signature

overall_design: White adipose tissue from three Rosiglitazone treated KO mice were arrayed against a pool of white adipose tissue from vehicle treated wildtype B6 animals.  All samples were hybridized as fluor-reverse pairs, and then the data between the pairs was combined to form a single experiment.  Three repeats were performed to increase the power to detect expression changes and to reduce effects of experimental noise.

first: Eric

last: Schadt

first: Pek

middle: Y

last: Lum


title: Differential gene expression in mice liver following bulaquine administration

summary:
Global gene expression in livers of bulaquine treated mice was compared with control mice at different time points.
Keywords: Time course

overall_design: Mice (Mus musculus) belonging to control and bulaquine-administered groups (n=4) were sacrificed. The livers were snap frozen in liquid nitrogen, and subsequently stored at -80ºC till further use. Liver samples were homogenized and total RNA was extracted with TRI reagent. 25 ug of total RNA was converted into labeled cDNA using CyScribe first strand cDNA labeling kit. The Cy5 and Cy3 labeled cDNAs were resuspended in Cyscribe Hyb buffer containing 10ug/ml sheared Salmon sperm DNA and 10ug/ml Yeast tRNA. The labeled samples were hybridized to the arrays and incubated for 16-18hrs at 42ºC. Fluorescent array images were collected for both Cy3 and Cy5 with molecular dynamics III scanner supported with ImageQuant v5.0. Image intensity data were extracted and analyzed with ArrayVision v8.0 analysis software. Background corrected data was LOWESS normalized and log ratios were calculated using Avadis v4. Replicate experiments were carried out in dye swap manner for each time point.


title: Microarray based Gene expression profiling of Cervical Cancer in India

city: Navi Mumbai

first: Chandan

last: Kumar

state: Maharashtra

address: Kharghar Node

department: ACTREC

institute: Tata Memorial Center

summary:
This is a study to identify clinically relevant molecular signatures of gene expression in cervical cancer samples fro m patients in India
Keywords: Cancer versus normal study

overall_design: Cervical cancer and non-malignant cervical cancer tissues are obtained from consenting patients following hospital ethics committee approved protocols. The samples are profiled against universal Human reference RNA from Stratagene on 19K EST microarrays from Microarray Center, University Health Network, Toronto, Canada. Cancer samples are compared against normal samples and outliers belonging to identifiable clusters, following unsupervised hierarchical clustering are validated by other biochemical means such as real time PCR and western blot.

last: Kumar-Sinha

first: Cheryl

last: Travasso

first: Mansi

last: Samarth


title: Microarray-based classification of a consecutive series of 121 childhood acute leukemias

status: Public on Apr 15 2007

city: Lund

first: Thoas

middle: Anna

address: Lund University Hospital

country: Sweden

institute: Dept of Clinical Genetics

summary:
Gene expression analyses, were performed on 121 consecutive childhood leukemias (87 B-lineage acute lymphoblastic leukemias (ALLs), 11 T-cell ALLs, and 23 acute myeloid leukemias; AMLs), investigated during an 8-year period at a single center. The supervised learning algorithm k-nearest neighbor (k-NN) was utilized to build gene expression predictors that could classify the ALLs/AMLs according to clinically important subtypes with high accuracy. Validation experiments in an independent data set verified the high prediction accuracies of our classifiers. B-lineage ALLs with uncharacteristic cytogenetic aberrations or with a normal karyotype displayed heterogeneous gene expression profiles, resulting in low prediction accuracies. Minimal residual disease status (MRD) in T-cell ALLs with a high (>0.1%) MRD at day 29 could be classified with 100% accuracy already at the time of diagnosis. In pediatric leukemias with uncharacteristic cytogenetic aberrations or a normal karyotype, unsupervised analysis identified two novel subgroups: one consisting mainly of cases remaining in complete remission (CR) and one containing a few patients in CR and all but one of the patients who relapsed. This study of a consecutive series of childhood leukemias confirm and further extend previous reports demonstrating that global gene expression profiling provides a valuable tool for genetic and clinical classification of childhood leukemias.
Keywords: supervised classification, Pediatric leukemia, AML, B-ALL, T-ALL, Normal bone marrow

overall_design: Bone marrow (BM; n=108) or peripheral blood (PB; n=13) samples from 121 children with ALL (87 B-lineage and 11 T-cell) or AML (n=23) were obtained at the time of diagnosis. In addition, six normal BMs (NBMs) were obtained from healthy donors. RNA extraction, amplification, labeling, hybridization, scanning, post-hybridization washing and feature analysis were performed as described (Andersson et al., 2005, Leukemia). The quality of total and amplified RNA was assessed using an Agilent 2100 BioAnalyzer (Agilent Technologies, Palo Alto, CA). As a reference, aRNA from the Universal Human Reference (Stratagene, La Jolla, CA) was used.

