title: Ets2 is required for trophoblast stem cell self renewal

status: Public on Nov 07 2007

first: Roy

last: Williams

address: 3030 Science Park Road

department: Regenerative Medicine

institute: The Scripps Research Institute

summary:
The Ets2 transcription factor is essential for the development of the mouse placenta and for generating signals for embryonic mesoderm and axis formation.   Using a conditional targeted Ets2 allele, we show that Ets2 is essential for trophoblast stem  (TS) cells self renewal.   Inactivation of Ets2 results in slower growth, increased expression of a subset of differentiation associated genes and decreased expression of several genes implicated in TS self renewal.  Among the direct TS targets of Ets2 is Cdx2, a key master regulator of TS cell state.  In addition other Ets2 responsive genes include Pace4, Errb, Socs2 and Bmp4. Thus Ets2 contributes to the regulation of multiple genes important for maintaining the undifferentiated state of TS cells and as candidate signals for embryonic development.
Keywords: cell type comparison

overall_design:
We analysed  RNA with Illumina MouseRef-8 and MouseRef-6 Expression BeadChips using the manufacturers BeadArray Reader and collected primary data using the supplied Scanner software. Data analysis was done in three stages. First, expression intensities were calculated for each gene probed on the array for all hybridizations (24 in total) using illumina’s Beadstudio#1 software. Second, intensity values were quality controlled and normalized: quality control was carried out by using the illumina Beadstudio detection P-value set to <0.01 as a cutoff. This removed genes which were effectively absent from the array (that is, were not detected). After this step, the initial gene set was reduced to 14888 genes. All the arrays were then normalized using the normalize.quantiles routine from the Affy R-package in Bioconductor. This procedure accounted for any variation in hybridization intensity between the individual arrays.  An assessment of several different normalization techniques using the Bioconductor maCorrPlot routine suggested that normalize.quantiles was the most appropriate for the data.
Finally, these normalized data were imported into GeneSpring and analysed for differentially expressed genes. The groups of biological replicates were described to the software and significantly differentially expressed genes determined on the basis of t-tests and fold difference changes in expression level.
GroupA (8 replicates): AdenoGFP Ets2 f/f.
GroupB (7 replicates) Adeno Cre Ets2 f/f.
GroupC (3 replicates) TS cells Ets2 f/f.
GroupD (4 replicates) TS differ Ets2 f/f. DM medium
GroupE (2 replicates) SB teated DM cells Ets2 f/f .

Here we describe the generation of an Ets2 conditional allele and show that Cre-mediated Ets2 inactivation causes a defect in the self renewal of TS cells. Inactivation of Ets2 results in changes in gene expression normally associated with differentiation caused by growth factor withdrawal.  Among the genes sensitive to Ets2 inactivation are Pace4, Cdx2 and BMP4.  Ets2 is essential to maintain expression of multiple genes necessary for TS cell self renewal.
To facilitate study of Ets2 function in different cell types, we generated by gene targeting a conditional Ets2 allele that could be inactivated by Cre-mediated recombination.  Two loxP sites were engineered into the targeting vector to flank exon 9 and 10, a major part of the conserved DNA binding domain that is essential for Ets2 function.  Additionally, a Pgk-neo cassette flanked by Flip recombinase recognitions sites (frt) was inserted into intron 8, which together with a thymidine kinase gene inserted upstream of exon 6, provided both positive and negative selection for homologous recombination.  Twenty-seven targeted ES cell clones were identified from a total of 132 clones screened by Southern blot analysis with a 3’ flanking probe and PCR screening for the presence of both loxP sites.  After karyotyping, two clones were selected for injection into blastocysts and one transmitted the targeted allele, designated Ets2flox-neo, through the germ line.  The Ets2flox-neo mice were phenotypically wild type and transmitted the targeted allele at the expected Mendelian frequency.
An epiblast-specific Cre line, Mox2-Cre (MORE), was used to recombine the Ets2flox allele .  The resulting deletion mutant, designated Ets2db2 due to its similarity to the previously described Ets2db1, caused recessive embryonic lethality around E8 . Ets2db2/db2 embryos at E7.5 were very similar to the previously described Ets2db1/db1 mutants, including much smaller concepti and unusual cone-shaped yolk sacs. We further tested the viability of embryos containing a combination of the Ets2A72 hypomorphic knock-in allele and Ets2db2.  As expected, Ets2A72/db2 embryos died at mid-gestation like previously described Ets2A72/db1  embryos while their Ets2A72/flox littermates developed normally (Table 1).
The combination of Ets2flox and the Cre recombinase of MORE mice can be used as an alternative to tetraploid embryo aggregation because wild-type function will remain in primitive endoderm and trophectoderm derivatives that are devoid of Cre activity.  We tested whether the recessive lethality of Ets2db2 could be rescued when one allele was generated in the MORE background.  Indeed, the wild-type function provided by one parental Ets2flox allele in the extraembryonic tissues was sufficient to support development of the otherwise largely homozygous Ets2db2 embryos.  The near complete inactivation of Ets2 in the somatic tissues of these mice was confirmed by PCR analysis that was capable of determining the Ets2flox:Ets2db2 ratio in genomic DNA.  Interestingly, these mice recapitulated the “waved” hair phenotype of Ets2db1/db1 mice rescued by tetaploid embryo aggregation.  The young animals were distinguished by wavy hair and curly whiskers, which became less apparent over time.  Skin sections revealed misaligned and dysplastic hair follicles, consistent with the observation in Ets2db1/db1 mice.  Both sexes of these MORE;Ets2db2/flox mice were fertile, and without obvious abnormality.

Gene expression changes associated with Ets2 inactivation.
RNA expression profiles of  Ets2flox/flox TS cells infected with Adenovirus expressing Cre recombinase were compared with those of TS cells infected with control virus and to TS cells induced to differentiate by growth factor withdrawal. The analysis of replicate samples from two different TS cell isolates confirmed reliable reproduction of gene expression changes. Unsupervised cluster analysis of individual samples revealed that inactivation of Ets2 under normal growth conditions caused only moderate changes in gene expression most closely related to control cultures either not infected or infected with virus not expressing Cre.  By contrast RNAs from differentiated TS cells (DM) clustered together and were distinguished from mouse embryo fibroblast RNAs (MEF) and undifferentiated TS cells (GM).   To identify individual candidate Ets2 target genes, grouped samples were analyzed.   While the changes in gene expression due to differentiation were greater than those due to inactivation of Ets2, most of the changes associated with Ets2 inactivation were in genes that also changed when TS cells were induced to differentiate.  89 of the top 100 changes in gene expression resulting from AdenoCre infection of Ets2flox/flox TS cells also changed significantly upon growth factor withdrawal induced differentiation. Thus Ets2 inactivation caused changes in gene expression associated with differentiation but these genes represented only a subset of differentiation dependent gene expression changes. Gene expression changes associated with infection with either virus were modest and did not overlap with either Ets2-dependent TS genes or differentiated dependent genes.  The fold changes in gene expression due to inactivation of Ets2 were less than cells induced to differentiate by growth factor withdrawal. . Among the Ets2 sensitive TS genes are: Errb, that has been previously implicated in ES cell self renewal ; Socs2, an inhibitor of intracellular signaling; Cdx2, a master regulator of trophoblast specification and self renewal  and Pace4, an extraembryonic protease that activates Nodal to promote TS cell maintenance.  In contrast to Pace4, Furin, a second protease implicated in Nodal processing , is unaffected by Ets2 inactivation (Figure 7C). Two Ets  transcription factors (Etv4 and Elf5) are down regulated upon TS cell differentiation. Ets2 inactivation resulted in decreased Etv4 RNA but had no effect on Elf5 RNA.  Four other members of the Ets family either changed little or increased upon differentation of TS cells

Fewer significant increases in gene expression were associated with Ets2 inactivation. RNAs for Wnt7a and Cldn23, a junctional protein, were increased in AdenoCre infected Ets2flox/flox TS cells.

first: Fang

last: Wen

middle: A

last: Tynan

first: Grace

last: Cecena

first: Jorge

last: Munera

first: Robert

last: Oshima


title: Molecular portrait of cisplatin induced response in human testis cancer cell lines based on gene expression profiles

status: Public on Oct 01 2007

city: Oslo

first: Nur

last: Duale

address: Lovisenbergg. 8, P.O.Box 4404 Nydalen,

department: Division of Climate and Environment Health

country: Norway

institute: Norwegian Institute of Public Health

summary:
Testicular germ cell tumors (TGCTs) are the most common tumors in young men. Fortunately, TGCTs respond very well to cisplatin -based chemotherapy and show a low incidence of acquired resistance compared to most somatic tumors. This high sensitivity also applies to more than 80% of all TGCTs with metastatic disease. The reasons for this particularly high sensitivity of male germ cell tumors to chemotherapy seem to be multifactorial. To study this phenomenon, we have addressed the issue of whether cisplatin produces germ cell tumor specific gene expression profiles using testicular germ cell tumor derived cell lines.
By means of microarray technology, we have analyzed cisplatin-induced gene expression in two well characterized human testicular germ cell tumor (TGCT) derived cell lines (833K and GCT27) which are sensitive to cisplatin treatment, and in a human colon carcinoma cell line (HCT116), to compare responses in germ cell tumor cell lines with those of tumor cells of somatic origin. We further evaluate the male germ cell tumor specificity, by mining available public databases and literature.
By using SAM analysis, we identified 1794 differentially expressed genes. Among these, 1180 genes were over-expressed in TGCT cells and under-expressed in HCT116 cells and 614 genes were down-regulated in TGCT cells and up-regulated in HCT116 cells after exposure to cisplatin. Functional classification of these genes showed that they participate in a variety of different and widely distributed functional categories and biochemical pathways.
Keywords: Testicular germ cell tumors (TGCT) Cisplatin, Microarray, cell type comparison, Gene expression

overall_design:
Description of the cell lines: The testis cancer cell lines 833K (metastasis, nonseminomatous germ cell tumour) and GCT27 (primary, nonseminomatous germ cell tumour), were generous gifts from John Masters. HCT116 (ATCC-CCL-247, Manassas, VA) is a colon-cancer derived cell line.
Treatment of cell cultures: Cells in exponential growth phase were exposed to cisplatin 24h after plating. Cells were harvested after 24h and 48h of exposure and prepared for further analysis. Mock treated cells used as control (referance)
Microarray analysis: Array slides, sample preparation, and hybridisation:
-Human oligo array slides with 21K genes produced at the Norwegian Microarray Consortium were used.
-Total RNA was isolated from the cell lines by GeneElute kit (Sigma). 20μg of total RNA was used to generate a probe for hybridization, using FairPlay kit from Stratagene. Hybridization was performed overnight at 42°C.
-The slides were scanned by Agilent microarray laser Scanner (Agilent, Palo Alto, CA).
Data analysis:The microarray images were analyzed using the GenePix 4.1 image analysis software (Axon Instruments). Flagged spots were filtered out and normalized using lowess normalization. All data filtering and normalization was performed in J-Express Pro v 2.7 (MolMine, Bergen, Norway). The missing values were estimated by the k-nearest neighbor imputation (k=15).
To minimize the experimental variations, the cells were treated in three independent experiments and the RNA isolated from these experiments were mixed and used as a template for the microarray assay. Each RNA sample was hybridized with two (some with three) arrays.

