Genome Oligo Microarray Research Articles

Abstract 4360: Loss of NOTCH1 is associated with decreased expression of hsa-miR-125a-5p in T-cell acute lymphoblastic leukemia: Implications for using NOTCH1 inhibition in chemotherapy

Abstract T-cell acute lymphoblastic leukemia (ALL) accounts for 15% of pediatric ALL cases and is associated with early relapse and inferior outcome. The poorer prognosis of T-ALL compared to B-precursor ALL may in part reflect the lack of unique features on which to base therapy. NOTCH1 mutations are of particular interest since these were reported in 37-71% of T-ALLs. The prognostic value of NOTCH1 mutations remains controversial as both favorable and unfavorable associations were reported, whereas in other studies there were no associations between NOTCH1 mutations and treatment outcome. NOTCH1 is a potentially attractive therapeutic target for T-ALL since constitutively activating effects of mutant NOTCH1 can be abolished with gamma secretase inhibitors (GSIs). Because of possible effects of GSIs on other cellular targets in addition to NOTCH1, we explored shRNA knockdown of NOTCH1 to identify novel NOTCH1-regulated genes that may serve as prognostic indicators or therapeutic targets in T-ALL. NOTCH1 expression was knocked down in Jurkat T-ALL cells using lentivirus expressing shRNAs for NOTCH1 or a non-targeted control (NTC) sequence. NOTCH1 knockdown was verified using western blots to measure activated NOTCH1 (ICN1) protein levels, and real-time RT-PCR (qPCR) to measure transcript levels of known NOTCH1 targets (e.g., HES1). Two clonal sublines (J.2-4 and J.2-7) were identified with significantly decreased expression of NOTCH1 compared to NTC. The J.2-4 and J.2-7 sublines showed minimal changes in cell growth, cell cycle progression and apoptosis. To characterize genotypic changes accompanying NOTCH1 knockdown, we performed microarray analysis with Agilent Whole Genome oligonucleotide microarrays and microRNA (miR) HumanV2 arrays. The miR array identified 20 miRs in J.2-4 and J.2-7 cells with altered expression compared to NTC greater than 1.5-fold (p<0.05) and ranging from 3-to10-fold. miRs hsa-Let-7e, hsa-miR-125a-5p and hsa-miR-99b, reportedly derived from a polycistronic transcript, were decreased 10-fold accompanying NOTCH1 knockdown. Using miR qPCR, we confirmed decreased levels of hsa-miR-125a-5p and hsa-miR-99b in the J.2-4 and J.2-7 sublines. From predictive algorithms, Mcl-1 is a predicted target of hsa-miR-125a-5p. On westerns, clones J.2-4 and J.2-7 showed significant increases in levels of Mcl-1 protein over NTC. In conclusion, we have developed novel T-ALL cell line models to study the impact of decreased NOTCH1 levels and activity independent of GSI treatment. Our results implicate NOTCH1 in regulating levels of hsa-miR-125a-5p with possible downstream effects on the anti-apoptotic protein, Mcl-1. Our findings suggest caution may be warranted in targeting NOTCH1 with GSIs in the therapy of T-ALL, reflecting the potential downstream impact on Mcl-1 levels resulting in decreased apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4360.

Abstract 3051: mRNA and microRNA expression profiles integrated with drug sensitivities of the NCI-60 human cancer cell lines

Abstract We present here new NCI-60 profiling studies of mRNA and miRNA expression using the 41,000-probe Agilent Whole Human Genome Oligo Microarray and the 15,000-feature Agilent Human miRNA Microarray V2. Expression levels were determined for ∼21,000 genes and 723 human miRNAs. The resulting data sets are freely available and searchable online at http://discover.nci.nih.gov in our CellMiner relational database package. The profiling included technical replicates, with six and eight cell lines assayed in quadruplicate for mRNA and miRNA, respectively. The remaining cell lines were assayed in duplicate. Our analysis indicates high reproducibility for both platforms and an essential biological similarity across the various cell types. The expression levels were integrated with our previously published 1,429-compound database of anticancer activity in the NCI screen. Large blocks of both mRNAs (∼2000) and miRNAs (∼200) with unidirectional correlation to ∼1300 drugs including 121 drugs with known mechanisms of action were identified. The data sets presented here will facilitate recognition of the groups of mRNAs, miRNAs and drugs that potentially affect and interact with one another. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3051.