first: Anna

last: Andersson

first: Cecilia

last: Ritz

first: David

last: Lindgren

first: Patrik

last: Edén

first: Carin

last: Lassen

first: Jesper

last: Heldrup

first: Tor

last: Olofsson

first: Johan

last: Råde

first: Magnus

last: Fontes

last: Porwit-MacDonald

first: Mikael

last: Behrendtz

first: Mattias

last: Höglund

first: Bertil

last: Johansson

last: Fioretos


title: Ectodysplasin regulates the lymphotoxin-beta pathway for hair differentiation

status: Public on Feb 06 2007

first: Minoru

last: Ko

address: 251 Bayview Blvd, Suite 100, 10C

summary:
To find developmental stage specific genes controled by EDA
Keywords: development or differentiation design,genetic modification design

overall_design: Triplicated for all samples

first: Chang-Yi

last: Cui

first: Tsuyoshi

last: Hashimoto

first: Sergei

middle: I

last: Grivennikov

first: Yulan

last: Piao

middle: A

last: Nedospasov

last: Schlessinger


title: Natural killer T-cell characterization through gene expression profiling

status: Public on Feb 01 2007

city: Berlin

first: Hans-Joachim

last: Mollenkopf

address: Charitéplatz 1

country: Germany

institute: Max-Planck-Institute for Infection Biology

summary:
Natural killer T (NKT) cells are a distinct lymphocyte lineage thought to operate primarily at the interface between the innate and adaptive immune response. Yet, their unique role in the immune system remains elusive. Whilst NKT cells show high similarities to other cells of the innate and adaptive immune system, they express unique functional features such as rapid, concomitant production of Th1 and Th2 cytokines upon TCR ligation. In order to portray gene expression of NKT cells and to analyze their complete functional potential, we performed comparative microarray analyses of naive NKT cells, naive NK cells as well as naive conventional CD4+ T cells (Th, CD4+CD25–) and naive regulatory CD4+CD25+ T cells (Treg ). Furthermore, we compared the gene expression profiles of naive versus alpha-galactosylceramide activated NKT cells to elucidate the gene set rapidly expressed upon activation. We describe profound gene expression differences between the different cell types as well as between naive and activated NKT cells allowing the identification of a unique gene expression profile of NKT cells. In addition to known NKT cell specific markers, a high number of genes were expressed and detected which had not been attributed to NKT cells. Notably, our analyses reveals that NKT cells are not only of Th1 and Th2 type but also fulfil criteria of Th17 cells. Hence, our data provide new insight into the genetic décor of NKT cells which will facilitate a better understanding of their versatile role during the immune response.
Keywords: NKT, NK, Th and Treg cell type comparison

overall_design: Microarray experiments were done as two-color hybridizations. Total RNA was extracted from single-cell suspensions. An amount of 4 µg total RNA was reverse transcribed with an oligo-dT-T7-promotor primer by a fluorescent linear amplification reaction (Agilent Technologies) and cDNA was labeled either with Cyanine 3-CTP and Cyanine 5-CTP (NEB Life Science Products, Ipswich, MA, USAFrankfurt, Germany) in a T7 polymerase amplification reaction according to the supplier’s protocol. In order to compensate specific effects of the dyes and to ensure statistically relevant data analysis, a color- swap dye-reversal was performed [Churchill, 2002 #100]. The RNA samples were labeled vice versa with the two fluorescent dyes (fluorescence reversal). After precipitation, purification and quantification with a Nanodrop ND-1000 spectrophotometer (Kisker), 1.25 µg of each labeled cRNA was mixed, fragmented and hybridized to an 8.4 K custom-made mouse array (AMADID 010646) according to the supplier`s protocol (Agilent Technologies). Scanning of microarrays was performed with 5 µm resolution using a DNA microarray laser scanner (Agilent Technologies). Features were extracted with an image analysis tool version A.6.1.1 (Agilent Technologies) using default settings. Data analysis was carried out on the Rosetta Inpharmics platform Resolver Version 5.0. Ratio profiles were combined in an error-weighted fashion with Resolver to create ratio experiments. A two fold change expression cut-off for ratio experiments was applied together with anti-correlation of ratio profiles rendering the microarray analysis set highly significant (P-value > 0.05), robust and reproducible

first: Marcus

last: Niemeyer

first: Stefan

middle: H

last: Kaufmann


title: Identify cis-regulatory modules for OCT4

status: Public on Jan 25 2007

city: Columbus

first: Victor

middle: X

last: Jin

state: OH

address: 333 West 10th Ave

department: Biomedical Informatics

institute: OSU Medical Center

type: Genome binding/occupancy profiling by genome tiling array

summary:
Recent ChIP-chip studies have revealed that many in vivo binding sites have a weak match to the consensus sequence for the transcription factor being analyzed. Possible explanations for these observations include a) the consensus site was derived from in vitro analyses and does not represent the preferred in vivo binding site and/or b) the factor is recruited to a weak binding site via interaction with a protein that binds nearby. To address these possibilities we have developed a motif discovery approach (ChIPMotifs) that incorporates a bootstrap re-sampling method to statistically infer the optimal cutoff threshold for a position weight matrix (PWM) of a motif identified from in vivo ChIP-chip data by ab initio motif discovery programs. Using OCT4 ChIP-chip data derived from genomic tiling arrays and the ChIPMotifs approach, we developed a refined OCT4 PWM. We then used the in vivo-derived PWM and a ChIPModules approach to identify transcription factors co-localizing with OCT4 in a testicular germ cell tumor (Ntera2 cells). We found that the consensus binding site for SRY, a transcription factor critical for testis development, co-localizes with the OCT4 PWM. To further characterize the relationship between OCT4 and SRY binding sites, we used ChIP-chip analysis of human promoter microarrays, and found that 49% of the top ~1000 OCT4 target promoters were also bound by SRY. This analysis represents the first identification of SRY target promoters. Our studies not only validate the ChIPMotifs and ChIPModules combinatorial approach but also identify a possible new regulatory partner of OCT4.
Keywords: ChIP-chip, computational approaches

overall_design: First performing ChIP-chip experiments, then find OCT4 binding motif, then identify regulatory modules using ChIPModules approach.

