Microarray-based Gene Expression Analysis Research Articles

Global transgenerational gene expression dynamics in two newly synthesized allohexaploid wheat (Triticum aestivum) lines

BackgroundAlteration in gene expression resulting from allopolyploidization is a prominent feature in plants, but its spectrum and extent are not fully known. Common wheat (Triticum aestivum) was formed via allohexaploidization about 10,000 years ago, and became the most important crop plant. To gain further insights into the genome-wide transcriptional dynamics associated with the onset of common wheat formation, we conducted microarray-based genome-wide gene expression analysis on two newly synthesized allohexaploid wheat lines with chromosomal stability and a genome constitution analogous to that of the present-day common wheat.ResultsMulti-color GISH (genomic in situ hybridization) was used to identify individual plants from two nascent allohexaploid wheat lines between Triticum turgidum (2n = 4x = 28; genome BBAA) and Aegilops tauschii (2n = 2x = 14; genome DD), which had a stable chromosomal constitution analogous to that of common wheat (2n = 6x = 42; genome BBAADD). Genome-wide analysis of gene expression was performed for these allohexaploid lines along with their parental plants from T. turgidum and Ae. tauschii, using the Affymetrix Gene Chip Wheat Genome-Array. Comparison with the parental plants coupled with inclusion of empirical mid-parent values (MPVs) revealed that whereas the great majority of genes showed the expected parental additivity, two major patterns of alteration in gene expression in the allohexaploid lines were identified: parental dominance expression and non-additive expression. Genes involved in each of the two altered expression patterns could be classified into three distinct groups, stochastic, heritable and persistent, based on their transgenerational heritability and inter-line conservation. Strikingly, whereas both altered patterns of gene expression showed a propensity of inheritance, identity of the involved genes was highly stochastic, consistent with the involvement of diverse Gene Ontology (GO) terms. Nonetheless, those genes showing non-additive expression exhibited a significant enrichment for vesicle-function.ConclusionsOur results show that two patterns of global alteration in gene expression are conditioned by allohexaploidization in wheat, that is, parental dominance expression and non-additive expression. Both altered patterns of gene expression but not the identity of the genes involved are likely to play functional roles in stabilization and establishment of the newly formed allohexaploid plants, and hence, relevant to speciation and evolution of T. aestivum.

Group VIA phospholipase A2is a target for vasopressin signaling in the thick ascending limb

Na(+)-K(+)-2Cl(-) cotransporter (NKCC2)-mediated NaCl reabsorption in the thick ascending limb (TAL) is stimulated by AVP via V2 receptor/PKA/cAMP signaling. This process is antagonized by locally produced eicosanoids such as 20-HETE or prostaglandin E(2), which are synthesized in a phospholipase A(2)-dependent reaction cascade. Using microarray-based gene expression analysis, we found evidence for an AVP-dependent downregulation of the calcium-independent isoform of PLA(2), iPLA(2)β, in the outer medulla of rats. In the present study, we therefore examined the contribution of iPLA(2)β to NKCC2 regulation. Immunoreactive iPLA(2)β protein was detected in cultured mTAL cells as well as in the entire TAL of rodents and humans with the exception of the macula densa. Administration of the V2 receptor-selective agonist desmopressin (5 ng/h; 3 days) to AVP-deficient diabetes insipidus rats increased outer medullary phosphorylated NKCC2 (pNKCC2) levels more than twofold in association with a marked reduction in iPLA(2)β abundance (-65%; P < 0.05), thus confirming microarray results. Inhibition of iPLA(2)β in Sprague-Dawley rats with FKGK 11 (0.5 μM) or in mTAL cells with FKGK 11 (10 μM) or (S)-bromoenol lactone (5 μM) for 1 h markedly increased pNKCC2 levels without affecting total NKCC2 expression. Collectively, these data indicate that iPLA(2)β acts as an inhibitory modulator of NKCC2 activity and suggest that downregulation of iPLA(2)β may be a relevant step in AVP-mediated urine concentration.

The arthritis severity locus Cia5a regulates the expression of inflammatory mediators including Syk pathway genes and proteases in pristane-induced arthritis