Abstract 1103: Linkage between endogenous Chk2 activation and p14ARF expression in p53-mutated cells revealed by proteomic and genomic analyses of the NCI60

Abstract Chk2 is a multi-substrate kinase involved in the DNA damage response and genomic stability. Chk2 inhibitors are currently being evaluated in cancer models, with the rationale of enhancing the activity of DNA damaging agents in p53-deficient tumors while protecting normal tissues. Part of the ATM-Chk2-p53 axis, Chk2 is overexpressed or activated in various malignant tumors. Here we determined Chk2 copy number, total and single-exon transcriptional levels, and total and activated (pT68) protein across the 60 cell lines of the NCI DTP screen. We correlated these parameters with p53 mutational status, miRNA expression (Agilent miRNA Microarray V2), and performed supervised transcript (Agilent Whole Human Genome Oligo Microarray) class comparison analysis between high and low pT68 Chk2 cell lines using BRB Array Tools 3.8. All the lines used for this purpose were known to be p53-mutated. Our phosphoproteomic analyses demonstrate that a number of cell lines have high endogenous Chk2 activation (ovarian OVCAR3 and OVCAR4, renal RFX-393, lung NCI-H322M, and EKVX, melanoma UACC-62 and −257, leukemia RPMI-8226 and breast BT-549) whereas the wild-type p53 tumors almost invariably show undetectable Chk2 activation. Expression of total Chk2 protein and mRNA were generally positively correlated to each other. Exon array analyses across the NCI-60 suggest exon 3 splicing in almost all Chk2 high expresser cell lines and premature truncation between exons 8 and 10 in the ovarian SKOV3 cells, which could be related to the low level of Chk2 expression in that cell line. One microRNA, hsa-miR-455-3p, showed a highly significant negative correlation with both Chk2 transcript levels and with total protein levels (Spearman's r =-0.48, p < 10-3 and r=-0.35, p < 0.01 respectively) and was predicted to hybridize to Chk2 mRNA by miRBase (www.mirbase.org). Among the 181 probes differentially expressed in cells with highly activated Chk2, 123 mapped to known gene IDs, and 58 were eligible for network analysis with Ingenuity Pathways Analysis 8.0 (Ingenuity Systems Inc.). Functions associated with the networks enriched in genes differentially expressed by high pT68 cells included: cell cycle, cell-to-cell signaling, cell growth and proliferation, cancer. Remarkably, the tumor suppressor CDKN2A (p14ARF) showed a 132-fold difference between the two groups considered (unpaired t test, two-tailed p < 10-7; false discovery rate < 10-7), being highly overexpressed in high pT68 Chk2 cell lines. Several EGFR-Ras-MAPK related genes were downregulated in high pT68 cell lines. Together, these data suggest that Chk2 is part of a multifaceted regulation network in cancer. Although traditionally considered as a low-penetrance tumor suppressor, it clearly plays more complex roles in tumor biology, possibly behaving as an activated oncogene in specific genomic contexts. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1103.

Abstract 5722: Effects of dietary omega-3 and omega-6 in the prevention of rat mammary cancer with tamoxifen

Abstract There is evidence that omega 3-rich diet strengthened the inhibitory effect of tamoxifen on the development of estrogen dependent tumors, pointing to omega 3 as a potential adjuvant to standard therapy (Manni A. et al. Cancer Prev Res, in press). However, the mechanistic basis of this observation is unknown. The objective of the present work is to analyze the genomic changes in chemically induced mammary tumors that developed in tamoxifen treated rats fed omega 3 or omega 6 fatty acids. Sprague Dawley rats, 21 days-old, were administered an i.p. injection of 1-Methyl-1-nitrosourea (MNU) 50 mg/kg body weight. The animals were distributed into 4 groups (G), and maintained under standard conditions and were given the following treatments for 8 weeks: G1 received a diet of 20% of corn oil (CO); G2 received a diet with 17% of fish oil (FO) and 3% CO; G3 received the CO diet and tamoxifen; G4 received FO diet and tamoxifen. Tamoxifen was administered s.c 100 μg/Kg b.w., 5 days/week. The animals were euthanized after 8 weeks. Mammary tumors were frozen in liquid nitrogen and stored at −80C. RNA from cribiform tumors was extracted and one-color Agilent 44K whole rat genome oligonucleotide microarray was performed in three tumor samples per group. Tamoxifen-treated groups (G3 and G4) were compared with their respective controls (G1 and G2). The genes were considered differentially expressed when the p value was lower or equal to 0.01 and had at least 1.50 fold change. A total of 488 genes were considered statistically different comparing G3 to G1 (G3/G1), whereas 206 genes were considered statistically different comparing G4 to G2 (G4/G2). Gene ontology analysis showed that 125 genes of G3/G1 comparison and 58 of G4/G2 comparison were related to cancer, apoptosis, cellular movement, cellular proliferation, inflammatory response or angiogenesis. In the G3 /G1 comparison, the expression pattern of genes related to cell proliferation and apoptosis was found to be divergent, as well as the genes related to cell proliferation in the G4/G2 comparison. In contrast, G4/G2 did not showed relevant genes expressed related to apoptosis. CO treatment in presence of tamoxifen (G3/G1) induced an up-regulation of C5r1, Hck, Il6Ra, Myo1f_predicted and Tlr6, and down-regulation of Mif, indicating an increase of the inflammatory host response. These changes were accompanied by up-regulation of Ets1, Tlr6 and Wdr44 genes controlling angiogenesis and cell migration, which can lead to metastasis. These changes were not observed in the FO and tamoxifen treated group. We conclude that an omega 6 rich-diet could modify the genomic profile of tamoxifen treated tumors making them more prone to an aggressive behavior or result in tamoxifen resistance. (Supported by the Susan G. Komen for the Cure, grant KG081632). The first two authors contributed equally to this work. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5722.