first: Henny

last: O'Geen

first: Sushma

last: Iyengar

first: Roland

last: Green

first: Peggy

middle: J

last: Farnham


title: Hematopoietic reconstitution by multipotent adult progenitor cells: precursors to long-term hematopoietic stem cells

status: Public on Jan 26 2007

city: Baltimore

institute: NIH

summary:
We used comparative transcriptome analysis to determine the relationship between rodent Multipotent Adult Progenitor Cells (MAPCs) and pluripotent embryonic stem cells (ESCs), or lineage restricted mesenchymal stem cells (MSCs) and neurospheres (NS). This comparison revealed a unique gene expression profile of MAPCs, that express transcripts of early endodermal and mesodermal but fewer ectodermal genes. In addition, MAPCs, but not MSCs or NS, express a number of genes expressed in ESCs including Oct-4 (Pou5f1) and other genes previously identified as ES cell associated transcripts and/or genes involved in maintenance of ESC selfrenewal capacity and pluripotency. However MAPCs do not express some key genes involved in maintaining selfrenewal and pluripotency in ESCs, including Nanog and Sox-2. Because MSC-like cells isolated under MAPC conditions are virtually identical to MSCs isolated in the presence of high serum, the MAPC signature is cell-type specific and not merely the result of differing culture conditions. Finally, this unique molecular signature was seen irrespective of the microarray platform used, and was highly similar for both mouse and rat MAPCs.
Keywords: cell type comparison design

overall_design: Two biological replicates were used for hybridizations.  cRNA from MAPCs, ESCs, and NS were added to a universal reference cRNA.

first: Fernando

last: Ulloa-Montoya

first: Benjamin

middle: L

last: Kidder

first: Karen

last: Pauwelyn

first: Lucas

last: Chase

first: Aernout

last: Lutton

first: Annelies

last: Crabbe

first: Alexei

last: Sharov

first: Yu-Lan

first: Wei-Shou

last: Hu

first: Catherine

middle: M

last: Verfaillie


title: Diet Induced Obesity Signature in Mouse Liver Tissue

first: eric

last: schadt

summary:
The purpose of this experiment was to determine the murine liver expression traits that were changed in response to diet induced obesity.
Keywords: diet induced obesity signature

overall_design: Liver tissue from five diet induced obesity (DIO) mice were arrayed against a pool of liver tissue from chow-fed wildtype B6 animals.  All samples were hybridized as fluor-reverse pairs, and then the data between the pairs was combined to form a single experiment.  Five repeats were performed to increase the power to detect expression changes and to reduce effects of experimental noise.


title: Transcriptional profile of breast cancer cell lines after treatment with docetaxel.

status: Public on Feb 09 2007

city: Bron

first: Hector

last: Hernandez-Vargas

state: Rhone

address: 36 bis Rue de la Batterie

country: France

institute: Genomics Consulting

summary:
Among microtubule-targeting agents, docetaxel has received recent interest owing to its good therapeutic index. Clinical trials have underlined its potential for the treatment of advanced breast cancer, although little is known about its molecular mode of action in this context. We characterized the molecular changes induced by docetaxel in two well-known human breast carcinoma cell lines. Two mechanisms of action according to drug concentration were suggested by a biphasic sensitivity curve, and were further validated by cell morphology, cell cycle and cell death changes. Two to four nanomolar docetaxel induced aberrant mitosis followed by late necrosis, and 100 nM docetaxel induced mitotic arrest followed by apoptosis. Passing through mitosis phase was a requirement for hypodiploidy to occur, as shown by functional studies in synchronized cells and by combining docetaxel with the proteasome inhibitor MG132. Transcriptional profiling showed differences according to cell line and docetaxel concentration, with cell cycle, cell death and structural genes commonly regulated in both cell lines. Although p53 targets were mainly induced with low concentration of drug in MCF7 cells, its relevance in the dual mechanism of docetaxel cytotoxicity was ruled out by using an isogenic shp53 cell line. Many of the genes shown in this study may contribute to the dual mechanism by which docetaxel inhibits the growth of breast cancer cells at different concentrations. These findings provide a basis for rationally enhancing docetaxel therapy, considering lower concentrations, and better drug combinations.
Keywords: Dose-response analysis

overall_design: MCF7 and MDA-MB-231 breast cancer cells lines were treated with low (2-4 nM) and high (100 nM) concentrations of docetaxel. After 8, 24, and 48 hours of exposure total RNA was extracted, amplified and hybridized onto CNIO Oncochip cDNA microarrays. Untreated control cells were used for normalization of duplicate arrays.