first: Gunnar

last: Brunborg

first: Birgitte

last: Lindeman


title: Diabetic mice 21 days after streptozotocin injection

status: Public on Sep 30 2007

city: Honolulu

first: John

last: Maresh

state: HI

address: 651 Ilalo

department: Medicine

country: USA

institute: University of Hawaii

summary: We used streptozotocin to induce a model of type I diabetes in transgenic mice that express green fluorescent protein under the control of an endothelial-specific promoter (Tie2 GFP). Three weeks after treatment, endothelial cells were isolated from animals with blood glucose > 350 mg/dl. Age-matched mice received sham intraperitoneal injections. Aortae from the root to the renal bifurcation were rapidly processed by mincing and proteolytic digestion followed by fluorescent activated cell sorting to yield endothelial cell populations of >95% purity. RNA was isolated from >50,000 endothelial cells and subjected to oligodT amplification prior to transcriptional analysis on microarrays displaying the Operon V3 collection of long oligonucleotides representing 32,000 murine transcripts. Dysregulated transcripts were confirmed by real-time PCR, and included glycam1, angptl4, slc36a2, cidea, adam5, ces3, adipsin and adiponectin. By comprehensively examining cellular gene responses in vivo in a whole animal model of type I diabetes, we have identified novel regulation of key endothelial transcripts that likely contribute to the metabolic, angiogenic and pro-inflammatory responses which accompany diabetes.

overall_design:
Mice homozygous for the Tie2-GFP transgene (Tg[TIE2GFP]287Sato, stock number 003658) were obtained from Jackson labs (Bar Harbor, ME) and bred for these experiments.  Males were used at 6-10 weeks of age. Controls were siblings of the treated animals.   All procedures were approved by the Institutional Animal Care Committee of the University of Hawaii.

Induction of Diabetes
Following an overnight fast, a single 180 mg/kg dose of streptozotocin (STZ, Sigma, St. Louis, MO) was injected into the peritoneum. Control animals received injections of sterile saline.  Animals were maintained on normal chow and cages changed daily.  At the time of sacrifice, glucose levels in cardiac blood were assessed by glucometer (Accucheck, Becton Dickinson).   Diabetic, STZ-treated animals (with a glucose level >350 mg/dL) and controls were processed on the same day.  Two independent experiments both performed 21 days after STZ-injection.
Animals were euthanized by CO2 asphyxiation.  The aortas from the iliac bifurcation to the aortic root were excised by dissection and freed of any adherent tissue.  The luminal blood was removed and the aortas were sliced into 2 mm segments.  The aortic segments pooled from 3-4 animals were suspended in 5 ml of Dulbecco’s PBS with dextrose 2 mg/ml. The suspension was combined with 5 ml of prewarmed PBS to contain 5mg/ml type II collagenase (Worthington), and deoxyribonuclease I (300 units), agitated continuously at 37°C on a shaking platform, and triturated 10 times every 10 minutes for a total digestion period of 40min to generate a single cell suspension. The cell suspension was maintained at 0-4ºC throughout the remainder of the isolation, which lasted 2-3 hours total. The suspension was combined with 10 ml of 10% FBS in DMEM and cells were collected by centrifugation and resuspended in 10 ml of PBS. This suspension was then filtered through a sterile 40 um mesh filter to remove undigested tissue fragments.  Following centrifugation, the pellet was resuspended in 3.5 ml PBS containing 0.5 mM EDTA, 30U/ml DNAse I, 3% FBS, and 2 mg/ml dextrose. The suspension was once again filtered through a 40 um mesh filter.
Aortic cell suspensions were sorted using a MoFlo from Dako Cytomation (Carpinteria, CA) or the FacsAria from Becton Dickinson.  Cells were excited by a 488 nm laser and GFP signals collected via the FL1 channel (510 to 550 nm). Positive cells (were collected directly into Trizol (Invitrogen, Carlsbad CA) and processed according to the manufacturer’s protocol.  Glycogen was added to facilitate precipitation of the sub-microgram quantities of RNA thus obtained.
RNA was purified further using MicroRNEasy system (Qiagen,) according to the manufacturer’s protocol.  The Qubit system (Invitrogen) was used to fluorometrically quantify the resultant RNA.Up to 20 ng RNA isolated from cells was then subjected to two cycles of amplification as described [9] using the Message Amp kit (Ambion, Austin, TX) according to manufacturer’s instructions. This produced approximately 5-30 ug of amplified cRNA after two rounds of amplification.

Microarray Analysis
2 ug of cRNA each from diabetic and control were labeled directly with Cy3 and Cy5 fluorophores with the MicroMax labeling kit (Perkin  Elmer) according to the manufacturer’s instructions and hybridized overnight to microscope slides containing Operon V3 long oligo array.  Following stringency washing, slides were scanned using the Molecular Devices 4000B (CA). Replicate hybridizations were performed (with Cy3-Cy5 dye-swapped) and were analyzed with Genepix and Acuity software from Molecular Devices.

middle: G

first: Ralph

middle: V

last: Shohet


title: Schizosaccharomyces pombe ployg mutant cells expression profile.

status: Public on Sep 28 2007

city: Singapore

first: Zhaoqing

last: Chu

address: 60, Biopolis Streat

country: Singapore

institute: Genome Institute of Singapore

summary:
Expression profiles of polg mutant cells reveal that many genes encoding proteins involved in cell wall biogenesis and stress response are induced, suggesting that polg mutant cells attempt to maintain growth potential and undergo extensive oxidative metabolism.  Conversely, many genes encoding proteins involved in ribosome biogenesis and respiration are repressed, indicating that cells depleted of mtDNA are adapted to grow slowly in absence of mitochondrial function.
Keywords: Schizosaccharomyces pombe ployg mutant cells vs wild type cells.

overall_design: We analyzed 5 arrays of polyg mutant cells vs wild type cells.

first: Jianhua

last: Liu


title: Intraprostatic Androgens and Androgen-Regulated Gene Expression Persist Following Testosterone Suppression

status: Public on Oct 25 2007

city: Seattle

first: Denise

last: Mauldin

state: WA

address: 1100 Fairview Ave N D4-100

department: Human Biology

institute: Fred Hutchinson Cancer Research Center

summary:
Introduction: Androgen deprivation therapy (ADT) remains the primary treatment for advanced prostate cancer (PCa). The efficacy of ADT has not been rigorously evaluated by demonstrating suppression of prostatic androgen activity at the target tissue and molecular level. We determined the efficacy and consistency of medical castration in suppressing prostatic androgen levels and androgen-regulated gene-expression.

Design: Androgen levels and androgen-regulated gene-expression (by microarray-profiling, qRT-PCR and immunohistochemistry) were measured in prostate samples from a clinical trial of short-term castration (1 month) using the GnRH-antagonist, Acyline, vs. placebo in healthy men.  To assess the effects of long-term ADT, gene-expression measurements were evaluated at baseline and after 3, 6 and 9 months of neoadjuvant ADT in prostatectomy samples from men with localized PCa.

Results: Medical castration reduced tissue androgens by 75% and reduced the expression of several androgen-regulated genes (NDRG1, FKBP5, TMPRSS2). However, many androgen-responsive genes, including the androgen receptor (AR) and PSA, were not suppressed after short-term castration, nor after 9 months of neoadjuvant ADT. Significant heterogeneity in PSA and AR protein expression was observed in PCa samples at each time-point of ADT.

Conclusions: Medical castration based on serum testosterone levels cannot be equated with androgen ablation in the prostate microenvironment. Standard androgen deprivation does not consistently suppress androgen-dependent gene-expression. Suboptimal suppression of tumoral androgen activity may lead to adaptive cellular changes allowing PCa cell survival in a low androgen environment. Optimal clinical efficacy will require testing of novel approaches targeting complete suppression of systemic and intracrine contributions to the prostatic androgen microenvironment.
Keywords: prostate, androgen, Acyline, microarray, immunohistochemistry

overall_design:
Laser Capture Microdissection and RNA Amplification. Prostate samples embedded in OCT were used for laser capture microdissection (LCM). Approximately 2000-3000 epithelial cells per sample were collected from 8µm sections using the Arcturus Veritas™ Laser Capture Microdissection System according to the Arcturus HistoGene LCM Frozen Section Staining Kit Protocol (Mountain View, CA). Total RNA was isolated using the Arcturus Picopure™ Kit, and the samples were treated with DNAse using the Qiagen RNase-Free DNase Set (Qiagen Inc, Valencia, CA). Total RNA was subjected to two rounds of linear amplification using the Ambion MessageAmpII Kit (Ambion Inc, Austin, TX), quantitated in a Gene-Spec III spectrophotometer (Hitachi, Tokyo) and aRNA integrity evaluated using gel electrophoresis. RNA was extracted from microdissected benign epithelial cells from all subjects in the Acyline trial (placebo, castrate , and castrate +T; n=4 in each group), and from microdissected malignant epithelial cells from a randomly selected subset of patients in the neoadjuvant ADT trial (at 0, 3-6, and 6-9 months of treatment; n=3 in each group).

cDNA Microarray Hybridization and Analysis. cDNA probe pairs were prepared from samples obtained in the Acyline trial by amino-allyl reverse transcription using 2 µg of amplified RNA from the microdissected samples and 2µg of amplified RNA from a benign prostate reference standard. The reference was created by pooling equal amounts of RNA amplified from the microdissected placebo-treated prostate epithelial samples. Samples from the neoadjuvant trial of ADT were prepared using 2 µg of amplified RNA from the microdissected samples and 30 µg of total RNA from a reference RNA pool comprised of total RNA isolated from LNCaP, DU145, and PC3 prostate cancer cell lines.  Probes were labeled with either Cy5 or Cy3 fluors (Amersham Bioscience, Piscataway, NJ) and competitively hybridized to custom cDNA microarrays spotted in duplicate with approximately 6,700 unique cDNA clones from the Prostate Expression Database (PEDB) as previously described (Nelson PS, Pritchard C, Abbott D, and Clegg N. The human (PEDB) and mouse (mPEDB) Prostate Expression Databases. Nucleic Acids Res 2002;30:218-220. AND Pritchard CC, Hsu L, Delrow J, and Nelson PS. Project normal: defining normal variance in mouse gene expression. Proc Natl Acad Sci U S A 2001;98:13266-13271).

