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 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.