BackgroundCia5a is a locus on rat chromosome 10 that regulates disease severity and joint damage in two models of rheumatoid arthritis, collagen- and pristane-induced arthritis (PIA). In this study, we aimed to identify cellular and molecular processes regulated by Cia5a using microarray-based gene expression analysis of synovial tissues from MHC identical DA (severe erosive disease) and DA.F344(Cia5a) congenics (mild non-erosive disease) rats.ResultsSynovial tissues from six DA and eight DA.F344(Cia5a) rats were analyzed 21 days after the induction of PIA using the Illumina RatRef-12 BeadChip (21,922 genes) and selected data confirmed with qPCR. There was a significantly increased expression of pro-inflammatory mediators such as Il1b (5-fold), Il18 (3.9-fold), Cxcl1 (10-fold), Cxcl13 (7.5-fold) and Ccl7 (7.9-fold), and proteases like Mmp3 (23-fold), Mmp9 (32-fold), Mmp14 (4.4-fold) and cathepsins in synovial tissues from DA, with reciprocally reduced levels in congenics. mRNA levels of 47 members of the Spleen Tyrosine Kinase (Syk) pathway were significantly increased in DA synovial tissues compared with DA.F344(Cia5a), and included Syk (5.4-fold), Syk-activating receptors and interacting proteins, and genes regulated by Syk such as NFkB, and NAPDH oxidase complex genes. Nuclear receptors (NR) such as Rxrg, Pparg and Rev-erba were increased in the protected congenics, and so was the anti-inflammatory NR-target gene Scd1 (54-fold increase). Tnn (72-fold decrease) was the gene most significantly increased in DA.ConclusionsAnalyses of gene expression in synovial tissues revealed that the arthritis severity locus Cia5a regulates the expression of key mediators of inflammation and joint damage, as well as the expression of members of the Syk pathway. This expression pattern correlates with disease severity and joint damage and along with the gene accounting for Cia5a could become a useful biomarker to identify patients at increased risk for severe and erosive disease. The identification of the gene accounting for Cia5a has the potential to generate a new and important target for therapy and prognosis.

Blockade of TGF-β Signaling by the TGFβR-I Kinase Inhibitor LY2109761 Enhances Radiation Response and Prolongs Survival in Glioblastoma

Glioblastoma multiforme (GBM) is a highly aggressive primary brain tumor that tends to be resistant to the ionizing radiotherapy used to treat it. Because TGF-β is a modifier of radiation responses, we conducted a preclinical study of the antitumor effects of the TGF-β receptor (TGFβR) I kinase inhibitor LY2109761 in combination with radiotherapy. LY2109761 reduced clonogenicity and increased radiosensitivity in GBM cell lines and cancer stem-like cells, augmenting the tumor growth delay produced by fractionated radiotherapy in a supra-additive manner in vivo. In an orthotopic intracranial model, LY2109761 significantly reduced tumor growth, prolonged survival, and extended the prolongation of survival induced by radiation treatment. Histologic analyses showed that LY2109761 inhibited tumor invasion promoted by radiation, reduced tumor microvessel density, and attenuated mesenchymal transition. Microarray-based gene expression analysis revealed signaling effects of the combinatorial treatments that supported an interpretation of their basis. Together, these results show that a selective inhibitor of the TGFβR-I kinase can potentiate radiation responses in glioblastoma by coordinately increasing apoptosis and cancer stem-like cells targeting while blocking DNA damage repair, invasion, mesenchymal transition, and angiogenesis. Our findings offer a sound rationale for positioning TGFβR kinase inhibitors as radiosensitizers to improve the treatment of glioblastoma.

TOSO-Deficient B Cells Show An Impaired NF-κB and Migration Signaling In Vivo

Abstract 1779 Background:Our group firstly demonstrated that TOSO (FAIM3) is over-expressed in CLL compared to healthy B cell subsets as well as other B cell lymphomas. Furthermore, we detected a significant correlation of high TOSO expression to high lymphocyte count, unmutated IgVH status and Binet C, which are all markers for poor prognosis. TOSO has been described as pro-survival gene also in other settings. However, its mode of action is discussed controversially.Therefore, we aimed to elucidate the role of TOSO in B-cell specific gene expression by creating a knockdown mouse model. CD40 ligation and B cell receptor (BCR) activation influences TOSO expression and the fact that transcriptional regulation of TOSO is still unknown, we were eager to determine transcriptional factors that are directly responsible for the alterable TOSO levels. Methods:Faim3-floxed C57BL/6 FLP deleter mice were crossbred with CD19 specific Cre recombinase expressing mice. B-cells from the TOSOCD19−/− (KO) mice were isolated and gene expression was analyzed via mRNA based Illumina microchip array. Convincing results were verified by flow cytometry and blood count was carried out in addition.To determine the promoter region of TOSO, three overlapping DNA fragments (containing either NF-κB, Bcl-6 or both binding sites) upstream of the transcription start site of the first TOSO exon were cloned into a luciferase reporter vector lacking a promoter. Those constructs were transfected into HeLa cells. After 24 hours luciferase assays were performed. The involvement of NF-κB in the regulation of TOSO transcription was measured by TNFα stimulation of transfected cells prior to luminescence measurement. Targeted mutagenesis of the NF-κB binding site was performed to confirm the data. In addition, Bcl-6 expression vector was co-transfected for evaluation of repressing influence on TOSO expression. Results:In order to cover the functional part of TOSO, we generated a B-cell specific TOSOCD19−/− mouse model. Downstream effects of TOSO were validated via microarray-based gene expression analysis. Results displayed a clear clustering of deregulated genes compared to control mice. Nearly 400 genes showed expression alterations; genes involved in the NF-κB pathway and migration processes were downregulated in TOSOCD19−/−. These results were confirmed by flow cytometry analysis. The TOSO KO displayed also relevant effects on the hematopoietic system. Lymphocyte (p=0,0048), neutrophil (p=0,0007) and red blood cell counts (p=0,0051) were significantly decreased in the TOSOCD19−/− mice. Most important, the B-cell count was significantly reduced in TOSO-deficient settings (n=9; p=0,032).Since TOSO level seems to be so important for such fundamental pathways, investigation of gene expression regulation is mandatory. In situ analysis of the TOSO promoter region revealed NF-κB and Bcl-6 as promising results. Luciferase reporter assays including targeted mutagenesis confirmed the positive regulation of NF-κB and the repressing influence of Bcl-6 on TOSO expression significantly. Conclusions:We reveal for the first time a TOSO-dependent expression profile. We identified TOSO-dependent deregulated genes, which were involved in NF-κB signaling and migration, suggesting that TOSO represents an important factor in these pathways. Additionally, TOSO KO caused a decrease of peripheral B-cells in vivo. Furthermore, we identified NF-κB and Bcl-6 to regulate the TOSO expression in an opposite manner. Disclosures:No relevant conflicts of interest to declare.