first: Jose

last: Palacios

first: Gema

last: Moreno-Bueno


title: High-salt diet leads to differential gene expression in DOCA-treated mice

status: Public on Jan 01 2007

city: Chicago

first: Andrea

middle: A.

last: Domenighetti

state: IL

address: 345 East Superior St

institute: Northwestern University & Rehabilitation Institute of Chicago

summary:
In the present study we made use of the (1-renin) DOCA-salt mouse model - which has been previously shown to develop cardiac and renal hypertrophy  - to evaluate the direct effects of high-salt diet on cardiac function and gene expression profiling. The comparison between low-salt and high-salt DOCA-treated mice will reveal what genes are directly modulated by sodium in (normotensive) DOCA-treated mice.

Previous publications:

Wang Q, Hummler E, Nussberger J, Clement S, Gabbiani G, Brunner HR,
Burnier M. Blood pressure, cardiac, and renal responses to salt and
deoxycorticosterone acetate in mice: role of renin genes. J Am Soc
Nephrol. 2002;13:1509 –1516.

Wang Q, Domenighetti AA, Pedrazzini T, Burnier M. Potassium supplementation reduces cardiac and renal hypertrophy independent of blood pressure in DOCA/salt mice. Hypertension. 2005 Sep;46(3):547-54.
Keywords: comparative dose-response treatment (2 groups)

overall_design:
In this first series, six (7-8 week old) DOCA-treated mice were separated in 2 groups:

Group1 (Low-Salt)= LS + Nx + DOCA35 + tap
(LS= 0.5mg Na/g food; Nx= uninephrectomized; DOCA35= deoxycorticosterone acetate for 5 weeks; tap= normal drinking water)

Group2 (High-salt)= LS + Nx + DOCA35 + 1%NaCl
(LS= 0.5mg Na/g food; Nx= uninephrectomized; DOCA35= deoxycorticosterone acetate for 5 weeks; 1%NaCl= 1%NaCl in drinking water for 5 weeks)

cDNA high-density microarray assays were used to explore changes in cardiac expression of ~17’000 clones in hearts of Low-salt and High-salt treated DOCA-mice.

This series contains 3 biological replicates and one dye swap.

first: Qing

last: Wang

first: Michel

last: Burnier


title: Zfp90 Transgenic Signature in Mouse White Adipose Tissue

summary:
The purpose of this experiment was to determine the murine white adipose expression traits that were changed in response to over expressing Zfp90.
Keywords: single gene perturbation signature

overall_design: White adipose tissue from five wild type B6 mice and from three Zfp90 transgenic mice were arrayed against a pool of white adipose tissue from all mice.  All samples were hybridized as fluor-reverse pairs, and then the data between the pairs was combined to form a single experiment.


title: Alox5 Knockout Signature in Mouse White Adipose Tissue

summary:
The purpose of this experiment was to determine the murine white adipose expression traits that were changed in response to knocking Alox5 out.
Keywords: gene specific knockout signature

overall_design: White adipose tissue from five Alox5 KO mice were arrayed against a pool of white adipose tissue from wildtype B6 animals.  All samples were hybridized as fluor-reverse pairs, and then the data between the pairs was combined to form a single experiment.  Five repeats were performed to increase the power to detect expression changes and to reduce effects of experimental noise.

first: Eric

middle: E

last: Schadt

first: Margarete

last: Mehrabian

first: Aldons

last: Lusis

first: Hooman

last: Allayee

first: Pek

last: Lum


title: Whole-genome gene expression variation in 210 unrelated HapMap individuals

city: Geneva

first: Emmanouil

middle: T

last: Dermitzakis

address: 1 Rue Michel-Servet

department: Department of Genetic Medicine and Development

country: Switzerland

institute: University of Geneva Medical School

summary:
Extensive studies are currently being performed to associate disease susceptibility with one form of genetic variation, namely single nucleotide polymorphisms (SNPs). In recent years another type of common genetic variation has been characterised, namely structural variation, including copy number variations (CNVs). To determine the overall contribution of CNVs to complex phenotypes we have performed association analyses of expression levels of 14,925 transcripts with SNPs and CNVs in individuals who are part of the International HapMap project. SNPs and CNVs captured 83.6% and 17.7% of the total detected genetic variation in gene expression, respectively, but the signals from the two types of variation had little overlap. Interrogation of the genome for both types of variants may be an effective way to elucidate the causes of complex phenotypes and disease in humans.
Keywords: gene expression

overall_design:
RNA preparation:
Total RNA was extracted from lymphoblastoid cell lines of the 210 unrelated individuals of the HapMap (Coriell, Camden, New Jersey, United States). Two, one-quarter scale Message Amp II reactions (Ambion, Austin, Texas, United States) were performed for each RNA extraction using 200 ng of total RNA as previously described (Stranger et al. 2005).  1.5 µg of the cRNA was hybridized to an array.