Fluorescence array images were collected using a GenePix 4000B fluorescent scanner (Axon Instruments, Foster City, CA) and processed as we have previously described (True L, Coleman I, Hawley S, et al. A molecular correlate to the Gleason grading system for prostate adenocarcinoma. Proc Natl Acad Sci U S A 2006;103:10991-10996).  Changes in gene expression were evaluated using the Statistical Analysis of Microarray (SAM) program (http://www-stat.stanford.edu/_tibs/SAM/)  to perform a one-sample t-test assessing  the effect of Acyline treatment on gene expression (Tusher VG, Tibshirani R, and Chu G. Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A 2001;98:5116-5121.).  An FDR (false discovery rate) of less than 5% was considered significant. Quantile normalization was performed in Bioconductor (Gentleman RC, Carey VJ, Bates DM, et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 2004;5:R80) to allow single channel comparison of the absolute signal intensities in the experimental channel across different arrays in the same experiment.

first: Elahe

last: Mostaghel

first: Stephanie

middle: T

last: Page

first: Daniel

middle: W

last: Lin

first: Ladan

last: Fazli

first: Ilsa

middle: M

last: Coleman

first: Lawrence

middle: D

last: True

first: Beatrice

last: Knudsen

first: David

middle: L

last: Hess

first: Colleen

middle: C

last: Nelson

first: Alvin

last: Matsumoto

first: William

middle: J

last: Bremner

first: Martin

middle: E

last: Gleave

first: Peter

middle: S


title: A Microarray-based Analysis of Transcriptional Compartmentalization in the Alimentary Canal of Anopheles gambiae

city: St. Augustine

first: Marco

middle: V.

last: Neira

state: FL

address: 9505 Oceanshore Blvd.

department: The Whitney Laboratory

institute: University of Florida

summary:
Although the basic anatomical sub-divisions of the larval mosquito gut were established several decades ago, information regarding their exact physiological roles is rather scarce. Several studies have reported differences between larval gut compartments in various morphological and physiological aspects. Unfortunately, the fragmentary and incomplete nature of this information makes it hard to establish clear links to the specific and/or unique physiological roles of each gut region.
In this study, we attempted to identify transcripts significantly enriched in each  gut section of the 4th instar Anopheles gambiae larva. We took advantage of a commercially-available microarray platform containing ~14,000 different An. gambiae transcripts (Affymetrix GeneChip® Plasmodium/Anopheles array, Affymetrix Inc., Santa Clara, CA). Based on the information obtained, we discuss the putative physiological roles of each major compartment of the larval gut, and identify potential new targets for larval control strategies.
Keywords: tissue comparison

platform_organism:
Plasmodium falciparum
Anopheles gambiae

overall_design: We attempted to identify transcripts significantly enriched in each  gut section of the 4th instar Anopheles gambiae larva. Signal intensities of transcripts from each gut section were compared to those of a matching group of whole 4th instar larvae using t-tests. Transcripts showing an intensity value two-fold or higher (p < 0.05) in each gut section as compared to whole larvae were considered to be significantly enriched.

last: Neira Oviedo

first: Leslie

last: VanEkeris

first: Maria

last: Corena-Mcleod

first: Paul

last: Linser


title: Integrated epigenomic analyses of neuronal MeCP2 reveal a role for long-range interaction with active genes

status: Public on Dec 05 2007

city: Davis

first: Dag

last: Yasui

state: CA

address: 1 Shields Avenue

department: Medical Microbiology and Immunology

institute: University of California at Davis

type: Genome binding/occupancy profiling by genome tiling array

summary:
Mutations in MECP2 cause the autism-spectrum disorder Rett syndrome. MeCP2 is predicted to bind to methylated promoters and silence transcription. However, the first large-scale mapping of neuronal MeCP2-binding sites on 26.3 Mb of imprinted and nonimprinted loci revealed that 59% of MeCP2-binding sites are outside of genes and that only 6% are in CpG islands. Integrated genome-wide promoter analysis of MeCP2 binding, CpG methylation, and gene expression revealed that 63% of MeCP2-bound promoters are actively expressed and that only 6% are highly methylated. These results indicate that the primary function of MeCP2 is not the silencing of methylated promoters.
Keywords: ChIP-chip analysis

overall_design:
Experiments were performed using SH-SY5Y cells differentiated into a neuronal phenotype by 48 hour treatment with PMA. For custom genomic ChIP-chip analysis, chromatin from 3 biologic replicate cultures of SH-SY5Y neurons was crosslinked with formaldehyde, sonicated then immunoprecipitated with antibodies to MeCP2 and Pol2. Ligation mediated PCR amplified chromatin fragments were labeled then hybridized to a custom human genomic tiling array designed by Nimblegen along with differentially labeled total genomic DNA.
     To examine the relationship of MeCP2 promoter binding to gene expression, MeCP2 ChIP amplicons from two biologic replicates of SH-SY5Y cells were hybridized to Nimblegen 1.5kb human promoter arrays along with total genomic DNA. MeCP2 promoter binding levels were then compared with identically treated SH-SY5Y RNA transcript levels as assayed by expression profiling analysis using Affymetrix human U133A Plus 2.0 microarrays performed from 3 biologic replicates.
     To examine the relationship between MeCP2 binding and gene promoter methylation levels MeDIP analysis was performed again using SH-SY5Y cells differentiated with PMA. Genomic DNA was purified from SHSY-5Y cells, sonicated then immunoprecipitated with an anti-methylcytidine antibody. Selected fragments were then hybridized to Nimblegen 1.5kb human promoter arrays along with total genomic DNA. Methylcytidine levels of human promoters were then compared with MeCP2 binding levels determined in the analysis of MeCP2 promoter binding and gene expression levels.

middle: H

first: Sailaja

last: Peddada

first: Mark

last: Bieda

first: Roxanne

middle: O

last: Vallero

first: Amber

last: Hogart

first: Raman

middle: P

last: Nagarajan

first: Karen

middle: N

last: Thatcher

first: Peggy

last: Farnham

first: Janine

last: LaSalle


title: Determination of  a cell proliferation and chromosomal instability signature in anaplastic thyroid carcinoma

status: Public on Sep 21 2007

city: Phoenix

first: Michael

last: Bittner

state: AZ

address: 445 N. 5th Street

department: Computational Biology

institute: Translational Genomics Research Institute

summary:
A comparison of profiles of normal thryoid tissue (NT), papillary thyroid carcinoma tissue (PTC) and anaplastic thyroid carcinoma tissue (ATC) was carried out to identify expression patterns specifically associated with analplastic thyroid carcinoma
Keywords: Expression profile survey of normal tissue and tumor subtypes

overall_design: RNA from each of the 4 normal thyroids, 10 papillary thryoid carcinomas and 5 analplastic thryoid carcinomas was labeled (Cy5) and co-hybridized with RNA from a pool of throid cancer cell lines to an array of immobilized PCR products representing the inserts of human cDNA clones

first: Giuliana

last: Salvatore

first: Tito

last: Nappi

first: Paolo

last: Salerno

first: Yuan

last: Jiang

first: Corrado

last: Garbi

first: Francesco

last: Curcio

first: Pinuccia

last: Faviana

last: Miccoli

first: Fulvio

last: Basolo

first: Rosa

last: Melillo

first: Alfredo

last: Fusco

first: Massimo

last: Santoro


title: Gene-expression profiling of Saccharomyces cerevisiae irradiated by high-LET radiations

status: Public on Nov 01 2007

city: Tsukuba

first: Emiko

last: Kitagawa

state: Ibaraki

address: Onogawa 16-1

country: Japan

institute: National Institute of Advanced Industrial Science and Technology

summary:
Ionizing radiations are categorized by linear energy transfer (LET) into low-LET and high-LET.  High-LET is considered to have a higher relative biological effectiveness (RBE) than low-LET radiations.  However, the details of the effects have not been clearly determined.  The aim of this study was to characterize the difference between high-LET and low LET radiations.  The global effects of the three types of high-LET radiations (fast neutron, heavy ion (C) and thermal neutron) were compared with the low-LET radiation (gamma ray) using yeast DNA microarrays.  Highly induced genes by the three types of high-LET radiations were those genes related to oxidative stress.  Oxidative stress was one of the common factors associated with the four types of radiations.  Oxidative stress induced by high-LET radiations may be more serious than that induced by gamma rays.  Additionally, genes related to protein synthesis and the ubiquitin and proteasome system were detected.  This suggests that more protein damages can be induced by high-LET radiation that denatures the proteins in yeast cells.  The genes specifically altered by each type of high-LET radiation were also studied.
Keywords: stress response

overall_design: This series contains 4 kinds of irradiation-induced gene expression profiles. Triplicates hybridization was done in each irradiation exposure, and each array have high and low power scanned data respectively. All biological samples were collected independently.

first: Satomi

last: Murata

first: Hitoshi

last: Iwahashi


title: Integrated SV40 T/t-antigen Cancer Signature in Aggressive Breast, Prostate and Lung Carcinomas with Poor Prognosis 2

status: Public on Sep 01 2007

city: Bethesda

first: Jeffrey

last: Green

state: MD

address: Rm 4054, 37 Convent Drive

institute: NCI

summary:
Understanding the genetic architecture of cancer pathways that distinguishes subsets of human cancer is critical to developing new therapies that better target tumors based upon their molecular expression profiles.   In this study, we identify an integrated gene signature from multiple transgenic models of epithelial cancers intrinsic to the functions of the Simean virus 40 T/t-antigens that is associated with the biologic behavior and prognosis for several human epithelial tumors.  This genetic signature, composed primarily of genes regulating cell replication, proliferation, DNA repair and apoptosis, is not a general cancer signature. Rather, it is uniquely activated primarily in tumors with aberrant p53, Rb or BRCA1 expression, but not in tumors initiated through the overexpression of myc, ras, her2/neu, or Polyoma middle T oncogenes.  Importantly, human breast, lung and prostate tumors expressing this set of genes represent subsets of tumors with the most aggressive phenotype and with poor prognosis.  The T/t-antigen signature is highly predictive of  human breast cancer prognosis.  Since this class of epithelial tumors is generally intractable to currently existing standard therapies, this genetic signature identifies potential targets for novel therapies directed against these lethal forms of cancer.  Since the these genetic targets have been discovered using mammary, prostate, and lung T/t-antigen mouse cancer models, these models are rationale candidates for use in pre-clinical testing of therapies focused on these biologically important targets.
Keywords: Genetically engineered mouse (GEM) models of cancer, epithelial carcinoma, SV40 T/t-antigen, survival predictor

overall_design:
Gene expression profiles from the SV40 T/t-antigen mouse models were compared with respect to specimen type (normal vs. tumor tissue), location of tumor (mammary, lung, prostate, seminal vesicle), and background strain of mice (FVB vs. C57BL/6). A three-way ANOVA model with one interaction effect (type X location) was fitted. Cancer genes that differ among all four tumor locations were identified, as those that had a significant interaction effect at the 0.001 level and showed at least a 2-fold change between the maximal and minimal mean tumor/normal ratio over the different locations (2638 cDNA probes). Based on the ANOVA model, differentially expressed genes between normal and tumor specimens within each tumor location were also identified, as those genes whose expression were significant at the 0.001 level and were at least 2-fold different compared to the mean expression ratio. Overall 3004 unique array features were selected using ANOVA. Further selection was applied based upon identification of differentially expressed genes between normal and tumor tissue for the three epithelial tumors (mammary, lung and prostate). The SV40 T/t-antigen oncogene-specific signature included genes similarly differentially expressed in each epithelial tumor (153 cDNA clones). In contrast, genes were included in a tissue-specific SV40 T/t-antigen tumor signature if they were found to be differentially expressed between the tumor and normal samples exclusively for one location. Two-hundred and eighty three, 220 and 999 cDNA clones were identified as specifically dysregulated in mammary, lung and prostate tumors, respectively).
This set of the mammary mouse tumor models was used to demonstrate that the intrinsic SV40 T/t-antigen signature is not a feature of tumors initiated by other oncogenes or inactivation of supressor genes, but is most specific to tumors induced by T/t-antigen.

