Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling by array of human primary hepatocytes treated with trovafloxacin

ABSTRACT: Trovafloxacin is a broad spectrum antibiotic that inhibits the uncoiling of supercoiled DNA in various bacteria by blocking the activity of DNA gyrase and topoisomerase IV. Specific members of this drug family display high activity against eukaryotic type II topoisomerase, as well as cultured mammalian cells and in vivo tumor models. Trovafloxacin seems to have a higher affinity for eukaryotic polymerase II system than the other quinolone agents tested. This effect coupled with other factors, such as an inflammatory response, might result in a hepatotoxic reaction seen with drug. In this study we found genes regulated by trovafloxacin - induced and repressed - to be located much closer to each other than genes distributed randomly all over the genome (< 100 kbp). Experiment Overall Design: We calculated specifically the probability by which particular genes which were deregulated by the treatment of human hepatocytes with trovafloxacin will occur within DNA windows of different sizes as compared to the probability of all known mapped genes (RefSeq transcripts) of the human genome (NCBI RefSeq 19,360; build 36.2) to occur within the same DNA windows.

This experiment was reloaded in November 2010 after additional curation.

ORGANISM(S): Homo sapiens

SUBMITTER: Jeurgen Borlak

PROVIDER: E-GEOD-11941 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Topoisomerase II inhibition involves characteristic chromosomal expression patterns.

Reymann Susanne S Borlak Jürgen J

BMC genomics 20080708

<h4>Background</h4>The phenomenon of co-localization of transcriptionally upregulated genes showing similar expression levels is known across all eukaryotic genomes. We recently mapped the Aroclor 1254-regulated transcriptome back onto the genome and provided evidence for the statistically significant co-localization of regulated genes. They did, however, not always show similar expression levels, and many of the regulated genes were, in fact, repressed.<h4>Results</h4>In this study, we were abl ...[more]

PMID: 18611269

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Project description:It is well-known that isolation and cultivation of primary hepatocytes causes major gene expression alterations. In the present genome-wide, time resolved study of cultivated human and mouse hepatocytes, we made the observation that expression changes in culture strongly resemble alterations in liver diseases. Hepatocytes of both species were cultivated in collagen sandwich and in monolayer conditions. Genome-wide data were also obtained from human NAFLD, cirrhosis, HCC, and hepatitis B virus infected tissue as well as mouse livers after partial hepatectomy, CCl4 intoxication, obesity, HCC and LPS. A strong similarity between cultivation and disease induced expression alterations was observed. For example, expression changes in hepatocytes induced by one day cultivation and one day CCl4 exposure in vivo correlated with R=0.615 (P<0.001). Interspecies comparison identified predominantly similar responses in human and mouse hepatocytes but also a minority of genes that responded differently. Unsupervised clustering of altered genes identified three main clusters: (1) downregulated genes corresponding to mature liver functions, (2) upregulation of an inflammation/RNA-processing cluster, (3) upregulated migration/cell-cycle associated genes. Gene regulatory network analysis highlights overrepresented and deregulated HNF4 and CAR (cluster 1), KrÃ¼ppel-like factors MafF and ELK1 (cluster 2) as well as ETF (cluster 3) among the interspecies conserved key regulators of expression changes. Interventions ameliorating but by far not abrogating cultivation-induced responses include removal of non-parenchymal cells, generation of the hepatocytesâ own matrix in spheroids, supplementation with bile salts and siRNA mediated suppression of key transcription factors. In conclusion, the study shows that gene regulatory network alterations of cultivated hepatocytes resemble those of inflammatory liver diseases and should therefore be considered and exploited as disease models.

Project description:The liver is one of most important organs in our bodies. It performs many essential functions including metabolism, synthesis, secretion, detoxification, and storage. Hepatocytes are the principal cell type in the liver and are involved in multiple liver-specific functions. There have been several efforts to develop in vitro culture systems capable of maintaining hepatocyte-specific phenotype over long time periods. In hepatic tissue engineering, two commonly used culture systems are the collagen sandwich and monolayers of cells. ‚àö√á¬¨‚Ä†In this study, genome-wide gene expression profiles of primary hepatocytes were measured over an 8-day period for each cell culture system using Affymetrix GeneChips and analyzed via Gene Set Enrichment Analysis (GSEA), which is a powerful method to elicit biologically meaningful information from microarray data at the level of gene sets. Results indicate that the gene expression in hepatocytes in collagen sandwich cultures gradually diverges from that in monolayer cultures. Gene sets up-regulated in collagen sandwich cultures include those associated with liver metabolic and synthetic functions. ‚àö√á¬¨‚Ä†These functions are associated with lipid, amino acid, carbohydrate, and alcohol metabolism and bile acid synthesis. Nuclear receptors are up-regulated in collagen sandwiches 24 hours after seeding. Signals transmitted from these receptors may cause the up-regulation of other processes in subsequent days. Cytochrome-P450 monooxygenase expression was initially down-regulated but exhibited up-regulation after 72 hours. Our results provide a baseline for further explorations into the systems biology of engineered liver mimics as well as 2D and 3D co-cultures of primary hepatocytes and non-parenchymal cells. To better understand differences in quality of in vitro growth of rat hepatocytes between culture on a monolayer of collagen gel and sandwiched between two layers of gel, we measured gene expression in hepatocytes in these two culture conditions in triplicate for four time points: 1 day, 2 days, 3 days, and 8 days of culturing. Overall, we obtained Affymetrix microarray data for 24 samples, divided to 12 samples from monolayer and double layer cultures each, each of which are divided into four time points with 3 sample replicates.

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Dataset Information

Transcription profiling by array of human primary hepatocytes treated with trovafloxacin

Publications

Topoisomerase II inhibition involves characteristic chromosomal expression patterns.

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