title: Gene expression changes associated with progression and response in chronic myeloid leukemia

status: Public on Feb 21 2006

city: Rahway

first: Olivia

last: Fong

state: NJ

department: Molecular Profiling

institute: Merck & Co.

summary:
Chronic myeloid leukemia (CML) is a hematopoetic stem cell disease with distinct biological and clinical features. The biological foundation of the stereotypical progression from chronic phase through accelerated phase to blast crisis is poorly understood. We used DNA microarrays to compare gene expression in 91 cases of CML in chronic (42 cases), accelerated (17 cases), and blast phases (32 cases). Three thousand genes were found to be significantly (p<10-10) associated with the progression from chronic to blast phase. A comparison of the gene signatures of chronic, accelerated, and blast phases suggest that the progression of chronic phase CML from chronic advanced phase (accelerated and blast crisis) CML is a two-step rather than a three-step process, with new gene expression changes occurring early in accelerated phase before the accumulation of increased leukemia blast cells. The genetic signature of advanced phase CML is similar to that of normal CD34+ cells; however, progression also involved novel genes not expressed in normal CD34+ cells. Especially noteworthy is deregulation of the WNT/b-catenin pathway, the decreased expression of both JunB and Fos, and dysregulation of genes under the control of MZF1 and delta EF1 zinc finger transcription factors. Studies of CML patients who relapsed after initially successful treatment with imatinib mesylate demonstrated a gene expression pattern closely related to advanced phase disease. Take together, these data suggest that CML progression begins relative early and before clinical and pathological detection, and features distinct genetic differences compared to normal hematpoetic cells that might provide diagnostic and therapeutic targets.
Keywords: disease state analysis

overall_design: Samples from different phases of CML were hybridized against the pool of chronic phases of samples.

first: Jerald

last: Radich

first: Hongyue

last: Dai

first: Mao

last: Mao

first: Vivian

last: Oehler

first: Jan

last: Schelter

first: Brian

last: Druker

last: Sawyers

first: Neil

last: Shah

first: Wendy

last: Stock

first: Cheryl

last: Willman

first: Stephen

last: Friend

first: Peter

middle: S

last: Linsley


title: Identification of biomarkers for tumor endothelial cell proliferation through gene expression profiling

status: Public on Sep 13 2005

city: Seattle

first: Amit

middle: V

last: Kulkarni

state: WA

address: 401 Terry Ave N

department: Genomics

country: USA

institute: Rosetta Inpharmatics / Merck Pharmaceuticals

summary:
Extensive efforts are underway to identify anti-angiogenic therapies for the treatment of human cancers. Many proposed therapeutics target vascular endothelial cell growth factor (VEGF) or the kinase insert domain receptor (KDR/VEGFR-2/FLK-1), the mitogenic VEGF receptor tyrosine kinase expressed by endothelial cells. Inhibition of KDR catalytic activity blocks tumor neo-angiogenesis, reduces vascular permeability, and, in animal models, inhibits tumor growth and metastasis. Using a gene expression profiling strategy in rat tumor models, we identified a set of six genes that are selectively overexpressed in tumor endothelial cells relative to tumor cells, and whose pattern of expression correlates with the rate of tumor endothelial cell proliferation. In addition to being potential targets for anti-angiogenesis tumor therapy, the expression patterns of these genes or their protein products may aid the development of pharmacodynamic assays for small molecule inhibitors of the KDR kinase in human tumors.
Keywords: Endothelial cell proliferation