Genome-wide transcription profiling identifies the mechanisms of LFA-1 contributing to CD8+ T Cell Activation and immune signal network (50.26)

Abstract Our previous data demonstrated that the function of LFA-1 is to enhance TCR signaling through the immunological synapse and deliver distinct signal in CD8+ T cell activation. However, the detailed molecular pathways that regulate these processes and global impact on immune functions are poorly defined. The purpose of this genomic study is to explore and understand the LFA-1 and TCR signaling network with CD8+ T cell transcription profiling. CD8+ T cells from C57BL/6 mice and LFA-1-KO mice before and after stimulation were applied to the Mouse Whole Genome Oligo Microarray. Statistic analysis identified a list of LFA-1-associated genes differentially expressed in activated LFA-1-KO CD8+ T cells. Pathway analysis indicated that most of these genes are enriched in IL-10 signaling/ p38 MAPK signaling/ cell cycle regulation/ notch signaling/ NK signaling/ chemokine signaling. Furthermore, biological function analysis implicated these genes enriched in the functions of antigen presentation/ cell-mediated immune response/ inflammatory response/ cellular development/ cell-to-cell signaling and interaction/ immune cell trafficking/ cellular growth and proliferation/ inflammatory disease/ lymphoid tissue structure and development. Our results indicated that LFA-1 plays an important role in the immune signal network and has a global impact on immune system, and provided a complete genome-wide database to further characterize the molecular mechanisms of LFA-1 during T cell activation.

Gene expression analysis of Mas‐related gene receptors in the non‐inflamed and inflamed murine ileum

Due to the lack of data on the expression of Mas‐related gene receptors (Mrgs), a family of G protein‐coupled orphan receptors, we investigated their expression in the non‐inflamed and inflamed murine ileum. We performed a gene expression analysis using the Agilent Whole‐Mouse Genome Oligo Microarray. Relative expression levels of 20 Mrgs sequenced in mouse, were analysed using Real‐Time PCR, and immunohistochemical analyses with antibodies against MrgE and F were performed. Microarray analyses resulted in ~3000 and ~5000 differentially expressed genes in the acute phases of intestinal schistosomiasis and TNBS‐induced ileitis, respectively. No altered gene expression of any of the Mrgs was detected, whereas Real‐Time PCR revealed significant increased expression of MrgB1 and D during intestinal schistosomiasis and of MrgA2 during ileitis. The expression of MrgE, F, G, H and X1 was significantly reduced during ileitis. MrgX2 was found to be only expressed during inflammation. MrgE and F were both detected in a subpopulation of enteric neurons. In conclusion, the results indicate that, in accordance to what has been proposed earlier, some Mrgs appear to be involved in intestinal inflammation.

Gene expression profiles of human promyelocytic leukemia cell lines exposed to volatile organic compounds

Benzene, toluene, o-xylene, ethylbenzene, trichloroethylene and dichloromethane are the most widely used volatile organic compounds (VOCs), and their toxic mechanisms are still undefined. This study analyzed the genome-wide expression profiles of human promyelocytic leukemia HL-60 cells exposed to VOCs using a 35-K whole human genome oligonucleotide microarray to ascertain potential biomarkers. Genes with a significantly increased expression levels (over 1.5-fold and p-values <0.05) with six VOCs were then classified with gene ontology and KEGG pathway annotation. At IC 20 doses identified genes were functionally categorized as being involved in cytokine–cytokine receptor interactions and the toll-like receptor signaling pathway, whereas exposure at IC 50 doses identified genes associated with the p53 signaling pathway, apoptosis, and natural killer cell-mediated cytotoxicity pathway. Functionally important immune response- and apoptosis-related genes were further validated by real-time RT-PCR. The results showed that IFIT1, IFIT2, IFIT3, USP18, INFGR2, PMAIP1, GADD45A, NFKBIA, TNFAIP3, and BIRC3 genes altered their expression profiles in a dose-dependent manner. Similar expressions profiles were also found in human erythromyeloblastoid leukemia K562 cells and in human leukemic monocyte lymphoma U937 cells. In conclusion, both gene expression profiles and gene ontology analysis have elucidated potential gene-based biomarkers and provided insights into the mechanism underlying the response of human leukemia cell lines to VOC exposure.

