Temporal gene expression analysis of monolayer cultured rat hepatocytes

The use of cultured primary hepatocytes within toxicology has proven to be a valuable tool for researchers, however, questions remain with regard to f unctional differences observed in these hepatocytes relative to the intact liver. Cultured hepatocytes have typically been described as dedifferentiat ed, a classification based upon the investigation of a few key cellular pro cesses or hepatocellular markers. In the present study, parallel expression monitoring of approximately 8700 rat genes was used to characterize mRNA c hanges over time in hepatocyte cultures using Affymetrix microarrays. We is olated and labeled mRNA from whole rat livers, hepatocyte-enriched cell pel lets, and primary cultured hepatocytes (4, 12, 24, 48, and 72 h postplating ), and hybridized these samples to microarrays. From these data, several pa irwise and temporal gene expression comparisons were made. Gene expression changes were confirmed by RT/PCR and by performing replicate experiments an d repeated hybridizations using a rat toxicology sub-array that contained a 900-gene subset of the 8700-gene rat genomic microarray. PCR data qualitat ively reproduced the temporal patterns of gene expression observed with mic roarrays. Cluster analysis of time course data using self-organizing maps ( SOM) revealed a classic hepatocyte dedifferentiation response. Functional g rouping of genes with similar transcriptional patterns showed time-dependen t regulation of phase I and phase II metabolizing enzymes. In general, cyto chrome P450 mRNA expression was repressed, but expression of phase II metab olizing enzymes varied by class (upregulation of glucuronidation, downregul ation 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 assoc iated with the cellular cytoskeleton and extracellular matrix was observed in accord with physical changes in cell shape and connectivity associated w ith cellular adhesion. Finally, many transcriptional changes of genes invol ved in critical checkpoints throughout the hepatocyte cell cycle and differ entiation process were observed. In total, these data establish a more comp rehensive 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 t his study can be found in the Supporting Information.