Gene expression quantification:
To assay transcript levels in the cell lines, we used Illumina's commercial whole genome expression array, Sentrix Human-6 Expression BeadChip (Illumina, San Diego, California, United States).  These arrays utilize a bead pool with ~48,000 unique bead types (one for each of 47,294 transcripts, plus controls), each with several hundred thousand gene-specific 50mer probes attached.

On a single BeadChip, six arrays were run in parallel. Each bead type (probe) is present on a single array on average 30 times. Each of the two IVT reactions from the 210 samples was hybridized to two arrays each, so that each cell line had four replicate hybridizations. cRNA was hybridized to arrays, and subsequently labelled with Cy3-streptavidin (Amersham Biosciences, Little Chalfont, United Kingdom) and scanned with a Bead Station (Illumina) as previously described (Stranger et al. 2005).

Normalization:
With the Illumina bead technology, a single hybridization of RNA from one cell line to an array produces on average approximately 30 intensity values for each of 47,294 bead types. These background-corrected values for a single bead type are subsequently summarized by Illumina software and output to the user as a set of 47,294 intensity values for each individual hybridization. In our experiment, each cell line was hybridized to 4 arrays, thus resulting in 4 reported intensity values (as averages of the values from the 30 beads per probe) for each of the 47,294 bead types (Dunning et al. 2006). To combine data from our multiple replicate hybridizations, raw data were read using the beadarray R package(Dunning et al. 2006) and then normalized on a log scale using a quantile normalization method (Bolstad et al. 2003) across replicates of a single individual, followed by a median normalization method across individuals of a single population. These normalized values (for each probe, across replicates for each individual) are used in subsequent analyses.

first: Barbara

last: Stranger

first: Matthew

middle: S

last: Forrest

first: Mark

last: Dunning

last: Ingle

first: Claude

last: Beazley

first: Natalie

last: Thorne

first: Richard

last: Redon

first: Christine

last: Bird

first: Anna

last: de Grassi

first: Charles

last: Lee

first: Chris

last: Tyler-Smith

first: Nigel

last: Carter

first: Stephen

middle: W

last: Scherer

first: Simon

last: Tavare

first: Panagiotis

last: Deloukas

last: Hurles


title: miRNA let-7a-3 overexpression in lung cancer cell line A549

status: Public on Jan 15 2007

city: Heidelberg

first: Ruprecht

last: Kuner

address: Im Neuenheimer Feld 460

department: Molecular Genetics

institute: German Cancer Research Center and National Center of Tumor Diseases

summary:
MicroRNAs (miRNAs) are small non-coding RNAs that repress their target mRNAs by complementary base pairing and induction of the RNAi pathway. It has been shown that miRNA expression can be regulated by DNA methylation and it has been suggested that altered miRNA gene methylation might contribute to human tumorigenesis. In this study, we show that the human let-7a-3 gene on chromosome 22q13.31 is associated with a CpG island. Let-7a-3 belongs to the archetypal let-7
miRNA gene family DNA methylation and was found to be methylated by the DNA methyltransferases DNMT1 and DNMT3B. The gene was heavily methylated in normal human tissues, but hypomethylated in some human lung adenocarcinomas. Let-7a-3 hypomethylation facilitated epigenetic reactivation of the gene and elevated expression of let-7a-3 in a human lung cancer cell line resulted in enhanced tumor phenotypes and oncogenic changes in transcription profiles.
Keywords: genetic modification

overall_design: Gene expression analysis comprised 4 microarray hybridizations: 4 let-7a-3 transfected cell culture replicates and 4 parental cell culture replicates (controls). Each let-7a-3 expressing replicate was hybridized against a control replicate of the same cell line including a dye swap design. Rawdata gpr files were processed using software ArrayMagic (Buness et al. 2005) including VSN normalization available on Bioconductor open source software platform. Differentially expressed genes between let-7a-3 transfected and control cell lines were analyzed by LIMMA (Wettenhall and Smyth, 2004) considering biological replicates and dye swap design.

first: Bodo

last: Brueckner

first: Carlo

last: Stresemann

first: Frank

last: Lyko

first: Holger

last: Sultmann

first: Annemarie

last: Poustka

first: Andreas

last: Buness

first: Markus

last: Ruschhaupt


title: Identification of genes regulated in adrenal glands of Ftz-F1 transgenic mice

city: Valbonne

first: Enzo

last: LALLI

address: 660 route des Lucioles

department: CNRS UMR 7275

institute: IPMC

summary:
SF-1, a transcription factor belonging to the nuclear receptor superfamily, has a pivotal role for adrenogonadal development in humans and mice. A constant feature of childhood adrenocortical tumors (ACT) is SF-1 amplification and overexpression. Using an inducible cellular system, here we show that SF-1 overexpression increases human adrenocortical cell proliferation through opposing effects on cell cycle and apoptosis. SF-1 overexpression also selectively modulates steroidogenesis, reducing cortisol and aldosterone secretion. We identified a novel pro-apoptotic factor for adrenocortical cells, NOV/CCN3, whose levels are significantly reduced by SF-1 overexpression in human adrenocortical cells and are also reduced in primary adrenal tumors. Moreover, Sf-1 overexpression triggers adrenocortical hyperplasia and tumor formation in mice. These tumors express gonadal markers and activated Stat3. Our studies reveal the critical role of SF-1 gene dosage for adrenocortical tumorigenesis and constitute a rationale for the development of drugs targeting SF-1 transcriptional activity for ACT therapy.
Keywords: comparative gene expression

overall_design:
Gene expression profiles were analyzed by two-color microarrays in transgenic mice from line #56 heterozygous or homozygous for the transgene, male or female and of different ages. Profiles from each transgenic mouse were directly compared to a sex- and age-matched wild-type littermate.