Inactivation of the GacA response regulator in Pseudomonas fluorescens Pf‐5 has far‐reaching transcriptomic consequences

The GacS/GacA signal transduction system is a central regulator in Pseudomonas spp., including the biological control strain P. fluorescens Pf-5, in which GacS/GacA controls the production of secondary metabolites and exoenzymes that suppress plant pathogens. A whole genome oligonucleotide microarray was developed for Pf-5 and used to assess the global transcriptomic consequences of a gacA mutation in P. fluorescens Pf-5. In cultures at the transition from exponential to stationary growth phase, GacA significantly influenced transcript levels of 635 genes, representing more than 10% of the 6147 annotated genes in the Pf-5 genome. Transcripts of genes involved in the production of hydrogen cyanide, the antibiotic pyoluteorin and the extracellular protease AprA were at a low level in the gacA mutant, whereas those functioning in siderophore production and other aspects of iron homeostasis were significantly higher in the gacA mutant than in wild-type Pf-5. Notable effects of gacA inactivation were also observed in the transcription of genes encoding components of a type VI secretion system and cytochrome c oxidase subunits. Two novel gene clusters expressed under the control of gacA were identified from transcriptome analysis, and we propose global-regulator-based genome mining as an approach to decipher the secondary metabolome of Pseudomonas spp.

LFA-1 Regulates CD8 + T Cell Activation and Immune Signal Network.

Abstract 1641Poster Board I-667LFA-1 regulates T cell activation and signal transduction through the immunological synapse. TCR stimulation rapidly activates LFA-1, which provides unique LFA-1-dependent signals to promote T cell activation. We found LFA-1 directly participates in Erk1/2 signaling upon TCR stimulation in CD8+ T cells. The presence of LFA-1, not ligand binding, is required for the TCR-mediated Erk1/2 signal pathway. LFA-1-KO T cells have defects in sustained Erk1/2 signaling and TCR/CD3 clustering, which subsequently prevents MTOC re-orientation, cell-cycle progression and mitosis. LFA-1 regulates the TCR-mediated Erk1/2 signal pathway in the context of immunological synapse for recruitment and amplification of Erk1/2 signal. In addition, LFA-1 ligation with ICAM-1 generates an additional Erk1/2 signal, which synergizes with the existing TCR-mediated Erk1/2 signal to enhance T cell activation. We demonstrated that the function of LFA-1 is to enhance TCR signaling through the immunological synapse and deliver distinct signal in CD8+ T cell activation.Based on our results, we proposed a model of TCR-mediated and LFA-1-mediated Erk1/2 signal pathways in CD8+ T cell activation. However, the detailed molecular pathways that regulate these processes and global impact on immune functions are poorly defined. With the launching of The Immunological Genome Project, we have generated data with CD8+ T cell expression array to explore and understand the LFA-1 and TCR signaling network. GeneChip hybridization and analysis CD8+ T cells from C57BL/6 mice and LFA-1-KO mice were collected before and after stimulation. Microarray experiments were carried out using the “ Mouse Whole Genome Oligo Microarray Kit” from Agilent. Differentially expressed gene lists between samples were considered significant if their p values were <0.0001 and their fold-change >1.8. The results are summarized below: 1) 641 genes were up-regulated and 174 were down-regulated in unstimulated LFA-1-KO CD8+ T cells comparing to these in unstimulated WT CD8+ T cells; 2)1036 genes were up-regulated and 406 were down-regulated in activated LFA-1-KO CD8+ T cells comparing to these in activated WT CD8+ T cells after 1 hours stimulation with unstimulated CD8+ T cells as control; 3)636 genes were up-regulated and 354 were down-regulated in activated LFA-1-KO CD8+ T cells comparing to these in activated WT CD8+ T cells after 8 hours stimulation with unstimulated CD8+ T cells as control.To estimate which pathways are significantly enriched among the genes that are differentially expressed, we used a database IPA (Ingenuity Pathways Analysis) software. Signaling pathways enriched by the genes differentially expressed in activated LFA-1-KO CD8+ T cells vs WT CD8+ T cells after 8 hours stimulation generated the most highly enriched differentially expressed genes in signaling pathways displayed below: 1) Pattern Recognition Receptors in Recognition of Bacteria and Viruses; 2) Activation of IRF by Cytosolic Pattern Recognition Receptors; 3) Aryl Hydrocarbon Receptor Signaling; 4) IL-10 Signaling; p38 MAPK Signaling; 5) LPS/IL-1 Mediated Inhibition of RXR Function; Cell Cycle: G2/M DNA Damage Checkpoint Regulation; 6) Notch Signaling; Natural Killer Cell Signaling; 7) Cell Cycle: G1/S Checkpoint Regulation and Chemokine Signaling. Furthermore, we examined the biological functions enriched by the genes differentially expressed in activated LFA-1-KO CD8+ T cells vs WT CD8+ T cells. The LFA-1-KO CD8+ T cells vs WT CD8+ T cells after 8 hours stimulation generated the most highly enriched differentially expressed genes in following biological functions: Immunological Disease; Antigen Presentation; Cell-mediated Immune Response; Humoral Immune Response; Inflammatory Response; Cellular Development; Cell-To-Cell Signaling and Interaction; Hematological System Development and Function; Immune Cell Trafficking; Cellular Growth and Proliferation; Inflammatory Disease; Cellular Movement; Hematopoiesis Cell Death; Hematological Disease; Lymphoid Tissue Structure and Development; Infectious Disease; Tissue Development and Cancer. Our results indicate that LFA-1 plays an important role in the immune signal network and has a global impact on immune system. DisclosuresNo relevant conflicts of interest to declare.

