Differentiation of human hepatoma cells during confluence as revealed by gene expression profiling Angelica Butura a , Inger Johansson a , Kerstin Nilsson b , Lars Wa ¨rnga ˚rd b , Magnus Ingelman-Sundberg a,* , Ina Schuppe-Koistinen b a Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet, SE-171 77 Stockholm, Sweden b Molecular Toxicology, Safety Assessment, AstraZeneca R&D, SE-141 85 So ¨derta ¨lje, Sweden Received 27 June 2003; accepted 28 October 2003 Abstract Certain human hepatocarcinoma cells undergo differentiation when grown at confluence. In order to understand the basis for this differentiation, we investigated the phenotypic changes occurring during confluent growth of the human hepatoma B16A2 cell line. The global gene expression profile of B16A2 cells grown during confluence for 5 weeks was investigated using microarrays containing complementary sequences corresponding to approximately 10,000 genes, and compared with profiles of adult human liver and HepG2 cells. The major part of gene products detected were shared by all three systems and the hepatoma cell lines expressed surprisingly high levels of liver-enriched transcription factors. During confluence of B16A2 cells, the majority of transcriptional changes monitored were directed towards the phenotype of adult human liver in vivo, although the changes accounted for less than 10% of those necessary to acquire a native hepatic phenotype. Several markers of liver differentiation and regeneration were changed in similar manner as observed in developing liver and during liver regeneration. In conclusion, the data indicate that differentiation in vitro of the B16A2 cell line during confluence partially resembles that of hepatic differentiation and regeneration in vivo, implying a partial normalization of a low differentiated phenotype. # 2003 Elsevier Inc. All rights reserved. Keywords: Microarray; Liver-enriched transcription factor; Cytochrome P450; Wnt signaling; PCA analysis; Cluster analysis 1. Introduction The liver is unique in its ability to regenerate after injury. It has been postulated that the process of regeneration is similar to that of the differentiation of the hepatic lineage from the ventral mesoderm during embryogenesis [1]. Cell lines derived from either embryonic or malignant liver generally express few hepatic functions [2]. However, the process of hepatic differentiation has been difficult to mimic in any in vitro system and primary hepatocytes in culture rapidly loose their phenotype. Knowledge about the processes involved in differentiation of hepatocytes is thus central for being able to develop hepatocyte derived in vitro systems containing highly differentiated cells, originating from, e.g. stem cells or transformed hepatoma cells, for use in studies of hepatic functions. The hepatocytes contain the majority of phase I and phase II enzymes in the body responsible for detoxification of xenobiotics [3]. Drug-induced hepatotoxicity, often caused secondary to the metabolic activation of the parent compound, is common and can cause fulminate hepatitis, hepatic failure and death. It limits clinical use of several pharmacologically active potential drugs and causes with- drawal of drugs from the market [4,5]. In vitro systems that could predict the potential hepatotoxic effects, and unsui- table pharmacokinetic properties of drug candidates with improved accuracy would therefore facilitate drug devel- opment and make efficient tools in rational drug design [6,7]. Primary human hepatocytes constitute a common model for in vitro toxicological testing. However, the decline in activity over time of drug metabolizing enzymes after plating of primary hepatocytes is rapid and substantial Biochemical Pharmacology 67 (2004) 1249–1258 0006-2952/$ – see front matter # 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bcp.2003.10.033 * Corresponding author. Tel.: þ46-8-52487735; fax: þ46-8-337327. E-mail address: magnus.ingelman-sundberg@imm.ki.se (M. Ingelman-Sundberg). Abbreviations: CYP, cytochrome P450; HL, human liver; PC, principal component; PCA, principal component analysis; LETF, liver-enriched transcription factor.