first: Kristin

middle: K

last: Deeb

first: Aleksandra

last: Michalowska

first: Cheol-yong

last: Yoon

first: Scott

last: Krummey

first: Mark

last: Hoenerhoff

first: Claudine

last: Kananaugh

first: Ming-chung

last: Li

last: Demayo

first: Ilona

last: Linnoila

first: Chu-xia

last: Deng

first: Eva

last: Lee

first: Daniel

last: Medina

first: Joanna

last: Shih


title: Integrated SV40 T/t-antigen Cancer Signature in Aggressive Breast, Prostate and Lung Carcinomas with Poor Prognosis 1

overall_design: Gene expression profiles from the SV40 T/t-antigen mouse models were compared with respect to specimen type (normal vs. tumor tissue), location of tumor (mammary, lung, prostate, seminal vesicle), and background strain of mice (FVB vs. C57BL/6). A three-way ANOVA model with one interaction effect (type X location) was fitted. Cancer genes that differ among all four tumor locations were identified, as those that had a significant interaction effect at the 0.001 level and showed at least a 2-fold change between the maximal and minimal mean tumor/normal ratio over the different locations (2638 cDNA probes). Based on the ANOVA model, differentially expressed genes between normal and tumor specimens within each tumor location were also identified, as those genes whose expression were significant at the 0.001 level and were at least 2-fold different compared to the mean expression ratio. Overall 3004 unique array features were selected using ANOVA. Further selection was applied based upon identification of differentially expressed genes between normal and tumor tissue for the three epithelial tumors (mammary, lung and prostate). The SV40 T/t-antigen oncogene-specific signature included genes similarly differentially expressed in each epithelial tumor (153 cDNA clones). In contrast, genes were included in a tissue-specific SV40 T/t-antigen tumor signature if they were found to be differentially expressed between the tumor and normal samples exclusively for one location. Two-hundred and eighty three, 220 and 999 cDNA clones were identified as specifically dysregulated in mammary, lung and prostate tumors, respectively).

first: Francesco


title: Invasion control by a positive feedback loop mechanism Smad4-null cells

city: Beijing

first: yu

last: jian

state: Beijing

address: Qinghuayuan

department: Medical Systems Biology Research Center

country: China

institute: Tsinghua University

summary:
New findings demonstrate that transcriptional factors alternative to Smad4 can bind to Smad2/3 and mediate different transcriptional effects. In this study, we detected constitutively phosphorylation of Smad2/3 in Smad4-null pancreatic cancer cell line BxPC-3. Both pan-specific TGF-β-neutralizing antibody and specific TGF-β type I receptor (TβR-I) inhibitor, SB-431542, can decrease steady-state p-Smad2/3 levels. Moreover, exogenous TGF-β strongly stimulated translocation of phosphorylated Smad2/3 (p-Smad2/3) into the nucleus. Therefore, we identified TGF-β-responsive genes using genome-wide oligonucleotide microarrays and confirmed their dependency on Smad2/3 by the combination of RNA interference (RNAi) and microarray assay. The major finding from our microarray analysis was that of the 262 target genes seen to be regulated via TGF-β induction, 87 were differentially transcriptionally controlled by Smad2/3 signaling and 175 were Smad2/3-independent. Our results showed that integrin β6 was transcriptionally up-regulated via TGF-β induction in a Smad3-dependent manner, which was validated by real-time RT-PCR and western blot. We also provide evidence that αVβ6 integrin can activate TGF-β-Smad2/3 signaling. Thus, we for the first time suggest the positive feedback loop compose of TGF-β-Smad3 signaling and integrin β6. Functional analysis revealed that exogenous TGF-β can amplify the invasive property of Smad4-deficient pancreatic cancer cells; however, TGF-β-neutralizing antibody, specific TβR-I inhibitor, and anti-αVβ6 integrin antibody can reduce it. Therefore, integrin β6 mediated the invasion of BxPC-3 cells induced by TGF-β signaling.
Keywords: cell type comparison

overall_design: Large-scale microarray analysis was employed for comparison between the responses of wild-type BxPC-3 cell line and each knockdown cell line, with and without TGF-β induction, to identify target genes.

first: Jian

last: Yu

first: Liang

last: Zhang


title: Human bronchial epithelial cells_Passage 3 vs. passage 0

status: Public on Aug 31 2007

city: San Francisco

first: Yuanyuan

last: Xiao

address: 185 Berry Street, Lobby4, Suite 5700

department: Epidemiology and Biostatistics

institute: UCSF

summary:
Squamous metaplasia is common in smokers and is associated with airway obstruction in chronic obstructive pulmonary disease (COPD). A major mechanism of airway obstruction in COPD is thickening of the small airway walls. We asked whether squamous metaplasia actively contributes to airway wall thickening through alteration of epithelial-mesenchymal interactions in COPD. Using immunohistochemical staining, airway morphometry and fibroblast culture of lung samples from COPD patients, genome-wide analysis of a model of squamous metaplasia, and in vitro modeling of human airway epithelial-mesenchymal interactions, we have produced evidence that squamous metaplasia, through the increased secretion of IL-1, induces a fibrotic response of adjacent airway fibroblasts. We identify a pivotal role for integrin-mediated TGF-b activation in amplifying squamous metaplasia and driving IL-1-dependent profibrotic mesenchymal responses. Finally, we show that squamous metaplasia correlates with increasing severity of COPD and fibroblast expression of the integrin avb8, which is the major mediator of airway fibroblast TGF-b activation, correlates with disease severity and small airway wall thickening in COPD.
Keywords: genome-wide differential expression study

overall_design: A dye-swap design of three two-channel arrays

first: Jun

last: Araya

first: Stephanie

last: Cambier

first: Paul

last: Wolters

first: David

last: Jablons

first: Arthur

last: Hill

first: Walter

last: Finkbeiner

first: Kirk

last: Jones

first: Courtney

last: Broaddus

first: Dean

last: Sheppard

first: Andrea

last: Barzcak

last: Erle

first: Stephen

last: Nishimura


title: Suz12 ChIP-chip using Avi-6k-hu

city: Chicago

first: Jindan

state: IL

address: 303 E. Superior St.

department: Medicine - Hem/Onc

institute: Northwestern University

summary:
The polycomb group (PcG) protein, EZH2, possesses oncogenic properties for which the underlying mechanism is unclear. In this set of experiments, we sought to identify a robust set of Suz12-occupied gene promoters in prostate cancer cell lines. Genome-wide location analysis (ChIP-chip) of SUZ12 was performed in both the PC3 and LNCaP prostate cancer cell lines using an IgG ChIP-chip as a control.
Keywords: ChIP-chip

overall_design: There are total 3 hybridizations, including IgG ChIP-chip of PC3 cells, Suz12 ChIP-chip of PC3 and LNCaP cells.

first: Arul

last: Chinnaiyan


title: INDUCTION OF PANCREATIC ISLET CELL DIFFERENTIATION BY THE NEUROGENIN-NEUROD CASCADE

status: Public on Aug 29 2007

first: Michael

middle: Scott

last: German

address: 513 Parnassus, HSW 1090

department: Diabetes Center

institute: University of California San Francisco

summary:
Neurog3 and NeuroD1 share the ability to induce endocrine differentiation when expressed ectopically in vivo (Apelqvist et al., 1999; Schwitzgebel et al., 2000; Grapin-Botton et al., 2001) and in vitro  (Heremans et al., 2002; Gasa et al., 2004), suggesting that they target a common set of genes. To test this hypothesis, we directly compared in the same cells the effects of Neurog3 and NeuroD1 on global gene expression patterns.

Normalized M and A values [limmaGUI output] for the neurogenin3 (Ngn3) and neuroD1 (Nd1) experiments (individual or combined) have been linked below as Supplementary files.
Note that M=log2(test/ref), eg, log2(Nd1/Bgal); A=[log2(test*ref)]/2, eg, [log2(Nd1*Bgal)]/2. Additional measures of reliability/probability are also included within the supplementary files.
Keywords: comparative genomic hybridization, neurogenin3, neuroD, ductal cells, endocrine, differentiation

overall_design: Mouse pancreatic ductal cells (mPAC cells) were infected with equal titers of recombinant adenoviruses encoding human Neurogenin3 (AdCMV-NEUROG3) or mouse NeuroD1 (AdCMV-NeuroD1), and 48 hrs later their transcriptomes were compared with that of control cells infected with an adenovirus encoding betagalactosidase (beta-gal; AdCMV-Bgal) using cDNA microarrays. Differences in the activation patterns of the two transcription factors were then determined by comparing the fold-change (FC) values of the pair-wise hybridizations Neurog3/Bgal and NeuroD1/beta-gal.

first: Rosa

last: GASA

first: Caroline

last: MREJEN

first: Francis

last: LYNN

first: Lidia

last: SANCHEZ

first: Katherine

last: YANG

first: Chin-Hsing

last: LING

first: Ramon

last: GOMIS

last: GERMAN

first: Peter

last: SKEWES-COX


title: Comparison of Normal Prostate Fibroblasts Overexpressing Cyclin D1 and Carcinoma Associated Fibroblasts

first: Denise

last: Mauldin

summary:
The cyclin D1 oncogene encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the Rb protein and promotes progression through G1 to S phase of the cell cycle. Several prostate cancer cell lines and a subset of primary prostate cancer samples have increased cyclin D1 protein expression. However, the relationship between cyclin D1 expression and prostate tumor progression has yet to be clearly characterized. This study examined the effects of manipulating cyclin D1 expression in either human prostatic epithelial or stromal cells using a tissue recombination model. The data showed that overexpression of cyclin D1 in the initiated BPH-1 cell line increased cell proliferation rate, but did not elicit tumorigenicity in vivo. However, overexpression of cyclin D1 in Normal Prostate Fibroblasts (NPF) that were subsequently recombined with BPH-1 did induce malignant transformation of the epithelial cells. The present study also showed that recombination of BPH-1 + cyclin D1 overexpressing fibroblasts (NPF cyclin D1) resulted in permanent malignant transformation of epithelial cells (BPH-1 NPF-cyclin D1 cells) similar to that seen with Carcinoma Associated Fibroblasts (CAFs). Microarray analysis showed that the expression profiles between CAFs and NPF cyclin D1 cells were highly concordant including cyclin D1 upregulation. These data indicated that the tumor-promoting activity of cyclin D1 may be tissue-specific.
Keywords: cyclin D1; stromal-epithelial interactions; prostate cancer; cDNA microarray

overall_design: NPF cyclin D1 cells were generated from NPFs, which were isolated from two different patient samples; CAFs were isolated from two different patient samples as well. RNA was isolated from NPFs, CAFs, and NPF cyclin D1 cells using total RNA isolation kit (Qiagen). Custom spotted cDNA microarrays were constructed as previously described (True, L., Coleman, I., Hawley, S., Huang, C. Y., Gifford, D., Coleman, R., Beer, T. M., Gelmann, E., Datta, M., Mostaghel, E., Knudsen, B., Lange, P., Vessella, R., Lin, D., Hood, L., and Nelson, P. S. A molecular correlate to the Gleason grading system for prostate adenocarcinoma. Proc Natl Acad Sci U S A, 103: 10991-10996, 2006) using a non-redundant set of 6,700 prostate-derived cDNA clones identified from the Prostate Expression Data Base (PEDB), a public sequence repository of expressed sequence tag data derived from human prostate cDNA libraries. Total RNA was amplified through one round of linear amplification using the MessageAmp aRNA kit (Ambion, Austin, TX). Sample quality and quantification was assessed by agarose gel electrophoresis and absorbance at A260. Cy3 and Cy5 labeled cDNA probes were made from 4 µg of amplified RNA. Two NPF cyclin D1 and two CAF samples (labeled with Cy3) were hybridized head-to-head with an NPF control sample labeled with Cy5. Probes were hybridized competitively to microarrays under a coverslip for 16 h at 63°C. Fluorescent array images were collected for both Cy3 and Cy5 by using a GenePix 4000B fluorescent scanner, and image intensity data were gridded and extracted using GenePix Pro 4.1 software. Differences in gene expression between NPF cyclin D1/NPF and CAF/NPF groups were determined using a two-sample t-test with Significance Analysis of Microarrays (SAM) software (http://www-stat.stanford.edu/_tibs/SAM/) with a False Discovery Rate (FDR) of ≤ 10% considered significant (Tusher, PNAS, 2001).  Similarities in gene expression between NPF cyclin D1/NPF and CAF/NPF groups were determined using a one-sample t-test in SAM with an FDR of ≤ 0.1% considered significant. These results were reduced to unique genes by eliminating all but the highest scoring clones for each gene.  A Pearson correlation coefficient was calculated in Excel to assess the strength of the linear relationship between NPF cyclin D1/NPF and CAF/NPF average log2 ratios.