Silencing of Cited2 and Akap12 genes in radiation-induced rat osteosarcomas

We have previously studied genomic copy number changes and global gene expression patterns in rat osteosarcomas (OS) induced by the bone-seeking alpha emitter 238Pu by comparative genomic hybridization (CGH) and oligonucleotide microarray analyses, respectively. Among the previously identified genes that were down-regulated in radiation-induced rat OS tumors, Cited2 (Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 2) and Akap12 (a kinase anchoring protein, also known as src-suppressed C-kinase substrate, SSeCKS) genes mapped to the most frequently lost regions on chromosome 1p. In the present study, relative copy number losses of Cited2 and Akap12 genes were observed in 8 of 15 (53%) and 10 of 15 (67%) tumors by quantitative PCR analysis. Loss of Cited2 and Akap12 in the tumors was confirmed at the levels of mRNA and protein expression by quantitative RT-PCR and immunoblot analyses, respectively. These results indicate that Cited2 and Akap12 are silenced in radiation-induced OS, and therefore are novel candidate tumor-suppressor genes of this tumor.

Evaluation of genes identified by microarray analysis in favorable neuroblastoma

The biological heterogeneity of neuroblastoma results in a varied outcome ranging from spontaneous regression to fatal tumor progression. Microarray expression profiling and genetic polymorphism arrays may help identify key genes that differ in aggressive neuroblastomas from those observed in tumors associated with a favorable outcome. Total RNA was extracted from 16 neuroblastomas obtained from patients who subsequently died of the disease and from 16 favorable neuroblastomas and analyzed using a human whole genome oligomicroarray (55K CodeLink). Genes overexpressed in favorable tumors were subsequently analyzed in 121 neuroblastoma tumors obtained before chemotherapy using real-time RT-PCR. And among these cases, expression levels of these genes were also analyzed in 20 tumors obtained after chemotherapy. Oligomicroarray analysis revealed the overexpression of 283 genes in favorable tumors that were associated with either regressing or maturing tumors. Three candidate genes, including DHRS3, NROB1, and CYP26A1, were selected that were significantly overexpressed in favorable tumors by quantitative real-time RT-PCR (P < 0.01). No cases with overexpression of all three genes showed poor outcomes. In 20 post-chemotherapeutic tumors, the expression levels of these genes increased in the cases where patients survived but decreased in the fatal cases. Using microarray expression profiling, we identified genes that exhibit altered gene expression in neuroblastoma tumors associated with a favorable outcome. These candidates warrant further study for use in risk assessment and/or as therapeutic targets in neuroblastoma.