Differential gene expression and a functional analysis of PCB-exposed children: Understanding disease and disorder development

The goal of the present study is to understand the probable molecular mechanism of toxicities and the associated pathways related to observed pathophysiology in high PCB-exposed populations. We have performed a microarray-based differential gene expression analysis of children (mean age 46.1months) of Central European descent from Slovak Republic in a well-defined study cohort. The subset of children having high blood PCB concentrations (>75 percentile) were compared against their low PCB counterparts (<25 percentile), with mean lipid-adjusted PCB values of 3.02±1.3 and 0.06±0.03ng/mg of serum lipid, for the two groups, respectively (18.1±4.4 and 0.3±0.1ng/ml of serum). The microarray was conducted with the total RNA from the peripheral blood mononuclear cells of the children using an Affymetrix platform (GeneChip Human genome U133 Plus 2.0 Array) and was analyzed by Gene Spring (GX 10.0). A highly significant set of 162 differentially expressed genes between high and low PCB groups (p value <0.00001) were identified and subsequently analyzed using the Ingenuity Pathway Analysis tool. The results indicate that Cell-To-Cell Signaling and Interaction, Cellular Movement, Cell Signaling, Molecular Transport, and Vitamin and Mineral Metabolism were the major molecular and cellular functions associated with the differentially altered gene set in high PCB-exposed children. The differential gene expressions appeared to play a pivotal role in the development of probable diseases and disorders, including cardiovascular disease and cancer, in the PCB-exposed population. The analyses also pointed out possible organ-specific effects, e.g., cardiotoxicity, hepatotoxicity and nephrotoxicity, in high PCB-exposed subjects. A few notable genes, such as BCL2, PON1, and ITGB1, were significantly altered in our study, and the related pathway analysis explained their plausible involvement in the respective disease processes, as mentioned. Our results provided insight into understanding the associated molecular mechanisms of complex gene–environment interactions in a PCB-exposed population. Future endeavors of supervised genotyping of pathway-specific molecular epidemiological studies and population biomarker validations are already underway to reveal individual risk factors in these PCB-exposed populations.

Krebshemmende Wirkung des ätherischen Salbei-Öls auf eine Plattenepithelzellkarzinom-Zelllinie der Mundhöhle (UMSCC1)