first: Yue

last: He

first: Omar

last: Franco

first: Karin

last: Williams

first: Harold

last: Love

first: Ilsa

last: Coleman

last: Nelson

first: Simon

last: Hayward


title: Integrated SV40 T/t-antigen Cancer Signature in Aggressive Breast, Prostate and Lung Carcinomas with Poor Prognosis

first: Jeffrey

last: Green

summary: Understanding the genetic architecture of cancer pathways that distinguishes subsets of human cancer is critical to developing new therapies that better target tumors based upon their molecular expression profiles.   In this study, we identify an integrated gene signature from multiple transgenic models of epithelial cancers intrinsic to the functions of the Simean virus 40 T/t-antigens that is associated with the biologic behavior and prognosis for several human epithelial tumors.  This genetic signature, composed primarily of genes regulating cell replication, proliferation, DNA repair and apoptosis, is not a general cancer signature. Rather, it is uniquely activated primarily in tumors with aberrant p53, Rb or BRCA1 expression, but not in tumors initiated through the overexpression of myc, ras, her2/neu, or Polyoma middle T oncogenes.  Importantly, human breast, lung and prostate tumors expressing this set of genes represent subsets of tumors with the most aggressive phenotype and with poor prognosis.  The T/t-antigen signature is highly predictive of  human breast cancer prognosis.  Since this class of epithelial tumors is generally intractable to currently existing standard therapies, this genetic signature identifies potential targets for novel therapies directed against these lethal forms of cancer.  Since the these genetic targets have been discovered using mammary, prostate, and lung T/t-antigen mouse cancer models, these models are rationale candidates for use in pre-clinical testing of therapies focused on these biologically important targets.

first: Kristin

last: Deeb

first: Aleksandra

last: Michalowska

first: Cheol-yong

last: Yoon

first: Scott

last: Krummey

first: Mark

last: Hoenerhoff

first: Claudine

last: Kananaugh

first: Ming-chung

last: Li

first: Francesco

last: Demayo

first: Ilona

last: Linnoila

first: Chu-xia

last: Deng

first: Eva

last: Lee

first: Daniel

last: Medina

first: Joanna

last: Shih


title: A ChIP-chip analysis of E2F family in normal and tumor cells reveals interchangeable roles of E2F family members

status: Public on Aug 22 2007

city: Columbus

first: Victor

middle: X

last: Jin

state: OH

address: 333 West 10th Ave

department: Biomedical Informatics

institute: OSU Medical Center

summary:
Using ChIP-chip assays, we examined the binding patterns of three members of E2F family in five different cell types.
Keywords: ChIP-chip

overall_design: We used ENCODE arrays to determine the loaction of E2F family, then identify their binding motifs in vivo. We used promoter arrays to assess the relationship of E2F family in five different cells including tumor and normal cells.

first: Xiaoqin

last: Xu

last: Bieda

first: Alina

last: Rabinovich

first: Mathew

last: Oberley

first: Roland

first: Peggy

last: Farnham


title: IRF2 regulates macrophages apoptosis through a STAT1/3 and Caspase-1-dependent mechanism

city: Gaithersburg

first: Enrique

last: Zudaire

address: 8717 Grovemont Circle

department: CCR

institute: NIH

summary:
Using microarray gene expression profiling of liver RNA samples rerived from IRF2+/+ and IRF2-/- mice treated with saline or LPS, we identified >40 genes that were significantly down-regulated in IRF2 -/- mice, including STAT3 which has been reported to regulate apoptosis.
Keywords: compound treatment design

overall_design: We compared gene expression in IRF2+/+ and IRF2-/- mice treated with Saline or LPS for 3 or 6 hours. Two repeats were done for the LPS treatments. The total number of arrays is 6.


title: Anopheles gambiae genome-wide gene expression in response to bloodmeal

status: Public on Aug 20 2007

city: Irvine

first: Osvaldo

last: Marinotti

address: University of California, Irvine

department: Molecular Biology & Biochemistry

institute: University of California, Irvine

summary:
With their genome sequenced, Anopheles gambiae mosquitoes now serve as a powerful tool for basic research in comparative, evolutionary and developmental biology. The knowledge generated by these studies is expected to reveal molecular targets for novel vector control and pathogen transmission blocking strategies. Comparisons of gene-expression profiles between adult male and nonblood-fed female Anopheles gambiae mosquitoes revealed that roughly 22% of the genes showed sex-dependent regulation. Blood-fed females switch the majority of their metabolism to blood digestion and egg formation within 3 h after the meal is ingested, in detriment to other activities such as flight and response to environment stimuli. Changes in gene expression are most evident during the first, second and third days after a blood meal, when as many as 50% of all genes showed significant variation in transcript accumulation. After laying the first cluster of eggs (between 72 and 96 h after the blood meal), mosquitoes return to a nongonotrophic stage, similar but not identical to that of 3-dayold nonblood-fed females. Ageing and/or the nutritional state of mosquitoes at 15 days after a blood meal is reflected by the down-regulation of 5% of all genes. A full description of the large number of genes regulated at each analysed time point and each biochemical pathway or biological processes in which they are involved is not possible within the scope of this contribution. Therefore, we present descriptions of groups of genes displaying major differences in transcript accumulation during the adult mosquito life. However, a publicly available searchable database (Anopheles gambiae Gene Expression Database at UC Irvine) has been made available so that detailed analyses of specific groups of genes based on their descriptions, functions or levels of gene expression variation can be performed by interested investigators according to their needs.
Keywords: response to bloodmeal

overall_design: 12 Samples analyzed, 3 replicates per sample, Affymetrix internal controls


title: Genome-wide Profiling of DNA Replication Timing in pombe.

status: Public on Aug 14 2007

first: Majid

last: Eshaghi

state: Singapore

address: Biopolis

department: Bilogical Investigation

summary:
DNA replication is initiated at multiple sites or origins enriched with AT-rich sequences at various times during the S-phase.  While current studies of genome-wide DNA replication profiles have focused on the timing of replication and the location of origins, the efficiency of replication/firing at various origins remains unclear.  In this study, we show different efficiencies of DNA replication at various loci by using ORF-specific DNA microarrays.  DNA copy-number increases as a function of time at individual loci are approximated to near-sigmoidal models for estimation of replication initiation and completion timings in HU-challenged cells.  Duplicating times (from initiation to completion) vary from loci to loci, partly contributing to various firing efficiencies at origins.  DNA replication timing profiles are strikingly similar to the reported patterns of enriched ssDNA, suggesting that majority stalled forks are restored for resumption of DNA replication.  Although the DNA replication timing profiles are disrupted in HU-challenged cds1? cells, ~85% of potential origins overlapped with those found in wild type cells, significantly, most of which represents inefficiently fired origins in wild type cells.  Together, our result indicates that replication checkpoint plays a role in monitoring efficient origins and thus maintaining global DNA replication patterns in HU-challenged cells.
Keywords: WT or Cds1 HU synchronized cells released in HU free media and harvested at different time points vs WT or Cds1 synchronized with HU for 3 hrs.

overall_design: We analyzed 32 arrays for WT and 38 arrays for Cds1 cells which were synchronized with HU and released in HU free media and harvested at different time points. At least two biological repeats were done for each time points.

first: Karuturi

last: R. Krishna M.

first: Juntao

first: Zhaoqing

last: Chu

first: Edison

last: T. Liu

first: JianhuaL

last: Liu


title: Effects of mycophenolic acid on human fibroblast proliferation, migration and adhesion in vitro and in vivo

status: Public on Jul 29 2007

city: Heidelberg

first: Hendrik

last: Reuter

department: Moleculare Genetics B060

country: Germany

institute: DKFZ

summary:
Mycophenolic acid (MPA) is a potent inhibitor of the inosine monophosphate dehydrogenase and commonly used as an immunosuppressive drug in transplantation. MPA inhibits proliferation of both T- and B-lymphocytes by guansoin depletion. Since fibroblasts rely on the de novo synthesis of guanosin nucleotides, it is assumed that MPA interacts with fibroblasts causing an increased frequency of wound healing problems. We show a downregulation of the cytoskeletal proteins actin, vinculin and tubulin in human dermal fibroblasts exposed to pharmacologic doses of MPA using microarray technology and western blot. This reduction in protein content is accompanied by a substantial derangement of the cytoskeleton in MPA-treated fibroblasts as documented by confocal microscopy. The dysfunctional fibroblast growth was validated by scratch test documenting impaired migrational capacity. The results of the cultured dermal fibroblasts were applied to skin biopsies of renal transplant recipients. Skin biopsies of patients treated with MPA expressed less tubulin and actin as compared to control biopsies which could explain potential wound healing problems post transplantation. The perspective of MPA-induced cytoskeletal dysfunction may go beyond wound healing disturbances and has potential beneficial effects on (renal) allografts with respect to scarring.
Keywords: Timecourse and MPA and/or Guanosin response

overall_design:
Human fibroblast are treated with Guanin and or Mycophenolic Acid (MPA). Samples were taken at different timepoints (20min, 1h, 12h,36h). Samples were amplified and hybridised against amplified RNA of Control human fibroblasts in dye-swaps to Human Oligo arrays.

