Differentiation of human hepatoma cells during confluence as revealed by gene expression profiling

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.

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 withdrawal of drugs from the market [4], [5]. In vitro systems that could predict the potential hepatotoxic effects, and unsuitable pharmacokinetic properties of drug candidates with improved accuracy would therefore facilitate drug development 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 [8], [9], [10] making them unsuitable for such purposes. Due to the lack of human liver (HL) material, human hepatoma cell lines are often used for in vitro studies. One of the commonly used, HepG2, does not express important levels of xenobiotic metabolizing enzymes. However, during recent years, the human hepatoma cell line B16A2 has been established, which in contrast to HepG2, expresses more drug metabolizing enzymes like CYP3A and CYP2E1. B16A2 also expresses liver-enriched transcription factors (LETFs), such as HNF-1, HNF-3 and HNF-4 like DNA binding activity [11], [12], [13].

As has been previously registered by mainly monitoring drug-metabolizing enzymes, the B16A2 cell line differentiates during culture under confluent conditions, thereby acquiring a more suitable phenotype for studies of drug metabolism [11], [13]. Hence, we considered it important to monitor the fundamental transformations in phenotype occurring during confluent growth of B16A2 cells, and in addition, to compare the B16A2 phenotype to that of adult HL and HepG2 cells. This was achieved using microarray techniques, bioinformatics and statistical analyses with complementary analyses carried out by real time PCR for important LETFs. Such a knowledge can be of value for developing other cells lines, possibly based on stem cell research, that would allow better in vitro models.

The results interestingly indicate that the changes in gene expression occurring during confluence of the B16A2 cell line, both with respect to genes undergoing increased or decreased expression, to the major part force them phenotypically towards that of HL. The phenotypic changes seen represent the influence of cell–cell interaction on differentiation in vitro of a hepatoma cell line, which partially resembles that of hepatic differentiation and regeneration in vivo.