Every year there are several hundred thousand new cases of oral cancer worldwide. Clinical oncology is still challenged by toxicity and side effects of multimodal therapy strategies in which is associated with poor prognosis for patients. There is an urgent necessity to develop novel therapy strategies. As the majority of anticancer drugs are of natural origin, natural products represent a valuable source for the identification and development of novel treatment options for cancer. The aim of this investigation was to study the cytotoxicity of Salvia officinalis L. (sage) essential oil. Salvia officinalis essential oil was gained by aqueous extraction from plant material and subsequently analyzed by gas chromatography. The cytotoxicity of the essential oil on the squamous human cell carcinoma cell line of the oral cavity (UMSCC1) was assessed with the XTT assay. These experiments revealed the half maximal inhibitory concentration (IC(50)) of the essential oil. It was used in the microarray-based analysis of gene expression of UMSSC1 cells. The results were submitted to a signaling pathway analysis. The main constituents of Salvia officinalis essential oil include the monoterpenes thujone, β-pinene, and 1,8-cineol. Low concentrations of the essential oil increased vitality of the UMSCC1 cells. Beyond the concentration of the IC(50) of 135 µg/ml, sage essential oil reduced UMSSC1 cells viability to a minimum. In the microarray gene expression analysis, genes involved in cancer, cellular growth and proliferation, cell death, cell morphology, cell cycle, gene expression, and DNA repair were the most prominent. The three most significantly regulated pathways by sage were aryl hydrocarbon receptor signaling, cell cycle (G1/S checkpoint) regulation, and p53 signaling. To the best of our knowledge, this study suggests for the first time the ability of Salvia officinalis essential oil to inhibit human HNSCC cell growth. The therapeutic potential of sage essential oil might exceed that of its common use in otorhinolaryngology.

The Hepatitis E Virus ORF3 Protein Regulates the Expression of Liver-Specific Genes by Modulating Localization of Hepatocyte Nuclear Factor 4

The hepatitis E virus (HEV) is a small RNA virus and the cause of acute viral hepatitis E. The open reading frame 3 protein (pORF3) of HEV appears to be a pleiotropic regulatory protein that helps in the establishment, propagation and progression of viral infection. However, the global cellular effects of this protein remain to be explored. In the absence of traditional in vitro viral infection systems or efficient replicon systems, we made an adenovirus based ORF3 protein expression system to study its effects on host cell gene expression. We infected Huh7 hepatoma cells with recombinant adenoviruses expressing pORF3 and performed microarray-based gene expression analyses. Several genes down regulated in pORF3-expressing cells were found to be under regulation of the liver-enriched hepatocyte nuclear factor 4 (HNF4), which regulates hepatocyte-specific gene expression. While HNF4 localizes to the nucleus, its phosphorylation results in impaired nuclear localization of HNF4. Here we report that pORF3 increases HNF4 phosphorylation through the ERK and Akt kinases, which results in impaired nuclear translocation of HNF4 and subsequently the down modulation of HNF4-responsive genes in pORF3-expressing cells. We propose that modulation of several hepatocyte specific genes by pORF3 will create an environment favorable for viral replication and pathogenesis.

Technical Variability Is Greater than Biological Variability in a Microarray Experiment but Both Are Outweighed by Changes Induced by Stimulation

IntroductionA central issue in the design of microarray-based analysis of global gene expression is that variability resulting from experimental processes may obscure changes resulting from the effect being investigated. This study quantified the variability in gene expression at each level of a typical in vitro stimulation experiment using human peripheral blood mononuclear cells (PBMC). The primary objective was to determine the magnitude of biological and technical variability relative to the effect being investigated, namely gene expression changes resulting from stimulation with lipopolysaccharide (LPS).Methods and ResultsHuman PBMC were stimulated in vitro with LPS, with replication at 5 levels: 5 subjects each on 2 separate days with technical replication of LPS stimulation, amplification and hybridisation. RNA from samples stimulated with LPS and unstimulated samples were hybridised against common reference RNA on oligonucleotide microarrays. There was a closer correlation in gene expression between replicate hybridisations (0.86–0.93) than between different subjects (0.66–0.78). Deconstruction of the variability at each level of the experimental process showed that technical variability (standard deviation (SD) 0.16) was greater than biological variability (SD 0.06), although both were low (SD<0.1 for all individual components). There was variability in gene expression both at baseline and after stimulation with LPS and proportion of cell subsets in PBMC was likely partly responsible for this. However, gene expression changes after stimulation with LPS were much greater than the variability from any source, either individually or combined.ConclusionsVariability in gene expression was very low and likely to improve further as technical advances are made. The finding that stimulation with LPS has a markedly greater effect on gene expression than the degree of variability provides confidence that microarray-based studies can be used to detect changes in gene expression of biological interest in infectious diseases.