For each experimental point, two technical replicates (dye-swaps) were examined.

Array #		        Age       Sex	Transgene copy #	Cy3		Cy5

16682			10 days	  M		1			WT		transgenic
16479			10 days	  M		1			transgenic	WT
16683			10 days	  M		1			WT		transgenic
16625			10 days	  M		1			transgenic	WT
16685			10 days   F		 1			 WT		 transgenic
16680			10 days   F		 1			 transgenic	 WT
16516			4 months M	        2	               WT		transgenic
16452			4 months M		2		       transgenic	WT
16517			4 months M		2		       WT		transgenic
16453			4 months M		2		       transgenic	WT
16518			4 months F		 2			WT		 transgenic
16475			4 months F		 2			transgenic	 WT

first: Mabrouka

last: Doghman

first: Tatiana

last: Karpova

first: Giovanna

last: Rodrigues

first: Malika

last: Arhatte

first: Juliana

last: De Moura

first: Luciane

middle: R

last: Cavalli

first: Virginie

last: Virolle

first: Sébastien

last: Grosso

first: Hélène

last: Thibout

first: Maryvonne

last: Laurent

first: Patrick

last: Auberger

first: Pascal

last: Barbry

first: Gerard

last: Zambetti

first: Bonald

middle: C

last: Figueiredo

first: Cécile

last: Martinerie

first: Leslie

last: Heckert

last: Lalli


city: Leicester

first: Jerry

last: Styles

state: Leicestershire

address: Robert Kilpatrick Building, Univ. Leicester

department: Cancer Studies and Molecular Medicine

country: United Kingdom

institute: University of Leicester

summary:
Failure of ligamentous support of the genital tract to resist intra-abdominal pressure is a plausible underlying mechanism for the development of pelvic organ prolapse, but the nature of molecular response of pelvic tissue support remains unknown. We hypothesized that the expression of genes coding for proteins involved in maintaining the cellular and extracellular integrity would be altered in cases of pelvic organ prolapse. Therefore, cDNA microarrays were used to examine the difference in transcriptional profile in RNA of primary culture fibroblasts subjected to mechanical stretch and those that remained static.
Keywords: Changes in transcription profile in pelvic organ fibroblasts in response to stretch

overall_design:
Source of fibroblasts:
Three tissue samples were collected from uterine specimens of abdominal hysterectomy performed on premenopausal Caucasian women with no history of uterine prolapse. The supra-cervical area was cut (5 mm thick slices) with a scalpel to obtain the medial ends of the cardinal ligament. Every patient had to sign a consent form preoperatively, allowing the use of tissues removed at surgery for research purposes. The local ethics committee has approved this research program.

Primary culture:
The specimens were placed immediately in a cold Hanks’ Balanced Salt Solution, HBSS, (Gibco, Paisley, Scotland, UK)
and transferred to the tissue culture laboratory for immediate processing. They were minced into one mm3 cubes, treated with 1% Collagenase type I (Gibco, Paisley, Scotland, UK) in HBSS, and incubated at 37oC for 2h with gentle rotation. The supernatant, containing the fibroblasts, was washed with HBSS and Medium 199 Earle’s MOD Salts (Gibco, Paisley, Scotland, UK) to which 1% antibiotic/antimycotic solution (10 000 U penicillin, 10 mg streptomycin, 25 μg Amphotericin B per ml, Sigma-Aldrich, Dorset, UK) and 15% heat-inactivated fetal bovine serum (HyClone, Logan, Utah, USA) were added. The cell suspension was spun down twice to remove collagenase. The pellets were re-suspended in growth medium, plated in 25 cm2 tissue culture flasks with Nunclon surface (Nalge Nunc International Corporation, Hereford, UK) and incubated at 37oC
in humidified air with 5% CO2. The medium was changed on alternate days and confluent fibroblasts cells were passaged into a 75 cm2 flask (Nalge Nunc International Corporation, Hereford, UK) using 0.1% trypsin-EDTA (Gibco, Paisley, Scotland, UK) for 10 min at 37oC.

Stretch experiments:
Preparation of cells for treatment: Fibroblasts from the 4th to 6th passages were seeded (5x104 cells/well) into 6-well BioFlex® flexible plates with Collagen-I coated elastomer membrane bottoms (FlexCell International Corporation, McKeesport, Pennsylvania, USA), and allowed to grow. They were serum-starved for 48h, by using 1% charcoal/dextran treated heat-inactivated fetal bovine serum (HyClone, Logan, Utah, USA)
added to the tissue culture medium to achieve quiescence, and then they were cultured with 15% of the same serum added to the tissue culture medium.

