Platforms for biomarker analysis using high-throughput approaches in genomics, transcriptomics, proteomics, metabolomics, and bioinformatics.

Global biological responses that reflect disease or exposure biology are kinetic and highly dynamic phenomena. While high-throughput DNA sequencing continues to drive genomics, the possibility of more broadly measuring changes in gene expression has been a recent development manifested by a diversity of technical platforms. Such technologies measure transcripts, proteins and small biological molecules, or metabolites, and respectively define the fields of transcriptomics, proteomics and metabolomics that can be performed at a cell-, tissue-, or organism-wide basis.

Transcriptomic analysis of pathways regulated by toll-like receptor 4 in a murine model of chronic pulmonary inflammation and carcinogenesis.

Background: Therapeutic strategies exist for human pulmonary neoplasia, however due to the heterogeneity of the disease, most are not very effective. The innate immunity gene, toll-like receptor 4 (TLR4), protects against chronic pulmonary inflammation and tumorigenesis in mice, but the mechanism is unclear. This study was designed to identify TLR4-mediated gene expression pathways that may be used as prognostic indicators of susceptibility to lung tumorigenesis in mice and provide insight into the mechanism.

Trans-10, cis-12-conjugated linoleic acid alters hepatic gene expression in a polygenic obese line of mice displaying hepatic lipidosis.

The trans-10, cis-12 isomer of conjugated linoleic acid (CLA) causes a rapid reduction of body and adipose mass in mice. In addition to changes in adipose tissue, numerous studies have reported alterations in hepatic lipid metabolism. Livers of CLA-fed mice gain mass, partly due to lipid accumulation; however, the precise molecular mechanisms are unknown.

Profile of estrogen-responsive genes in an estrogen-specific mammary gland outgrowth model.

Both ovarian and pituitary hormones are required for the pubertal development of the mouse mammary gland. Estradiol directs ductal elongation and branching, while progesterone leads to tertiary branching and alveolar development. The purpose of this investigation was to identify estrogen-responsive genes associated with pubertal ductal growth in the mouse mammary gland in the absence of other ovarian hormones and at different stages of development.

Estrogen receptor beta is required for optimal cAMP production in mouse granulosa cells.

Granulosa cells of preovulatory follicles differentiate in response to FSH, and this differentiation is augmented by estradiol. We have previously shown that FSH-mediated granulosa cell differentiation requires functional estrogen receptor-beta (ERbeta) by demonstrating that the granulosa cells of ERbeta(-/-) FSH-treated mice are unable to maximally induce expression of the LH receptor (an indicator of granulosa cell differentiation) compared with ERbeta(+/+) controls. As a result, FSH-primed ERbeta(-/-) granulosa cells exhibit a reduced response to a subsequent ovulatory dose of LH.

Genome wide transcriptional profiling in breast cancer cells reveals distinct changes in hormone receptor target genes and chromatin modifying enzymes after proteasome inhibition.

Steroid hormone receptors, like glucocorticoid (GR) and estrogen receptors (ER), are master regulators of genes that control many biological processes implicated in health and disease. Gene expression is dependent on receptor levels which are tightly regulated by the ubiquitin-proteasome system. Previous studies have shown that proteasome inhibition increases GR, but decreases ER-mediated gene expression.

Kruppel-like zinc finger protein Glis2 is essential for the maintenance of normal renal functions.

To obtain insight into the physiological functions of the Krüppel-like zinc finger protein Gli-similar 2 (Glis2), mice deficient in Glis2 expression were generated. Glis2 mutant (Glis2(mut)) mice exhibit significantly shorter life spans than do littermate wild-type (WT) mice due to the development of progressive chronic kidney disease with features resembling nephronophthisis. Glis2(mut) mice develop severe renal atrophy involving increased cell death and basement membrane thickening in the proximal convoluted tubules.

RNA polymerase is poised for activation across the genome.

Regulation of gene expression is integral to the development and survival of all organisms. Transcription begins with the assembly of a pre-initiation complex at the gene promoter, followed by initiation of RNA synthesis and the transition to productive elongation. In many cases, recruitment of RNA polymerase II (Pol II) to a promoter is necessary and sufficient for activation of genes.

Farnesol-induced apoptosis in human lung carcinoma cells is coupled to the endoplasmic reticulum stress response.

Farnesol (FOH) and other isoprenoid alcohols induce apoptosis in various carcinoma cells and inhibit tumorigenesis in several in vivo models. However, the mechanisms by which they mediate their effects are not yet fully understood. In this study, we show that FOH is an effective inducer of apoptosis in several lung carcinoma cells, including H460.

Selective regulation of bone cell apoptosis by translational isoforms of the glucocorticoid receptor.