sample_organism:
Homo sapiens
Rattus norvegicus

platform_organism:
Homo sapiens
Rattus norvegicus

overall_design: Total RNA isolated from cultured cells or tumor tissue samples was used to make fluorescently labeled cRNA that was hybridized to DNA oligonucleotide microarrays. Briefly, 4 µg of total RNA from an individual tumor sample or endothelial cell culture were used to synthesize dsDNA through RT. cRNA was produced by in vitro transcription and labeled postsynthetically with Cy3 or Cy5. Two populations of labeled cRNA, a reference population and an experimental population, were compared with each other by competitive hybridization to microarrays. For animal tumor studies, reference cRNA pools were made by pooling equal amounts of cRNA from each tumor in the appropriate vehicle-dosed group. Species-specific microarrays were used throughout this study. Human microarrays contained 23,916 oligonucleotide probes corresponding to individual genes or expressed sequence tags. Rat microarrays contained probes to 22,592 genes or expressed sequence tags. Oligonucleotide probe sequences were chosen to maximize gene specificity and minimize the 3' replication bias inherent in RT of mRNA. In addition, both microarray formats contained 1,000 control probes for quality control purposes. All oligonucleotide probes on the microarrays were synthesized in situ with inkjet technology (Agilent Technologies, Palo Alto, CA). After hybridization, arrays were scanned and fluorescence intensities for each probe were recorded. Ratios of transcript abundance (experimental to control) were obtained following normalization and correction of the array intensity data. Gene expression data analysis was done with the Rosetta Resolver gene expression analysis software (version 3.2, Rosetta Biosoftware, Seattle, WA). For each gene sequence present on the microarrays, statistical significance of differential gene expression was determined by calculating P values.

first: James

last: Hardwick

first: Yi

last: Yang

first: Chunsheng

last: Zhang

first: Bin

last: Shi

first: Rosemary

last: McFall

first: Elizabeth

middle: J

last: Koury

first: Susan

middle: L

last: Hill

first: Robert

last: Wasserman

last: Phillips

first: Edward

last: Weinstein

first: Nancy

middle: E

last: Kohl

first: Michael

last: Severino

first: John

middle: R

last: Lamb

first: Laura

last: Sepp-Lorenzino


title: Characterization of mammary tumors from Brg1 heterozygous mice

status: Public on Jan 18 2008

city: Chapel Hill

first: Charles

middle: M.

last: Perou

state: NC

address: 12-044 Lineberger Comprehensive Cancer Center CB# 7295

department: Professor of Genetics, and Pathology & Laboratory Medicine; Lineberger Comprehensive Cancer Center

institute: University of North Carolina at Chapel Hill

summary:
Mammalian SWI/SNF-related complexes have been implicated in cancer based on some of the subunits physically interacting with RB and other proteins involved in carcinogenesis. Additionally, several subunits are mutated or not expressed in tumor-derived cell lines. Strong evidence for a role in tumorigenesis in vivo, however, has been limited to SNF5 mutations that result primarily in malignant rhabdoid tumors (MRTs) in humans and MRTs as well as other sarcomas in mice. We previously generated a null mutation of the Brg1 catalytic subunit in the mouse and reported that homozygotes die during embryogenesis. Here, we demonstrate that Brg1 heterozygotes are susceptible to mammary tumors that are fundamentally different than Snf5 tumors. First, mammary tumors are carcinomas not sarcomas. Second, Brg1+/- tumors arise because of haploinsufficiency rather than loss of heterozygosity (LOH). Third, Brg1+/- tumors exhibit genomic instability but not polyploidy based on array CGH results. We monitored Brg1+/-, Brm-/- double-mutant mice but did not observe any tumors resembling those from Snf5 mutants, indicating that the Brg1+/- and Snf5+/- tumor phenotypes do not differ simply because Brg1 has a closely related paralog whereas Snf5 does not. These findings demonstrate that BRG1 and SNF5 are not functionally equivalent but protect against cancer in different ways. We also demonstrate that Brg1+/- mammary tumors have relatively heterogeneous gene expression profiles with similarities and differences compared to other mouse models of breast cancer.  The Brg1+/- expression profiles are not particularly similar to mammary tumors from Wap-T121 transgenic line where RB is perturbed. We were also unable to detect a genetic interaction between the Brg1+/- and Rb+/- tumor phenotypes. These latter findings do not support a BRG1-RB interaction in vivo.
Keywords: reference X sample

overall_design:
14 microarrays consisting of 12 unique Brg1+/- murine mammary
tumors