Molecular profiling and characterization of luminal-like and basal-like in vivo breast cancer xenograft models

The number of relevant and well-characterized cell lines and xenograft models for studying human breast cancer are few, and may represent a limitation for this field of research. With the aim of developing new breast cancer model systems for in vivo studies of hormone dependent and independent tumor growth, progression and invasion, and for in vivo experimental therapy studies, we collected primary mammary tumor specimens from patients, and implanted them in immunodeficient mice. Primary tumor tissue from 29 patients with breast cancer was implanted subcutaneously with matrigel in SCID mice, in the presence of continuous release of estradiol. The tumors were transferred into new animals when reaching a diameter of 15 mm and engrafted tumors were harvested for morphological and molecular characterization from passage six. Further, gene expression profiling was performed using Agilent Human Whole Genome Oligo Microarrays, as well as DNA copy number analysis using Agilent Human Genome CGH 244K Microarrays. Of the 30 primary tumors implanted into mice (including two implants from the same patient), two gave rise to viable tumors beyond passage ten. One showed high expression levels of estrogen receptor-α protein (ER) while the other was negative. Histopathological evaluation of xenograft tumors was carried out at passage 10–12; both xenografts maintained the morphological characteristics of the original tumors (classified as invasive grade III ductal carcinomas). The genomic profile of the ER-positive xenograft tumor resembled the profile of the primary tumor, while the profile obtained from the ER-negative parental tumor was different from the xenograft. However, the ER-negative parental tumor and xenograft clustered on the same branch using unsupervised hierarchical clustering analysis on RNA microarray expression data of “intrinsic genes”. A significant variation was observed in the expression of extracellular matrix (ECM)-related genes, which were found downregulated in the engrafted tumors compared to the primary tumor. By IHC and qRT-PCR we found that the downregulation of stroma-related genes was compensated by the overexpression of such molecules by the mouse host tissue. The two established breast cancer xenograft models showed different histopathological characteristics and profound diversity in gene expression patterns that in part can be associated to their ER status and here described as basal-like and luminal-like phenotype, respectively. These two new breast cancer xenografts represent useful preclinical tools for developing and testing of new therapies and improving our knowledge on breast cancer biology.

Genome-wide transcriptional profiling of mononuclear phagocytes recruited to mouse lungs in response to alveolar challenge with the TLR2 agonist Pam3CSK4

Compared with the Toll-like receptor 4 (TLR4) ligand LPS restricted to gram-negative bacteria, few studies have addressed induction of lung inflammation and concomitant leukocyte recruitment in response to TLR2 ligands. This study is the first report showing that selective TLR2 stimulation by its ligand Pam(3)-Cys-Ser-Lys-Lys-Lys-Lys-OH (Pam(3)CSK(4)) within the alveolar compartment promoted lung inflammation in mice and induced the migration of circulatory immune cells including mononuclear phagocytes into the inflamed alveolar space. By using the transgenic CX(3)CR1(+/GFP) mouse strain for high-purity sorting of circulating and alveolar recruited mononuclear phagocytes together with SMART preamplification and whole genome oligonucleotide microarray techniques, we found that alveolar trafficking of mononuclear phagocytes was associated with profound changes of their gene expression profiles (approximately 900 differentially regulated genes postrecruitment). In particular, alveolar recruited mononuclear phagocytes showed upregulated transcripts of genes encoding cytokines/chemokines and pattern recognition receptor (PRR)-associated molecules. Notably, we observed a dynamic change of the genetic program of recruited mononuclear phagocytes obtained from bronchoalveolar lavage fluid at different time points (24 vs. 48 h) post-Pam(3)CSK(4) challenge. In early alveolar recruited mononuclear phagocytes, mRNA levels of both proinflammatory (e.g., TNF-alpha, CCL2, and IL-6) and central anti-inflammatory/ proresolution [e.g., IL-1-receptor antagonist (IL-1RN), CD200 receptor (CD200R), IL-1 receptor-associated kinase (IRAK-M), IL-10, and Bcl-2-associated X protein (Bax)] mediators were found to be highly upregulated simultaneously. In corresponding cells recruited until later time points, transcript levels of anti-inflammatory/proresolution molecules persisted at the same level, whereas mRNA levels of proinflammatory mediators were found to decline. Collectively, our in vivo study identifies genetic programs by which alveolar recruited mononuclear phagocytes may contribute to the development and termination of pneumonia caused by gram-positive bacteria.

A microarray study of altered gene expression in colorectal cancer cells after treatment with immunomodulatory drugs: differences in action in vivo and in vitro

Thalidomide and lenalidomide are FDA approved for the treatment of multiple myeloma, and along with pomalidomide are being investigated in a variety of other cancers. Although these agents display immunomodulatory, anti-angiogenic and anti-apoptotic effects, little is known about the primary mode of therapeutic action in patients with cancer. This paper describes a microarray study of the in vitro and in vivo effects of these drugs, and contrasts the difference in gene profiles achieved in the two models. In the current study, Agilent whole mouse genome oligonucleotide microarrays (44 K) were used to examine alterations in gene expression of colorectal cancer cells after treatment. Venn analysis revealed a divergence of gene signature for pomalidomide and lenalidomide, which although similar in vitro, different in vivo. Several clusters of genes involved in various cellular processes such as immune response, cell signalling and cell adhesion were altered by treatment, and common to the three drugs. Notably, the expressions of linked genes within the Notch/Wnt signalling pathway, including kremen2 and dtx4, highlighted a possible novel mechanistic pathway for these drugs. This study also showed that gene signatures were not greatly divergent in the models, and recapitulated the complex nature of these drugs. Overall, these microarray studies highlighted the diversity of this class of drug, which have effects ranging from cell signalling to translation initiation.