Chronic cyclical mechanical stretch: The Flexercell Stress Unit (FlexCell International Corporation, McKeesport, Pennsylvania, USA) provides the equipment for the application of stretch to cultured cells in vitro. The plates are housed within a manifold and subjected to a pre-calibrated computer-controlled vacuum stretch (Hsieh et al., 2000, Stanley et al., 2000). The cells were exposed to stretch amplitude of (21%)-relaxation (0%) cycles of 12h each, at a frequency of 1 Hz, for 96h, at 37oC in humidified air with 5% CO2. The corresponding BioFlex® static plate was kept in the same incubator.
Changes in gene expression were analyzed using three cDNA arrays, consisting of pairs of arrays forward labeled with Cy5 and Cy3 dyes, hybridizing RNA from stretched fibroblasts against controls.  All controls were pooled.  Microarray hybridization and analysis were performed as previously described (Turton et al., 2001). Changes in gene expression were analysed hybridizing RNA from treated cells against pooled controls. In this study Cy3 was added to control samples and Cy5 to treated samples and was referred to as forward labelling

first: Ayman

last: Ewies

first: Farook

last: Al-Azzawi

first: Mona

last: El-Shafie

first: Jin

last: Li

first: Adrian

last: Stanley

first: Bryan

last: Williams

first: John

last: Thompson

first: Ian

middle: N

last: White


title: Identification of genes regulated by overexpressed SF-1 in human adrenocortical cells

first: Enzo

last: LALLI

summary:
SF-1, a transcription factor belonging to the nuclear receptor superfamily, has a pivotal role for adrenogonadal development in humans and mice. A constant feature of childhood adrenocortical tumors (ACT) is SF-1 amplification and overexpression. Using an inducible cellular system, here we show that SF-1 overexpression increases human adrenocortical cell proliferation through opposing effects on cell cycle and apoptosis. SF-1 overexpression also selectively modulates steroidogenesis, reducing cortisol and aldosterone secretion. We identified a novel pro-apoptotic factor for adrenocortical cells, NOV/CCN3, whose levels are significantly reduced by SF-1 overexpression in human adrenocortical cells and are also reduced in primary adrenal tumors. Moreover, Sf-1 overexpression triggers adrenocortical hyperplasia and tumor formation in mice. These tumors express gonadal markers and activated Stat3. Our studies reveal the critical role of SF-1 gene dosage for adrenocortical tumorigenesis and constitute a rationale for the development of drugs targeting SF-1 transcriptional activity for ACT therapy.
Keywords: differential expression, transcription factor

overall_design:
Gene expression profiles were analyzed in two different H295R TR/SF-1 WT clones overexpressing SF-1 in a tetracycline-regulated fashion cultured in basal conditions or after three days of doxycycline treatment. For each condition, two biological replicates were examined.

Array #22354	Clone #1      replicate 1    basal Cy3/Dox Cy5
Array #22416	Clone #1      replicate 2    basal Cy5/Dox Cy3
Array #22446	Clone #2      replicate 1    basal Cy3/Dox Cy5
Array #22447	Clone #2      replicate 2    basal Cy5/Dox Cy3

first: Mabrouka

last: Doghman

first: Tatiana

last: Karpova

first: Giovanna

last: Rodrigues

first: Malika

last: Arhatte

first: Juliana

last: De Moura

first: Luciane

last: Cavalli

first: Virginie

last: Virolle

first: Sébastien

last: Grosso

first: Hélène

last: Thibout

last: Laurent

middle: P


title: Identification of salt stress regulated genes in tomato seedlings by SSH and microarray analysis

status: Public on Jan 09 2007

city: Ithaca

first: Zhangjun

last: Fei

state: NY

address: Tower Road

institute: Boyce Thompson Institute

summary:
High salinity is one of the most serious threats to crop production. To 1 better understand the molecular basis of plant responses to salt stress, we combined suppression subtractive hybridization (SSH) and microarray approaches to identify the potential important or novel genes involved in salt tolerance. First, SSH libraries were constructed for two cultivated tomato (Solanum lycopersicum) genotypes: LA2711, a salt tolerant cultivar, and ZS-5, a salt sensitive cultivar, to compare salt treatment and non-treatment plants. Then a subset of clones from these SSH libraries were used to construct a tomato cDNA array and microarray analysis was carried out to verify the expression changes of this set of clones upon salt treatment at various time points compared to the corresponding non-treatment controls. A totalof 201 non-redundant genes differentially expressed upon 30 min of salt stress treatment either in LA2711 or ZS-5 were identified from microarray analysis, most of which were not previously associated with salt stress. The diversity of the putative functions of these genes indicated that salt stress resulted in a complex response in tomato plants.
Keywords: gene expression, genotype, microarray, salt stress, SSH, tomato

overall_design: tomato seedling from two different genotypes, LA2711, a salt tolerant cultivar and ZS-5, a salt sensitive cultivar were treated with salt stress for 30 min, 2 hr and 6 hr. Total RNA extracted from root tissue of each treated plant was hybridized to the array together with RNA from control plant. Each hybridization was performed in duplicate by dye swap.

