10.1021/tx015518a.s576 Thomas K. Baker Thomas K. Baker Mark A. Carfagna Mark A. Carfagna Hong Gao Hong Gao Ernst R. Dow Ernst R. Dow Qingqin Li Qingqin Li George H. Searfoss George H. Searfoss Timothy P. Ryan Timothy P. Ryan Temporal Gene Expression Analysis of Monolayer Cultured Rat Hepatocytes American Chemical Society 2001 differentiation process RT phase II metabolizing enzymes hepatocyte cell cycle gene expression comparisons transcriptional patterns hepatocyte dedifferentiation response hepatocellular dedifferentiation Cultured hepatocytes cytochrome P 450 mRNA expression gene expression novel aspects hepatocyte cultures rat livers 8700 rat genes mRNA changes Cluster analysis Temporal Gene Expression Analysis PCR data glutathione metabolism hepatocyte adaptation Monolayer Cultured Rat Hepatocytes hepatocellular markers Functional grouping time course data Progressive induction transcriptional level Affymetrix microarrays gene expression changes expression monitoring extracellular matrix SOM cell shape transcriptional changes 2001-08-30 00:00:00 Figure https://acs.figshare.com/articles/figure/Temporal_Gene_Expression_Analysis_of_Monolayer_Cultured_Rat_Hepatocytes/3789912 The use of cultured primary hepatocytes within toxicology has proven to be a valuable tool for researchers, however, questions remain with regard to functional differences observed in these hepatocytes relative to the intact liver. Cultured hepatocytes have typically been described as dedifferentiated, a classification based upon the investigation of a few key cellular processes or hepatocellular markers. In the present study, parallel expression monitoring of approximately 8700 rat genes was used to characterize mRNA changes over time in hepatocyte cultures using Affymetrix microarrays. We isolated and labeled mRNA from whole rat livers, hepatocyte-enriched cell pellets, and primary cultured hepatocytes (4, 12, 24, 48, and 72 h postplating), and hybridized these samples to microarrays. From these data, several pairwise and temporal gene expression comparisons were made. Gene expression changes were confirmed by RT/PCR and by performing replicate experiments and repeated hybridizations using a rat toxicology sub-array that contained a 900-gene subset of the 8700-gene rat genomic microarray. PCR data qualitatively reproduced the temporal patterns of gene expression observed with microarrays. Cluster analysis of time course data using self-organizing maps (SOM) revealed a classic hepatocyte dedifferentiation response. Functional grouping of genes with similar transcriptional patterns showed time-dependent regulation of phase I and phase II metabolizing enzymes. In general, cytochrome P450 mRNA expression was repressed, but expression of phase II metabolizing enzymes varied by class (upregulation of glucuronidation, downregulation of sulfation). Potential metabolic targets for toxic insult, such as glutathione metabolism, gluconeogenesis, and glycolysis, were also affected at the transcriptional level. Progressive induction of several genes associated with the cellular cytoskeleton and extracellular matrix was observed in accord with physical changes in cell shape and connectivity associated with cellular adhesion. Finally, many transcriptional changes of genes involved in critical checkpoints throughout the hepatocyte cell cycle and differentiation process were observed. In total, these data establish a more comprehensive understanding of hepatocellular dedifferentiation and reveal many novel aspects of physiological and morphological hepatocyte adaptation. An assembly of all transcripts that demonstrated differential expression in this study can be found in the Supporting Information.