first: S

last: Bultman

first: Jason

last: Herschkowitz

middle: M

first: T

last: Magnuson


title: Genome wide identification of targets of the drosha-pasha/DGCR8 complex

status: Public on Dec 25 2008

city: Waltham

first: Joseph

last: Rodriguez

address: 415 South Street

department: Biology

institute: Brandeis University

type: Expression profiling by genome tiling array

type: Genome binding/occupancy profiling by genome tiling array

summary:
Drosha is a type III RNAse, which plays a critical role in miRNA biogenesis. Drosha and its double-stranded RNA-binding partner protein Pasha/DGCR8 likely recognize and cleave miRNA precursor RNAs or pri-miRNA hairpins co-transcriptionally. To identify RNAs processed by Drosha, we used tiling microarrays to examine transcripts after depletion of drosha mRNA with dsRNA in Drosophila Schneider S2 cells. This strategy identified 137 Drosha-regulated RNAs, including 11 putative pri-miRNAs comprising 15 annotated miRNAs. Most of the identified pri-miRNAs seem extremely large, >10 kilobases as revealed by both the Drosha knock down strategy and by RNA PolII chromatin IP followed by Drosophila tiling microarrays. Surprisingly, more than a hundred additional RNAs not annotated as miRNAs are under Drosha control and are likely to be direct targets of Drosha action. This is because many of them encode annotated genes, and unlike bona fide pri-miRNAs, they are not affected by depletion of the miRNA processing factor, dicer-1. Moreover, application of the evofold analysis software indicates that at least 25 of the Drosha-regulated RNAs contain evolutionarily conserved hairpins similar to those recognized by the Drosha-Pasha/DGCR8 complex in pri-miRNAs. One of these hairpins is located in the 5′ UTR of both pasha and mammalian DGCR8. These observations suggest that a negative feedback loop acting on pasha mRNA may regulate the miRNA-biogenesis pathway: i.e., excess Drosha cleaves pasha/DGCR8 primary transcripts and leads to a reduction in pasha/DGCR8 mRNA levels and Pasha/DGCR8 synthesis.

Keywords: time course, ChIP-chip

overall_design: S2 cells were depleted of drosha or dicer mRNA with dsRNA.  Total RNA was then converted to cDNA and hybridized to Affymetrix tiling arrays to identify pri-miRNAs and drosha regulated RNAs.  RNA PolII chromatin IP genomic DNA was also hybridized to Affymetix tiling arrays to support pri-miRNA targets from drosha knockdowns. Two replicates were used for each sample.

first: Sebastian

last: Kadener

first: Katharine

last: Abruzzi

first: Yevgenia

last: Khodor

first: Ken

last: Sugino

last: Marr

first: Sacha

last: Nelson

last: Rosbash


title: Prediction of toxicant-specific gene expression signatures following chemotherapeutic treatment of breast cell lines

status: Public on Sep 28 2004

summary:
Global gene expression profiling has demonstrated that the predominant cellular response to a range of toxicants is a general stress response.  This stereotyped environmental stress response commonly includes repression of protein synthesis and cell cycle regulated genes and induction of DNA damage and oxidative stress responsive genes.  Our laboratory has recently characterized the general stress response of breast cell lines derived from basal-like and luminal epithelium following treatment with doxorubicin (DOX) or 5-fluorouracil (5FU) and showed that each cell type has a distinct response.  However, we expected that some of the expression changes induced by DOX and 5FU were unique to each compound and might reflect the underlying mechanisms of action of these agents. Thus, we employed supervised analyses (Significance Analysis of Microarrays) to identify genes that showed differential expression between DOX- and 5FU-treated cell lines. We then used cross-validation analyses and identified genes that afforded high predictive accuracy in classifying samples into the two treatment classes.  To test whether these gene lists had good predictive accuracy in an independent data set, we treated our panel of cell lines with etoposide, a compound that is mechanistically similar to DOX.  We demonstrated that using expression patterns of 100 genes, we were able to obtain 100% predictive accuracy in classifying the etoposide samples as being more similar in expression to DOX- than 5FU-treated samples. These analyses also showed that toxicant specific gene expression patterns, similar to general stress responses, vary according to cell type.
Keywords = breast cancer
Keywords = class prediction
Keywords = doxorubicin
Keywords = etoposide
Keywords = 5-fluorouricil
Keywords = gene expression
Keywords = microarrays
Keywords: dose response