Methodology matters: IVF versus ICSI and embryonic gene expression

The use of assisted reproduction treatment, especially intracytoplasmic sperm injection (ICSI), is now linked to a range of adverse consequences, the aetiology of which remains largely undefined. Our objective of this study was to determine differences in gene expression of blastocysts generated by ICSI as well as ICSI with artificial oocyte activation (ICSI-A) versus the less manipulative IVF, providing fundamental genetic information that can be used to aid in the diagnosis or treatment of those adversely affected by assisted reproduction treatment, as well as stimulate research to further refine these techniques. Murine blastocysts were generated by ICSI, ICSI-A and IVF, and processed for a microarray-based analysis of gene expression. Ten blastocysts were pooled for each procedure and three independent replicates generated. The data were then processed to determine differential gene expression and to identify biological pathways affected by the procedures. In blastocysts derived by ICSI versus IVF, the expression of 197 genes differed ( P < 0.01). In blastocysts derived by ICSI-A versus IVF and ICSI-A versus ICSI, the expression of 132 and 65 genes differed respectively ( P < 0.01). Procedural-induced changes in genes regulating specific biological pathways revealed some consistency to known adverse consequences. Detailed investigation of procedure-specific dysfunction is therefore warranted. The use of reproductive manipulations including intracytoplasmic sperm injection (ICSI) has become increasingly linked to a range of adverse consequences, the cause of which remains largely undefined. Adverse effects include complications of pregnancy and parturition in the mother, as well as a range of birth defects in her offspring. The objective of this study was to determine differences in the gene expression of blastocysts generated by the major reproductive techniques: ICSI and ICSI with artificial oocyte activation (ICSI-A) versus the less manipulative IVF. In ICSI and ICSI-A, the head of a spermatozoon is directly injected into the mouse oocyte. Determination of differences in gene expression will provide novel information that can be used to aid in the diagnosis or treatment of those adversely affected by these procedures, as well as to stimulate research to further refine these techniques. Blastocysts were generated by performing ICSI, ICSI-A and IVF, using gametes collected from mice. The blastocysts were then processed for a microarray-based analysis of gene expression. Ten blastocysts were pooled for each procedure and three independent replicates generated. The data were then processed to determine differential gene expression and to identify biological pathways affected by procedure. Procedural-induced changes in the gene expression of blastocysts were observed. Classification of differentially regulated genes into biological pathways revealed consistency to known adverse consequences. Further investigation of procedure-specific treatment-induced dysfunction is needed.

Microarray-Based Analysis of Differential Gene Expression between Infective and Noninfective Larvae of Strongyloides stercoralis

BackgroundDifferences between noninfective first-stage (L1) and infective third-stage (L3i) larvae of parasitic nematode Strongyloides stercoralis at the molecular level are relatively uncharacterized. DNA microarrays were developed and utilized for this purpose.Methods and FindingsOligonucleotide hybridization probes for the array were designed to bind 3,571 putative mRNA transcripts predicted by analysis of 11,335 expressed sequence tags (ESTs) obtained as part of the Nematode EST project. RNA obtained from S. stercoralis L3i and L1 was co-hybridized to each array after labeling the individual samples with different fluorescent tags. Bioinformatic predictions of gene function were developed using a novel cDNA Annotation System software. We identified 935 differentially expressed genes (469 L3i-biased; 466 L1-biased) having two-fold expression differences or greater and microarray signals with a p value<0.01. Based on a functional analysis, L1 larvae have a larger number of genes putatively involved in transcription (p = 0.004), and L3i larvae have biased expression of putative heat shock proteins (such as hsp-90). Genes with products known to be immunoreactive in S. stercoralis-infected humans (such as SsIR and NIE) had L3i biased expression. Abundantly expressed L3i contigs of interest included S. stercoralis orthologs of cytochrome oxidase ucr 2.1 and hsp-90, which may be potential chemotherapeutic targets. The S. stercoralis ortholog of fatty acid and retinol binding protein-1, successfully used in a vaccine against Ancylostoma ceylanicum, was identified among the 25 most highly expressed L3i genes. The sperm-containing glycoprotein domain, utilized in a vaccine against the nematode Cooperia punctata, was exclusively found in L3i biased genes and may be a valuable S. stercoralis target of interest.ConclusionsA new DNA microarray tool for the examination of S. stercoralis biology has been developed and provides new and valuable insights regarding differences between infective and noninfective S. stercoralis larvae. Potential therapeutic and vaccine targets were identified for further study.

Characterization of post-surgical alterations in the bile duct-cannulated rat

The bile duct-cannulated (BDC) rat is a standard animal model used in ADME experiments. The aim of this study was to investigate post-surgical alterations that are relevant to ADME investigations in BDC rats compared with sham- and non-operated animals.Water and food intake was reduced in the animals’ post-surgery. This led to a lower body weight in operated animals. In BDC animals, aspartate aminotransferase (AST) levels in plasma were transiently elevated and total bile acid levels were reduced. Alpha1-acid glycoprotein (AGP) in plasma and the concentration of bile components in bile were elevated. Histopathology showed inflammation in the area of the cannulation between the liver and the small intestine. A microarray-based gene expression and RTq-PCR analysis identified altered expression for several genes involved in drug disposition including the down-regulation of cytochrome P450 enzymes. This led to reduced cytochrome P450 content in the liver and lower metabolic activity in microsomes from BDC and sham-operated rats compared with naïve animals.The results of the study suggest that the post-surgical inflammation leads to physiological changes relevant for drug absorption and disposition. These alterations should be accounted for in the interpretation of ADME studies in BDC animals.