first: Bo

last: Ouyang

first: Ting

last: Yang

first: Hanxia

first: Liang

last: Zhang

first: Yuyang

first: Junhong

first: Zhibiao

last: Ye


title: PPARa siRNA-Treated Expression Profiles Uncover the Causal Sufficiency Network for Compound-Induced Liver Hypertrophy

status: Public on Jan 20 2007

city: Rahway

first: Olivia

last: Fong

state: NJ

department: Molecular Profiling

institute: Merck & Co.

summary:
Approaches for discovering mechanisms of action and for identifying molecular biomarkers in biomedical research are evolving today, as the growing symbiosis with computational sciences becomes more widely appreciated than ever. In fact, the combination of various new technologies has been pushing forward both frontiers. Here, we present an example of the combined use of in vivo siRNA knock-down technology, genome-wide gene expression profiling, and computational reasoning to unveil regulatory causal relationships and the sufficiency network of identified genes for compound-induced toxicity. Unlike previously reported approaches, our method requires only one targeted perturbation for genome-wide de novo pathway discovery. Hence, our method can be directly applied to animal models in which it is still technically challenging to perform genome-wide genetic perturbation or RNAi screening. The independent application of our derived model to compounds with unrelated mechanisms of action suggests the existence of a universal molecular module that mediates liver hypertrophy. The resulting sufficiency network for induction of liver hypertrophy will have an immediate impact on the progress of toxicogenomics. When combined with phenotypic evaluation, our approach should help to unleash the full potential of siRNAs in systematically unveiling the molecular mechanisms of biological events.
Keywords: Regulatory pathway discovery by In vivo transcription modification

overall_design: Two different siRNAs targeting peroxisome proliferator-activated receptor a (Ppara), and two control siRNAs targeting secreted alkaline phosphatase (SEAP) and GL3 were delivered to the mouse liver by hydrodynamic tail vein injection . Four animals per group were treated 48 hours before livers were collected. RNAs were extracted and profiled on a custom-designed mouse oligonucleotide microarray with approximately 25,000 genes according to a published procedure. In total, the Ppara siRNA mouse expression profiles included eight profiles for the Ppara siRNAs targeted to two different fragments of the Ppara mRNA sequence, four profiles for the SEAP control siRNA, four profiles for the GL3 control siRNA, and eight profiles for a vehicle control (Ringer solution).

first: Xudong

last: Dai

first: Angus

last: De Souza

first: Hongyue

first: David

last: Lewis

first: Chang-kyu

last: Lee

first: Andy

middle: G

last: Spencer

first: Hans

last: Herweijer

first: Jim

last: Hagstrom

first: Peter

last: Linsley

first: Douglas

last: Bassett

first: Roger

last: Ulrich

first: Yudong

last: He


title: Gene Expression Profile identify a role for cyclooxygenase 2-depedent prostinoid generation in BMP6-induced

status: Public on Dec 31 2006

city: Chapel Hill

first: Monte

last: Willis

address: 103 Mason Farm Road

department: Pathology & Laboratory Medicine

institute: UNC

summary:
The bone morphogenetic protein (BMP) family of proteins participates in an underappreciated signaling pathway involved in regulating angiogensis in physiological and pathological conditions. Microarray profiles of BMP-responsive genes in the endothelium performed by our lab suggest important roles for cyclooxygenase-2 (Cox2) as a potential downstream regulator of BMP-induced angiogenesis. Cox2 is upregulated by BMP6 at both mRNA and protein levels. Inhibition of Cox2 blocks BMP6 induced endothelial cell proliferation, migration and tube formation activity. Microvessel outgrowth stimulated by BMP6 also required Cox2 activity. These results strongly support that Cox2 is an important downstream target for BMP6, and that prostaniod synthesis is essential for BMP6 induced endothelial cell activation. This further suggests that our microarray data is a reliable tool to identify unkown BMP6 targets in angiogenesis.
Keywords: BMP, angiogenesis, Cox2, MEC

overall_design: Mouse intraembryonic endothelial cells (MECs) were cultured as previously described.  Total RNA was extracted from MECs treated with BMP6 for 4, 12, or 24 hrs, or from mock-treated controls.  Universal mouse reference RNA was used as a reference, and was labeled with Cy3 florescence dye using the Fluorescent Linear Amplification Kit (Agilent Technologies, CA). Samples of MEC RNA were labeled Cy5, and equimolar mixtures of the two florescence dye-labeled probes were hybridized to Agilent 22k Mouse Development Oligo Microarrays (Agilent Technologies). Microarrays were scanned with a GenePix 4000B scanner (Axon Instruments) and images were quantified with GenePix Pro 5.0 Software (Axon Instruments). Each experimental group included independent triplicate biological replications.  Raw expression data was normalized via a modified quantiles local algorithm (X. He and C. Patterson, manuscript in preparation).  Differentially expressed genes were identified with a two-tailed type 2 Student t-test.  Cluster analysis and visualization using Java-TreeView were accomplished as previously described.

first: Rongqin

last: Ren

last: Charles

first: Chunlian

first: Yaxu

last: Wu

first: Hong

last: Wang

first: Beverly

last: Koller

first: Cam

last: Patterson