For GPR file names with _1, the test sample is labeled with Cy3.
For GPR file names with _1, the test sample is labeled with Cy3.

first: Christian

last: Morath

first: Meinhard

last: Hahn

first: Martin

last: Zeier

first: Peter

last: Lichter


title: Whole genome analysis for liver metastasis gene signitures in colorectal cancer

status: Public on Feb 01 2008

city: Seoul

first: DONG HYUK

last: KI

address: sinchon-dong 134

country: South Korea

institute: NBRC

summary:
Liver metastasis is one of the major causes of death in colorectal cancer (CRC) patients. To understand this process, we investigated whether the gene expression profiling of matched colorectal carcinomas and liver metastases could reveal key molecular events involved in tumor progression and metastasis. We performed experiments using a cDNA microarray containing 17,104 genes with the following tissue samples: paired tissues of 25 normal colorectal mucosa, 27 primary colorectal tumors, 13 normal liver and 27 liver metastasis, and 20 primary colorectal tumors without liver metastasis. To remove the effect of normal cell contamination, we selected 4,583 organ-specific genes with a false discovery rate (FDR) of 0.0067% by comparing normal colon and liver tissues using significant analysis of microarray, and these genes were excluded from further analysis. We then identified and validated 46 liver metastasis-specific genes with an accuracy of 83.3% by comparing the expression of paired primary colorectal tumors and liver metastases using prediction analysis of microarray. The 46 selected genes contained several known oncogenes and 2 ESTs. To confirm that the results correlated with the microarray expression patterns, we performed RT-PCR with WNT5A and carbonic anhydrase II. Additionally, we observed that 21 of the 46 genes were differentially expressed (FDR = 2.27%) in primary tumors with synchronous liver metastasis compared with primary tumors without liver metastasis. We scanned the human genome using a cDNA microarray and identified 46 genes that may play an important role in the progression of liver metastasis in CRC.
Keywords: gene expression profiling using cDNA microarray

overall_design: We performed 17K cDNA microarray with the amplified RNAs from the following tissue samples: normal colorectal mucosa, primary colorectal tumors, normal liver and liver metastasis tumors, and primary colorectal tumors without liver metastasis. Organ-specific genes in normal colon and liver tissues were excluded from the pre-filtered genes, and then we discovered and validated liver metastasis-specific genes commonly up-regulated in the primary colorectal tumors and liver metastasis tumors. To confirm the microarray data, we performed a RT-PCR of two genes (WNT5A and carbonic anhydrase II) in the primary colorectal tumors with and without liver metastases.

first: DONGHYUK

first: HYUNCHUL

last: CHUNG

first: CHANHEE

last: PARK

first: SEUNGHEE

last: KANG

first: GUIYOUN

last: LEE

first: NAMKYU

last: KIM

first: HEI-CHEUL

last: JEUNG

first: SUNYOUNG

last: RHA


title: Low Risk and High Risk Mouse Skin Carcinogenesis

status: Public on Aug 01 2007

first: Stuart Yuspa

last: Adam Glick

address: 9000 Rockville Pike

department: National Cancer Institute

institute: National Institutes of Health

summary:
Mouse skin carcinogenesis experiments suggest upon carcinogen treatment and promotion on mouse skin there arise at least two populations of tumors. One population has a higher incidence of progression from benign papilloma to a squamous cell carcinoma. Further studies have shown that a certain carcinogenesis protocol can select for these high risk tumors. These microarray experiments aim to reveal the genetic signatures of the low risk and high risk papilloma.
Keywords: Genetic Signatures

overall_design: These experiments were designed on a common reference platform. Chips were hybridized with cDNA labeled with two fluoro markers. One labeled with Cy5 and the other Cy3 and one of the probes would be derived from Clontech's mouse universal reference RNA.


title: Identification of downstream targets of the human papillomavirus E6 and E7 oncoproteins by RNA interference

status: Public on Jul 01 2007

first: Ruprecht

last: Kuner

address: Im Neuenheimer Feld 460

department: Molecular Genetics

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

summary:
Specific types of human papillomaviruses (HPVs) cause cervical cancer, the second most common tumor in females worldwide. Both cellular transformation and the maintenance of the oncogenic phenotype of HPV-positive tumor cells are linked to the expression of the viral E6 and E7 oncogenes. In order to identify downstream cellular target genes for the viral oncoproteins, we silenced endogenous E6 and E7 expression in HPV-positive HeLa cells by RNA interference (RNAi). Subsequently, we assessed changes of the cellular transcriptome by genome-wide microarray analysis. We identified, 648 genes wich were either downregulated (360 genes) or upregulated (288 genes) upon inhibition of E6/E7 expression. A large fraction of these genes is involved in tumour-relevant processes, such as apoptosis control, cell cycle regulation, or spindle formation. Others may represent novel cellular targets for the HPV oncogenes, such as a large group of C-MYC-associated genes involved in RNA splicing.
Keywords: siRNA mediated knockdown

overall_design: Hybridizations were performed on RZPD Unigene 3.1 cDNA microarrays (37.5K). Gene expression was analyzed by performing 6 microarray hybridizations, using three cell culture replicates treated with siRNA 18E7-3.5 or control siRNA siControl. Each replicate of the 18E7-3.5  experiments was hybridized against a replicate of the siControl experiment, including a technical dye swap.

first: Markus

last: Vogt

first: Holger

last: Sultmann

first: Andreas

last: Buness

first: Annemarie

last: Poustka

first: Felix

last: Hoppe-Seyler


title: Correlation analysis of genome-wide gene expression profiles by DNA microarray

status: Public on Jun 29 2007

first: Hye Jin

last: Jung

address: 134 Shinchon-dong,Seodaemun-gu

department: Biotechnology

institute: Yonsei University

summary:
We previously isolated a new angiogenesis inhibitor, terpestacin, from natural products. To investigate molecular mechanism of terpestacin for its anti-angiogenic activity, we identified a cellular binding protein of terpestacin using phage display biopanning analysis. Terpestacin specifically bound to the small molecular ubiquinone binding protein (QP-C), a 13.4 kD subunit of the mitochondrial Complex III. To explore the functional specificity of terpestacin at QP-C, we performed cDNA microarray analysis. The genome-wide gene expression profiling showed a significant phenotype correlation between terpestacin and QP-C genetic knock-down. These results demonstrate that QP-C is a biologically relevant target of terpestacin.
Keywords: Sample comparison

overall_design: We performed cDNA microarray analysis of transcriptional changes in HT1080 cells treated with terpestacin, antimycin A, or siRNA directed against human QP-C (siQP-C). The probe preparation and microarray hybridization was performed using the TwinChip Human-8K cDNA microarray. The signature correlation analysis was performed using both hierarchical clustering of gene expression profiles and signature correlation coefficient value (p). The comparison of gene expression profiles between different experimental conditions revealed that the signatures of siQP-C versus terpestacin correlated significantly (p = 0.62), while signatures of siQP-C versus antimycin A (p = -0.01) and terpestacin vs antimycin A (p < 0.1) exhibit no significant correlation. These results demonstrate that the cellular phenotype induced by terpestacin is significantly correlated with that of QP-C knock-down cells.

first: Hye-Jin

first: Joong-Sup

last: Shim

first: Jiyong

last: Lee

first: Young-Mi

last: Song

first: Ho-Jeong

last: Kwon


title: Expression data from Mus musculus Myc-null heart at E13.5

status: Public on Jun 30 2007

city: Los Angeles

first: Weiguang

last: Zhong

address: 675 Charles Young Drive

department: Cardiology

institute: UCLA

summary:
We created mice, which are deficient for Myc specifically in cardiac myocytes by crossing crossed Myc-floxed mice (Mycfl/fl) and MLC-2VCre/+ mice.  Serial analysis of earlier stages of gestation revealed that Myc-deficient mice died prematurely at E13.5-14.5.  Morphological analyses of E13.5 Myc-null embryos showed normal ventricular size and structure; however, decreased cardiac myocyte proliferation and increased apoptosis was observed.  BrdU incorporation rates were also decreased significantly in Myc-null myocardium.  Myc-null mice displayed a 3.67-fold increase in apoptotic cardiomyocytes by TUNEL assay.
We examined global gene expression using oligonucleotide microarrays. Numerous genes involved in mitochondrial death pathways were dysregulated including Bnip3L and Birc2.
Keywords: wildtype vs Myc-null

overall_design: Hearts were taken from wide type and Myc-null Mouse embryos at E13.5 under the dissecting scope.  Cardiac myocyte RNA was isolated using TRIZOL®Reagent  Total RNA (100 ng) was hybridized to the Sentrix® MouseRef-8 Expression BeadChip that contains probes for ~24,000 transcripts.  GeneChips were scanned using the Hewlett-Packard GeneArray Scanner G2500A.  The data were analyzed with Illumina Inc. BeadStudio version 1.5.0.34 and normalized by rank invariant method.

first: Ekatarina

last: Angelis

first: Katja

last: Schenke-Layland

first: WeiKe

last: Gong

first: Robb

last: MacLellan


title: Profiling of human PBMC exposed to M. leprae

city: Rio de Janeiro

first: Ulisses

middle: Gazos

last: Lopes

state: Rio de Janeiro

address: Cidade Universitaria- CCS

department: Molecular and Structural Biology

country: Brazil

institute: Federal University of Rio de Janeiro

summary:
Leprosy is major public health problem in developing countries. M. leprae infects human macrophages and Schwann cells leading to a chronic disease. Leprosy studies are constrained due to the lack of in vitro growth system for M. leprae.  Moreover, the overall  pathogenesis is poorly understood although the expression of some pro-inflammatory cytokines is associated with nerve damage that occurs during the infection. PBMC may participate of the host response to M. leprae infection leading to the inflammatory profile. We aimed to investigate the global human gene expression profile of human PBMC exposed to M. leprae for 4 H. Due to the reduced supply of M. leprae our study was restricted to three chips, including a dye-swap set. Our analysis revealed gene expression differences in cell receptors, NF-kB signaling and oxidative stress response. The results were confirmed by Real-time PCR and flow-cytometry and point to new insights into the molecular events resulted from host-pathogen interaction
Keywords: Modulation of human PBMC gene expression by M. leprae

overall_design: We analyzed 3 oligo-human chips, 10K elements each, spotted in duplicates. Two chips were replicas and one a dye-swap

first: Jorge

last: Ramirez

first: Alessandra

last: Saliba

middle: G


title: Sporadic and Post-Chernobyl Papillary Thyroid Cancers

status: Public on Jul 12 2007

city: Brussels

first: Vincent

last: Detours

address: 808 Route de Lennik

department: School of Medicine

country: Belgium

institute: Free University of Brussels

summary:
We compared the expression profiles of papillary thyroid tumors from the Chernobyl Tissues Bank (CTB) with tumors from French patients with no history of exposure to radiations.
Keywords: papillary thyroid cancer vs. patient-matched healthy adjacent thyroid

overall_design:
thyroid papillary cancer vs. patient-matched adjacent nontumor thyroid tissues.
-14 tumors from France
-12 tumors from Ukraine

first: Laurent

last: Delys


title: Genome-wide expression profiling of Drosophila adult heart organogenesis

city: MARSEILLE Cedex 9

first: Bruno

last: ZEITOUNI

address: Campus de Luminy Case 907

country: France

institute: CNRS

summary:
The abstract of the manuscript titled "Signalling pathways involved in adult heart formation revealed by gene expression profiling in Drosophila" is given below:
"Drosophila provides a powerful system for defining the complex genetic programs that drive organogenesis. Under control of the steroid hormone ecdysone, the adult heart in Drosophila forms during metamorphosis by a remodelling of the larval cardiac organ. Here, we evaluated the extent to which transcriptional signatures revealed by genomic approaches can provide new insights into the molecular pathways that underlie heart organogenesis. Whole-genome expression profiling at 8 successive time-points covering adult heart formation revealed a highly dynamic temporal map of gene expression through 13 transcript clusters with distinct expression kinetics. A functional atlas of the transcriptome profile strikingly points to the genomic transcriptional response of the ecdysone cascade, and a sharp regulation of key components belonging to a few evolutionary conserved signalling pathways. A reverse genetic analysis provided evidence that these specific signalling pathways are involved in discrete steps of adult heart formation. In particular, the Wnt signalling pathway is shown to participate in inflow tract and cardiomyocyte differentiation, while activation of the PDGF-VEGF pathway is required for cardiac valve formation. Thus, a detailed temporal map of gene expression can reveal signalling pathways responsible for specific developmental programs and provides here substantial grasp into heart formation."
Keywords: Time-course analysis

overall_design: Total RNAs were prepared from dissected cardiac tubes of Drosophila staged pupae at 8 successive time-points 21, 24, 27, 30, 33, 36, 42 and 48 hours after puparium formation covering adult heart organogenesis. For each time-point sample, 5 cardiac tubes were hand dissected from staged individuals. Four independent biological replicates were analyzed to confer a high reproducibility and statistical significance of the expression data. PolyA+ RNAs were linearly amplified (32 amplifications), labelled (32 with Cy3 and 32 with Cy5), and used for hybridization on Drosophila high-density oligonucleotide (INDAC) dual-channel microarrays (32 arrays) according to a loop-design dedicated to time-course experiments. Given the same number of arrays (8), a simple loop design is more efficient than a reference design: direct comparisons, smallest variance for log ratios, balancing varieties with dye-swapping (including technical replicates) and no reference sample needed (which has no intrinsic interest in our study). A graphical representation of the loop design used on this study is showed in the associated manuscript.