Abstract 877: Genetic variation in podoplanin in relation to glioma risk and outcome

Abstract Gliomas are highly invasive tumors that invariably disseminate throughout the brain. The mechanisms of glioma cell migration are still poorly understood. Human PDPN (eg. podoplanin) is a novel transmembrane glycoprotein and a lymphatic endothelial marker thought to play a role in wound healing and frequently upregulated in human neoplasia. Although its physiologic function is still unknown, the recent observation of restricted expression at the leading invasive edge of solid tumors in experimental systems has raised the possibility that PDPN plays a role in tumor cell migration and invasion. Microarray-based gene expression analysis has recently identified PDPN as a novel candidate gene in glioma pathogenesis (Clin Cancer Res. 2009 15:6541-50). In this study, we examined whether polymorphisms in the gene encoding PDPN (located at 1p36.21) are associated with glioma risk and outcome in a clinic-based case-control study encompassing 563 newly diagnosed (eg. nonrecurrent) glioma cases (including 324 WHO grade IV glioblastomas (GBM); 145 WHO grade II or III astrocytomas and 94 oligoastrocytomas and oligodendrogliomas) and 629 healthy controls with no history of brain cancer. DNA was isolated from saliva samples. A total of 22 candidate and haplotype tagging SNPs in PDPN including the 5’ and 3’ UTR region of PDPN were genotyped using the Illumina Goldengate assay. Logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CI) for glioma risk in relation to individual SNPs adjusting for age and gender. Proportional hazards regression was used to estimate age- and gender-adjusted hazard ratios (HR) for GBM-related death (203 deaths; median follow up: 11.2 months) according to PDPN genotypes. No consistent associations were observed for any examined PDPN variant with glioma risk, overall, or according to glioma histological subtype. In contrast, 3 intronic SNPs (rs92406, rs2487643 and rs4391657) in moderately high linkage disequilibrium (r2: 0.57-0.96) residing in a haplotype block spanning 8kb were significantly associated with GBM mortality with the strongest signal observed in rs4391657 (NT_004610.19:g.617369T&amp;gt;C) (per variant “T” allele HR: 1.62; 95% CI: 1.28-2.05; P=0.00006; allele frequency: 0.20). The magnitude of association was similar for deaths occurring within 12 months (per variant allele HR: 1.51; 95% CI: 1.13-2.02; P=0.005) and greater than 12 months (per variant allele HR: 1.84; 95% CI: 1.21-2.80; P=0.004) following GBM diagnosis. The median survival times were 14.4, 11.4 and 8.9 months in those carrying 0, 1, or 2 at-risk “T” alleles in PDPN rs4391657 (log rank P value 0.0018), irrespective of therapy. To our knowledge this is the first report of an association of a PDPN genetic variant predicting survival times in malignancy. Further studies are needed to confirm this observation and to identify the putative causal variant. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 877. doi:10.1158/1538-7445.AM2011-877

The homeobox gene HLXB9 is upregulated in a morphological subset of poorly differentiated hepatocellular carcinoma

The prognostic outcome for hepatocellular carcinoma (HCC) remains poor. Disease progression is accompanied by dedifferentiation of the carcinoma, a process that is not well understood. The aim of this study was to get more insight into the molecular characteristics of dedifferentiated carcinomas using high throughput techniques. Microarray-based global gene expression analysis was performed on five poorly differentiated HCC cell lines compared with non-neoplastic hepatic controls and a set of three cholangiolar carcinoma (CC) cell lines. The gene with the highest upregulation was HLXB9. HLXB9 is a gene of the homeobox genfamily important for the development of the pancreas. RT-PCR confirmed the upregulation of HLXB9 in surgical specimens of carcinoma tissue, suggesting its biological significance. Interestingly, HLXB9 upregulation was primary observed in poorly differentiated HCC with a pseudoglandular pattern compared with a solid pattern HCC or in moderate or well-differentiated HCC. Additional the expression of translated HLXB9, the protein HB9 (NCBI: NP_001158727), was analyzed by western blotting. Expression of HB9 was only detected in the cytoplasm but not in the nuclei of the HCC cells. For validation CC were also investigated. Again, we found an upregulation of HLXB9 in CC cells accompanied by an expression of HB9 in the cytoplasms of these tumor cells, respectively. In conclusion, homeobox HLXB9 is upregulated in poorly differentiated HCC with a pseudoglandular pattern. The translated HB9 protein is found in the cytoplasm of these HCC and CC. We therefore assume HLXB9 as a possible link in the understanding of the development of HCC and CC, respectively.