first: Melissa

middle: A

last: Troester

first: Katherine

last: Hoadley

first: Joel

last: Parker

first: Theresa

last: Sørlie

first: Brittney-Shea

last: Herbert

first: Jerry

last: Shay


title: Mutation of GATA3 in Human Breast Tumors

status: Public on May 24 2004

last: Perou

summary:
293T epithelial kidney cell line was infected with retrovirus containing pBabe puro empty plasmid or with pBabe puro plasmid containing either GATA3 wild type or mutation R367-L. Stable colonies were selected, grown, and their mRNAs harvested.  mRNA from three independent transfections of 293T cells with the empty plasmid was pooled and used as the reference RNA.  Dye flips were performed resulting in four arrays.

This series also contains two additional arrays (GSM13007 & GSM13008) not in the publication.
Keywords: other

last: Usary


title: Global gene expression profiling on renal scarring in rat model of pyelonephritis

status: Public on Feb 22 2007

city: Toyoake

first: Ichino

last: Manabu

state: Aichi

address: Kutsukake-cho Denngakugakubo 1-98

department: Medical

country: Japan

institute: Fujita Health University

summary:
Renal scarring is a serious complication of chronic pyelonephritis due to vesicoureteral reflux, and requires specific biomarker for indication of early intervention. We investigated global expression profile of kidney during renal scarring formation using rat pyelonephritis model. An inoculum of the DS-17 strain Escherchia coli was injected directly into renal cortex of Wister rat. Histologically, renal scarring developed during 3 and 4 weeks after injection. The time course expression profile of approximately 20,500 genes was analyzed using high density oligonucleotide microarray followed by validation using real time RT-PCR. Most of up-regulated genes during this experimental renal scarring were associated with immune and defense response, including cytokines, chemokines, their receptors, complements, and adhesion molecules as well as proteins related to extra cellular matrix. These genes were up-regulated as early as 1 week after injection when histological examination did not show fibrotic change yet, peaked at 2 weeks and gradually decreased thereafter. However, a subset of cytokine genes is persistently activated even in 6 weeks after the injection, which are involved in TGF-β related tissue regeneration pathway or IL-1β related pathway. The products of these genes may potentially be the source of novel non-invasive diagnostic or prognostic biomarkers for renal scarring.
Keywords: renal scarring, TGF-β , IL-1β

overall_design: Experimental pyelonephritis was produced by standard method of direct infection of rat kidney. In brief, animals were anesthetized with pentobarbital intraperitoneally. The kidneys were exposed through a midline abdominal incision. An inoculum of 1x109 colony-forming units / 0.1 ml of Escherchia coli was injected directly into bilateral renal cortex of Wister rat using 26-gauge needle. We used DS-17 or NU-14 strains, both of which has type 1 and P pili, and SEC strain, which has only type 1 pili. Control rats received isotonic saline instead of bacterial solution. Both test and control kidneys were harvested at 1, 2, 4 and 6 weeks after injection. The kidney samples (1x1x2 mm each) were obtained including the injection site, which was easily identified by surface color change. Then we cut the kidney into half; one was used for microscopic examination and the other half was immediately frozen in liquid nitrogen for later RNA extraction.