last: Zeitouni

first: Sébastien

last: Sénatore

first: Dany

last: Séverac

first: Cindy

last: Aknin

first: Michel

last: Sémériva

last: Perrin


title: Renal, brain and lung macrophages

status: Public on Jul 04 2007

city: St Lucia

first: Darrin

middle: F

last: Taylor

state: Qld

address: Research Rd

department: Genomics and Bioinformatics

country: Australia

institute: Institute for Molecular Bioscience, Uni of Qld

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

overall_design: Refer to individual Series

first: Kyra

last: Woods

first: Fiona

last: Rae

first: Tedjo

last: Sasmono

first: Naomi

last: Campanale

first: Dmitry

last: Ovchinnikov

first: Sean

middle: M

last: Grimmond

first: David

middle: A

last: Hume

first: Sharon

last: Ricardo

first: Melissa

last: Little


title: Model-based Analysis of 2-Color Arrays (MA2C)

status: Public on Jun 26 2007

city: Boston

first: Arjun

middle: Kumar

last: Manrai

state: MA

address: 44 Binney Street, Mail Stop LW225

department: Department of Biostatistics and Computational Biology

institute: Dana Farber Cancer Institute

summary:
High-density oligonucleotide tiling-microarrays are
currently providing a powerful tool for genome-wide in vivo DNA
footprinting assays, yielding unprecedented insights into tissuespecific
protein-DNA interactions and chromatin structure. Despite
the impressive advances, however, the technology still suffers from
numerous complications caused by background noise and probespecific
effects. A few computational methods modeling sequencerelated
probe effects are now available for Affymetrix tiling arrays, but
no counterpart is yet available for two-color arrays. A novel normalization method based on the GC content of probes is developed for two-color tiling-arrays. The proposed method, together with robust estimates of the model parameters, is shown to perform superbly on published data sets. Accompanying the normalization method, a robust algorithm for detecting peak regions is formulated and also shown to perform well compared to other approaches. The tools presented herein have been implemented for
NimbleGen tiling arrays as a stand-alone Java program, which can
also display various plots of statistical analysis for quality control of
experiments. Upon changing the file format, the program also works
on Agilent data.
Keywords: ChIP-chip

overall_design: 3 dual-channel samples, each comparing spiked-in DNA with genomic DNA

first: Jun

last: Song

first: Xiaole

last: Liu


title: Sex-, stage-, and tissue-specific alternative splicing in Drosophila melanogaster

status: Public on Jun 22 2007

city: Gainesville

first: Lauren

middle: M.

last: McIntyre

department: Dept. of Molecular Genetics and Microbiology

institute: University of Florida

summary:
We used long-oligonucleotide microarrays to investigate whether alternative splicing in Drosophila is regulated in a sex-, stage-, or tissue-specific manner.  To examine sex-specific splicing, we compared gene expression profiles of male and female pupae 12 hours after pupariation.  To examine stage-specific splicing, we compared expression profiles of mixed-sex, 0-24 hour old embryos and mixed-sex, 12 hour old pupae.  To examine tissue-specific splicing, we compared expression profiles of adult male heads and abdomens 24-48 hours after eclosion.  To examine tissue-specific splicing, we compared expression profiles of adult male heads and abdomens at 24-48 hours after eclosion.
Keywords: tissue-specific expression profiles

overall_design: Drosophila isogenic line WI89 was used.  Mixed-sex, mixed-stage embryos were harvested from plates on which females had been allowed to oviposit for 24 hours.  To obtain synchronized cohorts of pupae, male and female white prepupae were collected at 0-1 hour after pupariation and aged for 12 hours at 25C.  Mixed-sex pupal samples were generated by mixing equal amount of male and female pupal RNA.  Adult heads and abdomens were dissected from 24-48 hour old males.  mRNA was isolated and labeled without amplification.

first: Olga

last: Barmina

first: Artyom

last: Kopp

first: Sergey

middle: V

last: Nuzhdin

first: Marta

middle: L

last: Wayne

first: Lawrence

last: Harshman


title: Determining the number of individuals to pool

summary:
We compared 10, 20 and 40 animal pools to determine the best number of intact adults to pool
Keywords: Agilent microarray, pooling, Drosophila, intact adults

overall_design: 16 vials of flies were reared.  From each vial 5 individuals were selected.  For the 10 animal comparison 4 vials were used with each replicate being a pool of 5 animals over 2 independent vials.  For the 20 animal comparison 8 vials were used with each replicate being a pool of 5 animals over 5 independent vials and for the 40 animal comparison each replicate was a pool of 5 animals over 8 independent vials.  There are two independent replicates of each type for 6 samples which were hybridized on three chips in a loop design.


title: Immediate-Early and Delayed Primary Response Genes are Distinct in Function and Genomic Architecture

status: Public on Jul 03 2007

first: Geoffrey

last: Cooper

address: 5 Cummington St

department: Biology

institute: Boston University

summary:
Transcriptional profiling time course of serum deprived quiescent human T98G cells follwing platelet derived growth factor (PDGF) stimulation in the absence and presence of the translation inhibitor, cycloheximide.  RNA was harvested at 0.5, 2, and 4 hr following PDGF treatment.
Keywords: time course

overall_design: Two-color microarrays with dye-swap; 3 biological replicates per condition, independently grown and harvested. One replicate per array.

first: John

middle: W

last: Tullai

first: Michael

middle: E

last: Schaffer

first: Steven

last: Mullenbrock

first: Gabriel

middle: D

last: Sholder

first: Simon

last: Kasif


title: Expression data from adult male and female Drosophila melanogaster antenna, proboscis, and front leg

summary:
Sex-specific gene expression in sensory organs may play an important role in mating and foraging behavior.  We used long-oligonucleotide microarrays to compare gene expression profiles of males and females in three adult appendages that carry large numbers of chemosensory organs – antenna, proboscis, and front leg.
Keywords: tissue-specific expression profiles

overall_design: Drosophila isogenic line WI89 was used.  Adult male and female antennae (a3+arista), front legs (from distal tibia down), and proboscises were dissected at 1-2 hours after eclosion.  Total RNA was extracted and the mRNA fraction was amplified by reverse transcription followed by in vitro transcription with T7 RNA polymerase.


title: Determining the wash solution to use

status: Public on Jun 18 2007

summary:
We tested a new wash solution from Agilent
Keywords: Agilent microarray, pooling, Drosophila, intact adults

overall_design: Intact males and females for a single genotype were examined.  Two independent replicates of each were compared on two slides in a dye flip.


title: Fluid Shear Stress Up-regulates Vascular Endothelial Growth Factor Gene

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:
Fluid induced shear stress is widely recognized as an important biophysical signal in cell-cell mechanotransduction. To identify cellular signaling pathways that are regulated by fluid shear stress, we applied the unbiased approach of transcriptional profiling. Our cDNA array analysis detected that 1165 of the 6288 sampled unigenes were significantly affected by pulsatile fluid flow. GenMapp 2.1 analysis revealed pathways of genes regulated by shear stress: angiogenesis, blood vessel morphogenesis, regulation of endothelial cell proliferation and prostaglandin biosynthesis. Individual genes significantly up-/down-regulated by shear stress included vascular endothelial growth
factor A (VEGFa), cysteine rich protein 61 (CRY61), platelet derived growth factor, alpha (PDGFa), connective tissue growth factor (CTGF), Neuropilin 1 (NRP1), angiotensin II receptor, type 1a (AGTR1a) and fibroblast growth factor 1 (FGF1). Based on these findings, we hypothesize that fluid shear stress regulated VEGF most likely
stimulates MC3T3-E1 cells through autocrine/paracrine release and may provide a powerful recruitment signal for osteoclasts, endothelial cells and/or stem cells during bone remodeling.
Keywords: stress response

overall_design: Study the transcriptomic effects of the shear stress on cultured osteoblast-like MC3T3 cells. We used six sets of controls (C1, C2, C3, C4, C5, C6) and shear stressed samples (S1, S2, S3, S4, S5, S6) to ensure statistical relevance of the study, co-hybridized with six microarrays in 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.

first: Mia

last: Thi

first: Sanda

first: David

middle: C

last: Spray


title: Integrative Analysis of Genomic Aberrations Associated with Prostate Cancer Progression

status: Public on Jul 31 2007

city: Ann Arbor

first: Jung

last: Kim

state: MI

address: 1400 E. Medical Center Drive

institute: University of Michigan

summary:
Genome-wide copy number changes were monitored using array comparative genomic hybridization (aCGH) of laser-capture microdissected prostate cancer samples spanning stages of prostate cancer progression including precursor lesions, clinically localized disease and metastatic disease.
A total of 62 specific cell populations from 38 patients were profiled.
Keywords: Disease state analysis using array-based comparatavie genomic hybridization

overall_design:
Experiment Design:

- Type of experiment: This study used aCGH experiments to profile genome-wide copy number changes through the isolation of pure cell populations representing entire spectrum of prostate cancer disease using laser capture microdissection (LCM) and OmniPlex Whole Genomic (WGA) Amplification.
- Experimental factors: This study profiled various pure cell populations isolated by LCM from prostate tissue.
- The number of hybridizations performed in the experiment: A total of 62 hybridizations were performed.
- The type of reference used for the hybridizations: A single commercially available pool of normal human male genomic DNAs (Promega Inc.) was used as the reference in all hybridizations. For the reference sample, 100 ng of normal genomic DNA was converted into a WGA library in a volume of 15 ul and amplified by PCR following the manufacturer’s WGA amplification protocol. Hundred ng aliquots of the products were then subjected to a second WGA PCR amplification in the presence of amino-allyl dUTP, purified, and used for the hybridizations.
- Hybridization design: For all hybridizations, the experimental prostate sample isolated by LCM was labeled with Cy5 and the common reference of benign pooled prostate as described above was labeled with Cy3.
- Quality control steps taken: We purchased human genomic DNA samples having multiple copies (1 to 5) of X chromosome from NIGMS Human Genetic Mutant cell repository (http://www.nigms.nih.gov/Initiatives/HGCR/) and amplified. The hybridization result obtained from original samples and WGA-amplified samples were cross-compared. Also, genomic DNA from both epithelium and stroma of normal prostate tissue from a cadaveric donor, previously diagnosed with Down’s syndrome, was hybridized for the detection of a single copy gain in chromosome 21.
- The mRNA expression data is also available from GEO (GSE6099) and Oncomine (www.oncomine.org).