The indole-3-carbinol cyclic tetrameric derivative CTet inhibits cell proliferation via overexpression of p21/CDKN1A in both estrogen receptor-positive and triple-negative breast cancer cell lines

IntroductionIndole-3-carbinol (I3C), an autolysis product of glucosinolates present in cruciferous vegetables, and its dimeric derivative (3,3'-DIM) have been indicated as promising agents in preventing the development and progression of breast cancer. We have recently shown that I3C cyclic tetrameric derivative CTet formulated in γ-cyclodextrin (γ-CD) efficiently inhibited cellular proliferation in breast cancer cell lines. This study aims to analyze the mechanisms involved in the in vitro inhibition of cell proliferation and to evaluate the in vivo antitumor activity of CTet in a xenograft study.MethodsEstrogen receptor-positive MCF-7 and triple-negative MDA-MB-231 breast cancer cell lines were exposed to CTet to evaluate cell cycle perturbation (propidium iodide staining and cytofluorimetric acquisition), induction of autophagic morphological features (co-localization of LC3b autophagosome marker and LAMP2a lysosome marker by immunofluorescence) and changes in protein expression (immunoblot and microarray-based gene expression analyses). To test the in vivo efficacy of CTet, female athymic nude mice inoculated with MCF-7 cells were i.p. treated with 5 mg/kg/day of CTet for five days/week for two weeks and the tumor mass was externally monitored.ResultsCTet induced accumulation in G2/M phase without evidence of apoptotic response induction in both cell lines tested. In triple-negative MDA-MB-231 the autophagic lysosomal activity was significantly up-regulated after exposure to 4 μM of CTet for 8 hours, while the highest CTet concentration was necessary to observe autophagic features in MCF-7 cells. The inhibition of Akt activity and p53-independent p21/CDKN1A and GADD45A overexpression were identified as the main molecular events responsible for CTet activity in MCF-7 and p53-mutant MDA-MB-231 cells. In vivo, CTet administration was able to significantly inhibit the growth of MCF-7 xenotransplanted into nude mice, without adverse effect on body weight or on haematological parameters.ConclusionsOur data support CTet formulated with γ-CD as a promising and injectable anticancer agent for both hormone-responsive and triple-negative breast tumors.

Tools for managing and analyzing microarray data

The microarray-based analysis of gene expression has become a workhorse for biomedical research. Managing the amount and diversity of data that such experiments produce is a task that must be supported by appropriate software tools, which led to the creation of literally hundreds of systems. In consequence, choosing the right tool for a given project is difficult even for the expert. We report on the results of a survey encompassing 78 of such tools, of which 22 were inspected in detail and seven were tested hands-on. We report on our experiences with a focus on completeness of functionality, ease-of-use, and necessary effort for installation and maintenance. Thereby, our survey provides a valuable guideline for any project considering the use of a microarray data management system. It reveals which tasks are covered by mature tools and also shows that important requirements, especially in the area of integrated analysis of different experimental data, are not yet met satisfyingly by existing systems.

GOBO: Gene Expression-Based Outcome for Breast Cancer Online

Microarray-based gene expression analysis holds promise of improving prognostication and treatment decisions for breast cancer patients. However, the heterogeneity of breast cancer emphasizes the need for validation of prognostic gene signatures in larger sample sets stratified into relevant subgroups. Here, we describe a multifunctional user-friendly online tool, GOBO (http://co.bmc.lu.se/gobo), allowing a range of different analyses to be performed in an 1881-sample breast tumor data set, and a 51-sample breast cancer cell line set, both generated on Affymetrix U133A microarrays. GOBO supports a wide range of applications including: 1) rapid assessment of gene expression levels in subgroups of breast tumors and cell lines, 2) identification of co-expressed genes for creation of potential metagenes, 3) association with outcome for gene expression levels of single genes, sets of genes, or gene signatures in multiple subgroups of the 1881-sample breast cancer data set. The design and implementation of GOBO facilitate easy incorporation of additional query functions and applications, as well as additional data sets irrespective of tumor type and array platform.

Gene expression profiles in CHA3 and CHA4 human embryonic stem cells and embryoid bodies.