first: Mori

last: Terumi

first: Kusaka

last: Mamoru

first: Koroyanagi

last: Yoko

first: Ishikawa

last: Kiyohito

first: Shiroki

last: Ryoichi

first: Kowa

last: Hiroe

first: Kurahashi

last: Hiroki

first: Hoshinaga

last: Kiyotaka


title: Cell-type specific responses to chemotherapeutics in breast cancer

status: Public on Apr 18 2004

first: Charles

summary:
Recent studies have identified clinical subtypes of breast tumors that arise from different cell types and have different outcomes in response to chemotherapy.  Tumors derived from basal epithelium have a poorer prognosis than tumors derived from luminal epithelium.  To gain insight into differences underlying this disparity, we treated cell lines derived from basal epithelium (immortalized human mammary epithelial cells) and those derived from luminal epithelium (MCF-7 and ZR-75-1) with two chemotherapeutics commonly used in the treatment of breast cancer.  Treatment doses for doxorubicin (DOX) and 5-fluorouracil (5FU) were selected to cause comparable cytotoxicity across all four cell lines.  The predominant gene expression in each of the four cell lines was a general stress response, but distinct gene expression patterns were observed depending upon cell type.  Both cell types up-regulated DNA damage response genes such as p21waf1, but the response in the luminal cells was much more dramatic and included many p53-regulated genes.  Luminal cell lines down-regulated a large number of cell cycle regulators and other genes involved in cellular proliferation, while basal cell lines down-regulated a smaller set of genes, many of which are involved in cellular differentiation. These results were compared to gene expression data from tumor samples collected before and after treatment with DOX or 5FU/mitomycin C.  Similarities between the in vitro and in vivo responses validate this model for studying gene expression responses to chemotherapy in these cell types.  Understanding cell-type specific responses to chemotherapeutics will help in tailoring treatment to patients based upon tumor characteristics.
Keywords = breast cancer
Keywords = chemotherapy
Keywords = gene expression
Keywords = microarray
Keywords = stress response
Keywords: time-course

last: Troester


title: Immunohistochemical and Clinical Characterization of the Basal-like Subtype

status: Public on Oct 15 2004

summary:
3 fresh frozen Breast tumor from the University of North Carolina at Chapel Hill (UNC) were obtained from the UNC Tissue Procurement Facility under an IRB approved protocol. Each experimental sample was ssayed versus a common
reference sample that was a modified version of the Stratagene Human Universal Reference (which was further augmented with a 1/10 amount of MCF7 mRNA and 1/10 amount of ME16C mRNA)
Keywords = Agilent microarray
Keywords = EGFR
Keywords = keratin
Keywords: parallel sample

first: Torsten

middle: O

last: Nielsen

first: Forrest

middle: D

last: Hsu

first: Kristin

last: Jensen

first: Maggie

last: Cheang

first: Gamze

last: Karaca

first: Zhiyuan

last: Hu

first: Tina

last: Hernandez-Boussard

first: Chad

last: Livasy

first: Dave

last: Cowan

first: Lynn

last: Dressler

first: Lars

last: Akslen

first: Joseph

last: Ragaz

first: Allen

last: Gown

first: C. Blake

last: Gilks

first: Matt

last: Van de Rijn


title: Gene Expression Profiles of Head and Neck Squamous Cell Carcinoma (HNSCC) primary tumors

status: Public on Mar 24 2004

last: Perou

summary:
60 fresh frozen HNSCC from the University of North Carolina at Chapel Hill (UNC) were obtained from the UNC Tissue Procurement Facility under an IRB approved protocol. 55 tumor samples were collected from the primary tumor and five tumor samples were collected from a local recurrence at the primary site; one tumor also had a sample of the primary tumor and an associated lymph node metastasis . In addition, we profiled three normal tonsillar epithelium samples that were collected from three pediatric patients following routine tonsillectomy and four HNSCC tumor derived cell lines (UNC7, UMSCCA1, CAL27 and JHU022). Each experimental sample (tumor, normal or cell line) was assayed versus a “common reference” sample that was a pool of total RNA derived from 30 of the HNSCC samples. This tumor pool reference strategy has been successfully used in another profiling study. In total, 78 experiments were performed, which utilized three separate preparations of the common reference pool.