Samples used, extract preparation and labeling:

- The origin of the biological sample (for instance, name of the organism, the provider of the sample) and its characteristics: All samples used were of human origin. Tissues were from the radical prostatectomy series at the University of Michigan and from the Rapid Autopsy Program, which are both part of University of Michigan Prostate Cancer Specialized Program of Research Excellence Tissue Core. All samples were collected with informed consent of the patients and prior institutional review board approval. The table in the MIAME Checklist (Supplementary Methos) summarizes the samples used. For prostate cancer samples, the Gleason patterns present on the sample section used are indicated, as well as the Gleason patterns of the actual cells isolated by LCM. A unique case number is given. Descriptions of samples are given where applicable, including the location of metastatic foci.
- Manipulation of biological samples and protocols used: Laser Capture Microdissection (LCM) was performed from frozen tissue sections with the SL Microtest device using μCUT software (MMI). Approximately 10,000 cells were captured for each sample. Serial sections were used if cells could not be obtained from a single section. Genomic DNA was isolated from the cells using QIAamp DNA Mini Kit (Qiagen, Valencia, CA) and DNA concentration was determined using Quant-iT DNA Assay Kit, High Sensitivity (Invitrogen, Carlsbad, CA).
- Protocol for preparing the hybridization extract: For each sample, 100 ng of normal genomic DNA was converted into a WGA library in a volume of 15 ul and amplified by PCR following the manufacturer’s WGA amplification protocol. Hundred ng aliquots of the products were then subjected to a second WGA PCR amplification in the presence of amino-allyl dUTP and purified. Yields after all WGA PCR amplifications were between 10 to 20 μg per reaction and used for the hybridizations.
- Labeling protocol(s): For all WGA amplified genomic DNA samples with aminoallyl dUTP incorporated (4 μg), nuclease free water was added to 500 μl,  the sample was transferred to a Microcon YM-30 filter, and centrifuged at 12,000 rcf at RT for 12 minutes. The spin column was inverted in a new collection tube and centrifuged at 12,000 rcf for 2 min at RT. The amount of sample was measured and the volume was adjusted to 5 μl with nuclease free water. For labeling, 5 μl of 1 M sodium bicarbonate (pH 9.0) was added and allowed to incubate at RT for 10 min. Two μl of anhydrous DMSO were added to Alexa Fluor 647 (Cy5) and 555 (Cy3) dye packs (Invitrogen, Carlsbad, CA), mixed thoroughly, and 2 μl of the appropriate dye was added to each sample. The labeling mixture was incubated in the dark at RT for 1 hour. The reaction was stopped by the addition of 9 μl of 4 M Hydroxylamine and incubated in room temperature for 15 min. The Cy3 labeled and Cy5 labeled genomic DNA for each hybridization were mixed, and RNase free water was added to 100 μl, and purified using Qiaquick (Qiagen, Valencia, CA) spin columns.  The final eluted volume was 60 μl.
- External controls (spikes): Not utilized

Hybridization procedures and parameters:

- The protocol and conditions used during hybridization, blocking and washing: For hybridization, 40 μg of human Cot-1 DNA (Invitrogen, Carlsbad, CA) was added to 60 μl of labeled genomic DNA, the probe was transferred to a Microcon YM-30 filter and centrifuged at 12,000 rcf for 8 min at RT. The spin column was inverted in a new collection tube and centrifuged at 12,000 rcf for 2 min at RT. The eluted probe volume was adjusted to 18 μl with nuclease free water and the following were added: 2 μl of yeast tRNA (10 μg/μl, Invitrogen, Carlsbad, CA), 4 μl of Poly dA-Poly dT (10 μg/μl, Sigma-Aldrich, St. Louis, MO),  5.1 μl of 20X SSC and 0.9 μl of 10% SDS. The probe was denatured at 100° C for 3 min and centrifuged at 12,000 rcf for 10 sec. The probe was added directly to the microarray and a cover slip was placed carefully. Slides were hybridized overnight in DIE-TECH 1 or 5 slide hybridization chambers in a 65° C water bath. After hybridization, cover slips were removed by placing slides in 2x SSC / 0.03% SDS. Slides were then washed sequentially in the following order: 2x SSC / 0.03% SDS for 5 min, 1x SSC / 0.03% SDS for 5 min, and 0.2x SSC / 0.03% SDS for five minutes. Slides were given a final wash in 0.2x SSC for two minutes, and centrifuged dry at 500 rpm for 5 min.

Measurement data and specifications:

- Type of scanning hardware and software used: Microarrays were scanned using a GenePix 4000B scanner (Axon Instruments, Union City, CA).
- Type of image analysis software used: Images were gridded and spots were quantified using GenePix Pro 6.0 software (Axon Instruments, Union City, CA).
- A description of the measurements produced by the image-analysis software and a description of which measurements were used in the analysis: GenePix Pro 6.0 are described in the sample files. For all hybridizations, the log2 normalized Median of Ratios (as described below) was used.
- The complete output of the image analysis before data selection and transformation (spot quantitation matrices): The aCGH raw data files will be available from GEO. The mRNA expression data is already available from GEO (GSE6099) and Oncomine (www.oncomine.org) under the study name Tomlins_Prostate_Chinnaiyan-Lab_2.
- Data selection and transformation procedures: Arrays were auto-gridded by GenePix 6.0. Features flagged by GenePix as not found during grid alignment and areas of obvious defects were manually flagged and both were excluded from further analysis. The Median of Ratios (log2 of Cy5/Cy3) for each included feature was normalized using locally weighted regression (lowess) with a window of 0.6 using custom software written in Perl and R. To exclude unreliable features, features showing an average normalized Median of Ratios of > 1.5 or < 0.75 across a series of self-self hybridizations (including unamplified and WGA amplifi

middle: H

first: Saravana

last: Dhanasekaran

first: Rohit

last: Mehra

first: Arul

last: Chinnaiyan


title: Renal Cell Carcinoma - Papillary types 1, and 2b, Normal Kidney Tissue

status: Public on Jun 12 2007

city: Grand Rapids

first: Karl

last: Dykema

address: 333 Bostwick Ave. NE

institute: Van Andel Institute

summary:
Myc oncogenic signature in Papillary type 2b
Keywords: Gene Expression experiment

overall_design: Tumor samples compared to Normal renal tissue

middle: J


title: Normal CD34+ cells from unmobilized human peripheral blood stimulated by chemokine SDF1 alpha

status: Public on Jun 28 2007

city: Villejuif

first: Le Bousse

last: Marie-Caroline

address: 14, Avenue Paul-Vaillant Couturier

institute: Inserm

summary:
Headline: application of the oligo-microarray to the analysis of genes modulated by SDF1α chemokine in the human circulating hematopoietic CD34+ progenitors.

Summary: 1/ Introduction: The microarray analysis simultaneously explores the expression of thousands messager ribonucleic acids in the cell. The traditional method by reverse transcription requires 50 to 100 micrograms of total initial ribonucleic acids. The human circulating hematopoietic CD34+ progenitors are rare and expressed very few messager ribonucleic acids. SDF1α is a chemokine which binds a specific transmembrane receptor, the CXCR4, expressed on the human circulating hematopoietic CD34+ progenitors. A chemical gradient of SDF1α in the medullary micro-environment is implied in the regulation of the trans-endothelial migration of the hematopoietic CD34+ progenitors who leave bone marrow and then migrate towards peripheral blood. Apart from its role in the cellular mobilization, SDF1α also takes part in survival maintenance, cell cycle promotion and proliferation of hematopoietic progenitors. 2/ objectives: The objective of this work was to apply this technology to the differential analysis of genes modulated by SDF1α chemokine in the human circulating hematopoietic CD34+ progenitors. 3/ Material and methods: With regard to the biological application, CD34+ cells were incubated during sixteen hours in the presence or absence of SDF1α at the concentration of 0,5 ng/ml. For this, four experiments were carried out with CD34+ cells purified from the mononuclear cells obtained from blood of six to eight unmobilized healthy subjects. The total ribonucleic acids of untreated cells from the four experiments were pooled to synthesize a “control” microarray probe. In the same way, the total ribonucleic acids of SDF1α treated cells were used to synthesize a “SDF1α treated” microarray probe. The differential hybridization of an oligo-microarray slide (10 000 genes) was carried out simultaneously with the “control” and “SDF1α treated” probes. The bioinformatics studies and the use of classification algorithms on the results of this microarray analysis made it possible to classify the differential transcripts in intracellular functional groups. 4/ Results an discussion: Our results showed that a double linear amplification with T7 RNA polymerase of complementary retro-transcript desoxyribonucleic acids from total ribonucleic acids of human circulating hematopoietic CD34+ progenitor was necessary to obtain the probe quantity for hybridize an oligo-microarray. Indirect cyanin incorporation of the aminoallyl type during the last stage of in vitro transcription optimized the probe labelling and reduced the experimental skew during the microarray analysis. We showed that under our experimental conditions, SDF1α acted on the expression of genes mainly implied in cell cycle (mitosis induction and proliferation) and cell metabolism (energy and translation). Our microarray results, which were confirmed by quantitative RT-PCR validate the biological ones previously obtained and help us to a better understanding of the intimate regulation mechanism of gene expression modulated by SDF1α.

Key words: oligo-microarray, optimization, hematopoietic CD34+ progenitors, chemokine, SDF1α, genomic, transcriptome, cell cycle, quantitative RT-PCR, bioinformatics, principal component analysis, clustering.

Keywords: oligo-microarray, optimization, hematopoietic CD34+ progenitors, chemokine, SDF1α, genomic, transcriptome, cell cycle, quantitative RT-PCR, bioinformatics, principal component analysis, clustering.

first: Christophe

last: Desterke

first: MarieCaroline

last: LeBousseKerdiles

first: JeanJacques

last: Lataillade

first: Delphine

last: Gilges

first: Georges

last: Uzan


title: TGF-beta on breast cancer cell

status: Public on Jun 01 2007

city: bethesda

first: binwu

last: tang

address: 9000 rockville pike

department: nci

institute: nih

summary:
To understand the molecular mechanism of action of TGF-beta as a tumor suppressor in breast cancer. Comparison of gene expression between MCF10ACa1h breast tumor xenograft with or without an intact TGF-beta response.
Keywords: genetic modification design

overall_design: Nine tumors in each genotype group were arrayed twice and 1 additional tumor/group was arrayed once. For the replicate array for each tumor, reference and tumor RNA dyes were swapped compared with the initial array. Initial and replicate arrays were performed on different array print batches.

first: Binwu

last: Tang

first: Lalage

last: Wakefield

first: Mary

last: Vu

first: Naomi

last: Yoo

first: Joanna

last: Shih

first: Aleksandra

last: Michalowska