The establishment of the first human embryonic stem cells (hESCs) in 1998 provided a unique tool for studying human development. Although several Western embryoderived hESC lines are well characterized, the biological properties of Asian embryo-derived hESC lines remain unexamined. The aim of this study was to characterize Korean embryo-derived hESC lines and their differentiation potential. In this context, we conducted microarraybased differential gene expression analyses using two Korean embryo-derived hESC lines (CHA3 and CHA4) to identify undifferentiated and spontaneously differentiated (human embryoid body, or hEB) status. These two cell lines showed great similarity in gene expression. By comparing their expression patterns, we determined novel hESC-specific genes and transcriptomes that could serve as reliable hESC markers associated with the "stemness" phenotype. Additionally, we sought to identify hEB markers that could be used to determine the presence of differentiated cells in specific tissues, allowing for the purification of homogeneous cell populations or serving as indicators of hESC differentiation. Novel sets of 68 hESC-specific markers, 12 hESC-specific transcripts and 36 hEB markers were identified and shown by quantitative RTPCR to be similarly expressed in CHA3- and CHA4-hESC lines, as compared to the Western embryo-derived H9-hESC line. Furthermore, our data analysis revealed that the cell cycle, urea cycle, p53 signaling, and metabolism of amino groups are significantly implicated in the regulation of hESC differentiation. These results provide another unique set of hESC/hEB markers and foster a better understanding of the molecular mechanisms underlying hESC biology. These results may thus facilitate studies of human developmental events and provide information regarding Korean embryo-derived hESCs, which could be used to determine differences in developmental events between human races.

Inorganic arsenic alters expression of immune and stress response genes in activated primary human T lymphocytes

Inorganic arsenic, a carcinogenic environmental contaminant, exerts immunosuppressive effects on human T lymphocytes. In particular, interleukin-2 (IL2) secretion and T cell proliferation are reduced when peripheral blood mononuclear cells (PBMC) from individuals chronically exposed to arsenic are stimulated ex vivo with lectins such as phytohemaglutinin (PHA). However, it is not clear whether the metalloid directly acts on T cells or blocks monocyte-dependent accessory signals activated by PHA. We report that in vitro pre-treatment of PBMC with sodium arsenite (NaAs) reduces IL2 secretion and T cell proliferation induced by PHA, but does not prevent expression of monocyte-derived cytokines (IL1, IL6, TNFα) functioning as lymphocyte-activating factors. In addition, we found that NaAs delays induction of IL2 and IL2 receptor α chain (IL2RA) mRNA levels in human primary isolated T cells activated by PHA. Kinetic analysis showed that NaAs pre-treatment first inhibits, but thereafter markedly increases, induction of IL2 and IL2RA mRNA when T cells are stimulated with PHA for 8h and 72h, respectively. We conducted whole genome microarray-based analysis of gene expression in primary T cell cultures derived from independent donors. NaAs systematically and significantly up-regulated a set of 35 genes, including several immune and stress genes, such as IL13, granulocyte-macrophage colony stimulating factor, lymphotoxin α and heme oxygenase-1 (HO-1). Up-regulation of HO-1, a stress and immunosuppressive protein, was rapidly detectable, both in T cells and in PBMC treated with NaAs. Inhibition of the immunosuppressive activity of HO-1 in PBMC however failed to prevent NaAs-dependent inhibition of T cell proliferation induced by PHA. Our findings demonstrate that, at least in vitro, inorganic arsenic acts directly on human T cells and impairs their activity, probably independently of HO-1 expression and monocyte-related accessory signals.

Gene Expression Analysis of PCB Exposed Children: Understanding Toxicity and Disease Process

O-29A5-2 Background/Aims: To understand the molecular mechanism of PCB-related toxicities and the basics of pathophysiology in association with high PCB exposure, we studied the differential global gene expression profile of children (mean age, 45 month) of East European descent from Slovakian Republic in a well-defined study cohort. Methods: An unsupervised microarray based gene expression analysis was performed on the subset of children having high blood PCB concentration (>75 percentile) against their low PCB counterpart (<25 percentile). The mean lipid adjusted PCB values in 2 groups were 3.16 ± 1.2 and 0.06 ± 0.03 ng/g of serum lipid, respectively. Total RNA from peripheral blood mononuclear cells were isolated and the microarray was conducted using Affymetrix platform (GeneChip Human genome U133 Plus 2.0 Array). The data obtained from microarray were analyzed by Genespring GX10. Results: A highly significant set of 162 differentially expressed genes between high PCB and low PCB group was observed (P < 0.00001). The subsequent Ingenuity Pathway Analysis on this set of genes implicated different compromised molecular and cellular function in higher PCB exposure included: Cell-to-Cell Signaling and Interaction, Cellular Movement, Cellular Assembly and Organization, Cellular Function and Maintenance, and Carbohydrate Metabolism. The analysis conducted on children with high PCB exposure also indicated perturbed expression of genetic pathways related to cardiotoxicity, hepatotoxicity, and nephrotoxicity. These uncharacteristic effects on gene expression may play a role in development of major diseases and disorders in population, including cancer. Some of the relevant genes reported in relation to PCB-related toxicities were subsequently validated through quantitative RT-PCR. Conclusion: Our results provided a molecular insight in understanding the associated mechanism of this complex gene environment interaction. The future endeavor of supervised genotyping of pathway-specific molecular epidemiological study would reveal the individual risk factor in PCB exposure.