Experimental design and environmental parameters affect Rhodospirillum rubrum S1H response to space flight

In view of long-haul space exploration missions, the European Space Agency initiated the Micro-Ecological Life Support System Alternative (MELiSSA) project targeting the total recycling of organic waste produced by the astronauts into oxygen, water and food using a loop of bacterial and higher plant bioreactors. In that purpose, the alpha-proteobacterium, Rhodospirillum rubrum S1H, was sent twice to the International Space Station and was analyzed post-flight using a newly developed R. rubrum whole genome oligonucleotide microarray and high throughput gel-free proteomics with Isotope-Coded Protein Label technology. Moreover, in an effort to identify a specific response of R. rubrum S1H to space flight, simulation of microgravity and space-ionizing radiation were performed on Earth under identical culture set-up and growth conditions as encountered during the actual space journeys. Transcriptomic and proteomic data were integrated and permitted to put forward the importance of medium composition and culture set-up on the response of the bacterium to space flight-related environmental conditions. In addition, we showed for the first time that a low dose of ionizing radiation (2 mGy) can induce a significant response at the transcriptomic level, although no change in cell viability and only a few significant differentially expressed proteins were observed. From the MELiSSA perspective, we could argue the effect of microgravity to be minimized, whereas R. rubrum S1H could be more sensitive to ionizing radiation during long-term space exploration mission.

Chronically CNS-Injured Adult Sensory Neurons Gain Regenerative Competence upon a Lesion of Their Peripheral Axon

Several experimental manipulations result in axonal regeneration in the central nervous system (CNS) when applied before or at the time of injury but not when initiated after a delay, which would be clinically more relevant. As centrally injured neurons show signs of atrophy and degeneration, it raises the question whether chronically injured neurons are able to regenerate. To address this question, we used adult rodent primary sensory neurons that regenerate their central axon when their peripheral axon is cut (called conditioning) beforehand but not afterwards. We found that primary sensory neurons express regeneration-associated genes and efficiently regrow their axon in cell culture two months after a central lesion upon conditioning. Moreover, conditioning enables central axons to regenerate through a fresh lesion independent of a previous central lesion. Using in vivo imaging we demonstrated that conditioned neurons rapidly regrow their axons through a fresh central lesion. Finally, when single sensory axons were cut with a two-photon laser, they robustly regenerate within days after attaining growth competence through conditioning. We conclude that sensory neurons can acquire the intrinsic potential to regenerate their axons months after a CNS lesion, which they implement in the absence of traumatic tissue.

Analysis of whole genomic expression profiles ofHelicobacter pylorirelated chronic atrophic gastritis with IL‐1B‐31CC/‐511TT genotypes

Many studies have linked cytokine interleukin-1B gene polymorphisms to H. pylori-related gastric cancer development. The current study evaluated the characterization of whole genomic expression profiles of the premalignant condition: H. pylori-related chronic atrophic gastritis (CAG) with IL-1B-31CC/-511TT genotypes. IL-1B-31/-511 gene polymorphisms were determined by DNA sequences. RNA was extracted and expression profiles were performed using Agilent human whole genomic oligonucleotide microarrays (G4112F). The expression of three samples with H. pylori infection was compared to that of three samples without H. pylori infection from samples of six CAG patients, all with IL-1B-31CC/-511TT genotypes. Differentially expressed genes related to H. pylori-induced CAG with IL-1B-31CC/-511TT genotypes were screened and analyzed further by Gene Ontology (GO) and pathway. Validation of the microarray data was performed using qRT-PCR. A total of 124 differentially expressed genes and 32 GO term annotations were identified between H. pylori positive and negative groups in the six CAG samples with IL-1B-31CC/-511TT genotypes. The signaling pathways identified were oxidative phosphorylation and epithelial cell signaling in H. pylori infection. Five overlapping genes were contained in identified GO terms and pathways: ATP6V0B, NDUFS5, NDUFV2, ATP6V1F and ATP6V1G1. Comparisons of qRT-PCR data and the previously reported data with the results of gene chips support the validity of our microarray data. The H. pylori-related CAG with IL-1B-31CC/-511TT genotypes has shown to be the more malignant phenotype than H. pylori negative CAG with IL-1B-31CC/-511TT genotypes. Mitochondrial energy metabolism probably plays a crucial role as it is the molecular mechanism of host-bacterial interactions.

