The effect of aromatic hydrocarbon receptor on the phenotype of the Hepa 1c1c7 murine hepatoma cells in the absence of dioxin

Abstract

The aromatic hydrocarbon receptor (AhR) mediates biological responses to certain exogenous ligands, such as the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and has also been demonstrated to modulate the cell cycle and differentiated state of several cell lines independently of exogenous ligands. In this study, we used DNA microarray analysis to elucidate the profile of genes responsive to the expression of unliganded AhR by re-introducing AhR into an AhR-deficient mouse derivative (c19) of the mouse hepatoma cell line Hepa 1c1c7. 22 gene products were up-regulated and 8 were down-regulated two-fold or more in c19 cells infected with a retroviral vector expressing mouse AhR. Surprisingly, expression of genes involved in cell proliferation or differentiation were not affected by introduction of AhR. AhR also did not restore expression of the albumin gene in c19 cells. Introduction of AhR into c12, a similar AhR-defective mouse hepatoma cell line, also did not restore albumin expression, and furthermore, did not lead to changes in cellular morphology or cell cycle parameters. These observations fail to support the notion that unliganded AhR regulates proliferation and differentiation of liver-derived cells.

Keywords: Cyp1a1; DNA microarray; albumin; aromatic hydrocarbon receptor (AhR); cell cycle; morphology.

Figure 1

Immunoblot for AhR. Proteins were extracted from whole cells and subjected to immunoblot analysis using an anti-AhR antibody prepared in our laboratory.

Figure 2

Total RNA was isolated from the indicated cells and used for reverse transcription. The cDNAs were then subjected to real time PCR analysis. The result is an average from three real time PCR reactions with the same template. Standard deviations are shown. A, Expression of Cyp1a1. The mRNA levels of Cyp1a1 were normalized to that of the constitutively expressed 36B4 gene, encoding a ribosomal subunit. B, Detection of albumin mRNA. The mRNA levels of albumin were normalized to that of the constitutively expressed β-actin gene. Standard deviations are shown.

Figure 3

Morphology of wild-type, B mutants, and B mutants infected with pMFG-AhR and pCMMP-lacZ. The cells were cultured under normal conditions, visualized by phase contrast microscopy, and photographed