Glucocorticoids are widely used in the treatment of inflammatory and other diseases. However, high-dose or chronic administration often triggers troublesome side effects such as metabolic syndrome and osteoporosis. We recently described that one glucocorticoid receptor gene produces eight translational glucocorticoid receptor isoforms that have distinct gene-regulatory abilities.

Gene expression profiling reveals a regulatory role for ROR alpha and ROR gamma in phase I and phase II metabolism.

Retinoid-related orphan receptors alpha (ROR alpha) and gamma (ROR gamma) are both expressed in liver; however, their physiological functions in this tissue have not yet been clearly defined. The ROR alpha1 and ROR gamma 1 isoforms, but not ROR alpha 4, show an oscillatory pattern of expression during circadian rhythm. To obtain insight into the physiological functions of ROR receptors in liver, we analyzed the gene expression profiles of livers from WT, ROR alpha-deficient staggerer (sg) mice (ROR alpha(sg/sg)), ROR gamma(-/-), and ROR alpha(sg/sg)ROR gamma(-/-) double knockout (DKO) mice by microarray analysis.

Developmental exposure to diethylstilbestrol alters uterine gene expression that may be associated with uterine neoplasia later in life.

Previously, we described a mouse model where the well-known reproductive carcinogen with estrogenic activity, diethylstilbestrol (DES), caused uterine adenocarcinoma following neonatal treatment. Tumor incidence was dose-dependent reaching >90% by 18 mo following neonatal treatment with 1000 microg/kg/d of DES. These tumors followed the initiation/promotion model of hormonal carcinogenesis with developmental exposure as initiator, and exposure to ovarian hormones at puberty as the promoter.

Optimal sampling of rat liver tissue for toxicogenomic studies.

Different degrees of a toxic response between and within the various lobes of the liver have been observed in rodents following treatment with acetaminophen. This study was designed to compare 2 sampling methods of the rat liver for gene-expression analysis. Ten male Fischer 344/N rats, 12-14 weeks of age, were treated with vehicle (0.

Novel mRNA targets for tristetraprolin (TTP) identified by global analysis of stabilized transcripts in TTP-deficient fibroblasts.

Tristetraprolin (TTP) is a tandem CCCH zinc finger protein that was identified through its rapid induction by mitogens in fibroblasts. Studies of TTP-deficient mice and cells derived from them showed that TTP could bind to certain AU-rich elements in mRNAs, leading to increases in the rates of mRNA deadenylation and destruction. Known physiological target mRNAs for TTP include tumor necrosis factor alpha, granulocyte-macrophage colony-stimulating factor, and interleukin-2beta.

Modulatory role for retinoid-related orphan receptor alpha in allergen-induced lung inflammation.

Rationale: Nuclear receptors play a critical role in the regulation of inflammation, thus representing attractive targets for the treatment of asthma.

Objective: In this study, we assess the potential regulatory function of retinoid-related orphan receptor alpha (RORalpha) in the adaptive immune response using ovalbumin (OVA)-induced airway inflammation as a model.

Methods: Allergen-induced inflammation was compared between wild-type (WT) and staggerer (RORalpha(sg/sg)) mice, a natural mutant strain that is deficient in RORalpha expression.

Identification of potential target genes for RFX4_v3, a transcription factor critical for brain development.

Regulatory factor X4 variant transcript 3 (Rfx4_v3) gene disruption in mice demonstrated that interruption of a single allele (heterozygous, +/-) prevented formation of the subcommissural organ, resulting in congenital hydrocephalus, while interruption of two alleles (homozygous, -/-) caused fatal failure of dorsal midline brain structure formation. To identify potential target genes for RFX4_v3, we used microarray analysis to identify differentially expressed genes in Rfx4_v3-deficient mouse brains at embryonic day 10.5, before gross structural changes were apparent.

NABP1, a novel RORgamma-regulated gene encoding a single-stranded nucleic-acid-binding protein.

RORgamma2 (retinoid-related orphan receptor gamma2) plays a critical role in the regulation of thymopoiesis. Microarray analysis was performed in order to uncover differences in gene expression between thymocytes of wild-type and RORgamma-/- mice. This analysis identified a novel gene encoding a 22 kDa protein, referred to as NABP1 (nucleic-acid-binding protein 1).

Global gene expression associated with hepatocarcinogenesis in adult male mice induced by in utero arsenic exposure.

Our previous work has shown that exposure to inorganic arsenic in utero produces hepatocellular carcinoma (HCC) in adult male mice. To explore further the molecular mechanisms of transplacental arsenic hepatocarcinogenesis, we conducted a second arsenic transplacental carcinogenesis study and used a genomewide microarray to profile arsenic-induced aberrant gene expression more extensively. Briefly, pregnant C3H mice were given drinking water containing 85 ppm arsenic as sodium arsenite or unaltered water from days 8 to 18 of gestation.