Protumorigenic Role of HAPLN1 and Its IgV Domain in Malignant Pleural Mesothelioma

Tumor extracellular matrix (ECM) plays a crucial role in cancer progression mediating and transforming host-tumor interactions. Targeting the ECM is becoming an increasingly promising therapeutic approach in cancer treatment. We find that one of the ECM proteins, HAPLN1, is overexpressed in the majority of mesotheliomas. This study was designed to characterize the protumorigenic role of HAPLN1 in mesothelioma. Overexpression of HAPLN1 was assessed and validated on a large set of normal/mesothelioma specimens on the RNA and protein levels. We also analyzed DNA copy number alterations in the HAPLN1 genomic locus using the array-based comparative genomic hybridization representational oligonucleotide microarray analysis tool. Tumorigenic activities of the HAPLN1 domains were evaluated in vitro on mesothelioma cells transfected with HAPLN1-expressing constructs. We found that HAPLN1 is 23-fold overexpressed in stage I mesothelioma and confirmed it for 76% samples (n = 53) on RNA and 97% (n = 40) on protein levels. The majority of lung cancers showed no differential expression of HAPLN1. Analysis of DNA copy number alterations identified recurrent gain in the 5q14.3 HAPLN1 locus in approximately 27% of tumors. Noteworthy, high expression of HAPLN1 negatively correlated with time to progression (P = 0.05, log-rank test) and overall survival (P = 0.006). Proliferation, motility, invasion, and soft-agar colony formation assays on mesothelioma cells overexpressing full-length HAPLN1 or its functional domains strongly supported the protumorigenic role of HAPLN1 and its SP-IgV domain. Overexpression of HAPLN1 and its SP-IgV domain increases tumorigenic properties of mesothelioma. Thus, targeting the SP-IgV domain may be one of the therapeutic approaches in cancer treatment.

Effects of folate on the brain gene expression of Tg2576 transgenic mice treated with Alzheimer drug memantine: a pilot study using cDNA microarray profiling

Using Tg2576 transgenic mice that produce endogenous beta‐amyloid (Aβ) protein in brain, we have previously reported that high dose of folic acid supplemented to Alzheimer drug memantine (MT)‐treated AD mice significantly improved the neuronal functional performance on Morris water maze test. To further study effects of folate on the brain gene expression profile of MT‐treated transgenic mice, a total of 6 Aβ mice brains (3 from MT mice and 3 from folate+MT mice) were analyzed by cDNA microarrays. Total RNA isolation, cDNA synthesis and cy3 and 5 dye labeling were performed using standard protocols. The labeled samples were hybridized to Agilent Whole Mouse Genome Oligo Microarray containing 41,000+ annotated genes and transcripts. Expression data were analyzed using GeneSpring GX 10. Only genes with net expression changes of &gt; 2‐fold occurring among 20‐100th percentile of expression levels in each hybridized arrays were considered for further analysis. Compared to MT‐treated Aβ mice brains, folate+MT brains showed a generalized up‐regulation of 72 brain gene transcriptions involving in neurogenesis, memory, action potential production, neurotransmitter receptors, vasodilation, and immune modulation. Down regulations were only found in 9 transcripts. Validation of gene expressions by real‐time PCR and the significance of modulated expression profiles by folate will be further discussed.

Expression Analysis of Early Response-Related Genes in Rat Liver Epithelial Cells Exposed to Thioacetamide in vitro

Thioacetamide (TA) is a potent hepatotoxicant known to affect liver metabolism, inhibit mRNA transport and induce immune suppression. The genetic mechanism underlining this biological toxic compound is well understood using microarray technology. Thus, we used high-throughput rat genome oligonucleotide microarrays containing approximately 22,000 genes to investigate the genetic components of TA-related cytotoxicity in WB-F344 rat liver epithelial (WB-F344) cells. We treated cells with TA (two concentrations over five time periods, ranging from 1 to 24 hr), isolated total RNA at 1, 3, 6, 12 and 24 hr following TA treatment and hybridized the RNA to microarrays. Clustering analysis distinguished two groups of genes, early (1 and 3 hr) and late (6, 12 and 24 hr) phase genes. In total, 2,129 and 2,348 differentially-expressed genes were identified following treatment with low and high concentrations of TA, respectively. A common set of 1,229 genes that were differentially expressed following treatment with both low (1,000 muM) and high (10,000 muM) concentrations of TA had similar expression patterns. Interestingly, 1,410 genes at the low concentration and 1,858 genes at the high concentration were differentially expressed in the early phases, suggesting that these genes associated with the early response to TA may be useful as early markers of hepatotoxicity.