Keywords = Head and neck cancer
Keywords = gene expression profiles
Keywords = microarray
Keywords: parallel sample

first: Christine

middle: H

last: Chung

first: Joel

last: Parker

first: Junyuan

last: Wu

first: William

middle: K

last: Funkhouser

first: Dominic

last: Moore

first: Dong

last: Xiang

middle: W

last: Shockley

first: Mark

middle: C

last: Weissler

middle: G

first: Carol

last: Shores

first: Wendell

last: Yarbrough


title: Gene expression patterns associated with p53 status in breast cancer

status: Public on Feb 20 2007

first: Charles

summary:
Breast cancer subtypes identified in genomic studies have different underlying genetic defects.  Mutations in the tumor suppressor p53 occur more frequently in estrogen receptor (ER) negative, basal-like and HER2-amplified tumors than in luminal, ER positive tumors.  Thus, because p53 mutation status is tightly linked to other characteristics of prognostic importance, it is difficult to identify p53's independent prognostic effects. The relation between p53 status and subtype can be better studied by combining data from primary tumors with data from isogenic cell line pairs (with and without p53 function). In this study, the p53-dependent gene expression signatures of four cell lines (MCF-7, ZR-75-1, and two immortalized human mammary epithelial cell lines) were identified by comparing p53-RNAi transduced cell lines to their parent cell lines.  Cell lines were treated with vehicle only or doxorubicin to identify p53 responses in both non-induced and induced states.  Each cell line displayed unique patterns of gene expression, but cell type specific trends were evident.  A common gene expression signature associated with p53 loss across all four cell lines was identified.  This signature showed overlap with the signature of p53 loss in primary breast tumors and predicted relapse-free survival and overall survival in independent test data sets.
Keywords: untreated x treated

overall_design: We analyzed 48 arrays performed using 48 polyA RNA samples.  RNAs were collected from cell lines treated with an IC50 dose of doxorubicin hydrochloride or with a feeding control.  Each cell line had its own reference which represented the second sample on the dual channel array.  These untreated RNAs were prepared by pooling four harvests of that cell line at 60-80% confluence and 48h after feeding


title: Widespread siRNA "off-target" transcript silencing mediated by seed region sequence complementarity

status: Public on Sep 12 2006

city: Carlsbad

first: Aimee

last: Jackson

state: CA

address: 1896 Rutherford Rd

institute: Regulus Therapeutics

summary:
Transfected siRNAs and miRNAs regulate numerous transcripts that have only limited complementarity to the active strand of the RNA duplex.  This process reflects natural target regulation by miRNAs, but is an unintended (“off-target”) consequence of siRNA-mediated silencing.  Here we demonstrate that this unintended off-target silencing is widespread, and occurs in a manner reminiscent of target silencing by miRNAs.  A high proportion of unintended transcripts silenced by siRNAs showed 3’ UTR sequence complementarity to the seed region of the siRNA.  Base mismatches within the siRNA seed region reduced the set of original off-target transcripts but generated new sets of silenced transcripts with sequence complementarity to the mismatched seed sequence.  An inducible shRNA silenced a subset of transcripts that were silenced by an siRNA of the same sequence, demonstrating that unintended silencing is sequence-mediated and is independent of delivery method.  In all cases, off-target transcript silencing was accompanied by loss of the corresponding protein and occurred with similar dependence on siRNA concentration as silencing of the target transcript.  These results demonstrate that short stretches of sequence complementarity to the seed region of the siRNA are key to the silencing of unintended transcripts, and that this limits the specificity of siRNA-mediated transcript silencing.   Because these off-target events are sequence-dependent, inclusion of multiple independent siRNAs to the target of interest can help to distinguish true positives from false positives in functional genetic analyses.
Keywords: siRNA, RNAi, sequence alignment, off-target, seed region

overall_design: For details, please see A.L. Jackson, et al. 2006. RNA 12(7): 1179-1187.  We used consensus genelists for clustering.  Please see attached tables for genelists.

first: Julja

last: Burchard

first: Janell

last: Schelter

first: Nelson

last: Chau

first: Michele

last: Cleary

first: Lee

last: Lim

first: Peter

last: Linsley