Estren behaves as a weak estrogen rather than a nongenomic selective activator in the mouse uterus.

A proposed membrane-mediated mechanism of rapid nongenomic response to estrogen has been the intense focus of recent research. Estren, a synthetic steroid, is reported to act selectively through a rapid membrane-mediated pathway, rather than through the classical nuclear estrogen receptor (ER)-mediated pathway, to maintain bone density in ovariectomized mice without uterotropic effects. To evaluate the mechanism and physiological effects of estren, we studied responses in adult ovariectomized mice.

Functional genomic characterization of delipidation elicited by trans-10, cis-12-conjugated linoleic acid (t10c12-CLA) in a polygenic obese line of mice.

Gene expression was measured during t10c12-CLA-induced body fat reduction in a polygenic obese line of mice. Adult mice (n = 185) were allotted to a 2 x 2 factorial experiment consisting of either nonobese (ICR-control) or obese (M16-selected) mice fed a 7% fat, purified diet containing either 1% linoleic acid (LA) or 1% t10c12-CLA. Body weight (BW) by day 14 was 12% lower in CLA- compared with LA-fed mice (P < 0.

A qualitative assessment of direct-labeled cDNA products prior to microarray analysis.

Background: The success of the microarray process in determining differential gene expression of thousands of genes is dependent upon the quality and integrity of the starting RNA, this being particularly true of direct labeling via a reverse transcription procedure. Furthermore, an RNA of reasonable quality still may not yield reliable hybridization data if the labeling efficiency was poor.

Results: Here we present a novel assay for assessing the quality of directly labeled fluorescent cDNA prior to microarray hybridization utilizing the Agilent 2100 Bioanalyzer, which employs microfluidic technology for the analysis of nucleic acids and proteins.

Global uterine genomics in vivo: microarray evaluation of the estrogen receptor alpha-growth factor cross-talk mechanism.

Cross-talk between growth factor receptors and the estrogen receptor (ER) has been proposed as a signaling mechanism in estrogen target tissues, with ER(alpha) as a direct target of growth factor receptor-activated signals, leading to regulation of estrogen target genes and estrogen-like biological responses to growth factors. We evaluated whether global genomic changes in the mouse uterus in response to epidermal growth factor or IGF-I mimic those of estradiol (E2), reflecting the cross-talk mechanism. Overlapping responses to growth factors and E2 were expected in the wild type (WT) whereas no response was expected in mice lacking ER(alpha) (ER(alpha) knockout).

Analysis of ATP-binding cassette transporter expression in drug-selected cell lines by a microarray dedicated to multidrug resistance.

Discovery of the multidrug resistance protein 1 (MDR1), an ATP-binding cassette (ABC) transporter able to transport many anticancer drugs, was a clinically relevant breakthrough in multidrug resistance research. Although the overexpression of ABC transporters such as P-glycoprotein/ABCB1, MRP1/ABCC1, and MXR/ABCG2 seems to be a major cause of failure in the treatment of cancer, acquired resistance to multiple anticancer drugs may also be multifactorial, involving alteration of detoxification processes, apoptosis, DNA repair, drug uptake, and overexpression of other ABC transporters. As a tool for the study of such phenomena, we designed and created a microarray platform, the ABC-ToxChip, to evaluate relative levels of transcriptional activation among genes involved in the various mechanisms of resistance.

Identification of putative gene based markers of renal toxicity.

This study, designed and conducted as part of the International Life Sciences Institute working group on the Application of Genomics and Proteomics, examined the changes in the expression profile of genes associated with the administration of three different nephrotoxicants--cisplatin, gentamicin, and puromycin--to assess the usefulness of microarrays in the understanding of mechanism(s) of nephrotoxicity. Male Sprague-Dawley rats were treated with daily doses of puromycin (5-20 mg/kg/day for 21 days), gentamicin (2-240 mg/kg/day for 7 days), or a single dose of cisplatin (0.1-5 mg/kg).

Dexamethasone blocks the rapid biological effects of 17beta-estradiol in the rat uterus without antagonizing its global genomic actions.

Estrogens and glucocorticoids have opposing effects on the female reproductive tract, but the molecular basis for this antagonism is poorly understood. We therefore examined the biological and transcriptional programs induced by estrogens and glucocorticoids in the uterus of immature female rats. Estradiol 17beta (E2) rapidly induced morphological changes reminiscent of an acute inflammatory response, including infiltration of eosinophils, edema in the stroma and myometrium, and a decrease in the height of luminal epithelial cells, whereas dexamethasone (Dex) only altered stromal cell morphology.