Deriving murine transformed hepatocytes in primary culture: novel technique and potential utility

Abstract

Introduction: There has been a heavy reliance on the use of hepatoma cell lines to study the pathways of hepatocarcinogenesis. Cell lines are transformed, immortalized, and do not mimic the natural pathobiological characteristics of hepatocellular carcinoma (HCC). Aim: This study aims to develop novel techniques of deriving and culturing primary dysplastic hepatocytes (DHs) and HCC cells for interventional and functional studies. Methods: Primary hepatocytes (PHs) are derived from naïve male mice using the hepatic portal vein perfusion method; primary DHs and HCCs from diethylnitrosamine-injected mice are isolated via fluorescence activated cell sorting and mechanical disruption. Cell viability is determined using MTT assay and protein expression by immunoblotting. Cell cycle phase analysis is performed using propidium iodide staining followed by fluorescence activated cell sorting, while p53 and mdm2 chemical inhibition with pifithrin-α and Nutlin-3 are used, respectively. Results: While the viability of PHs is limited to 5 days on collagen-coated plates, primary DHs can be maintained in culture for up to 4 weeks with minimal proliferative activity. In contrast, primary HCC cells undergo active hepatocyte proliferation, evident by several passages and positive proliferating cell nuclear antigen expression. Cell cycle phase analyses confirm that majority of PHs are maintained in cell cycle arrest phase (G0/G1) with only 2 ± 0.02% cells in G2/M phase. However, sorted DHs display an augmented population of G2/M cells (30 ± 0.03%) and such populations are enhanced in primary HCCs (56 ± 0.1%). Pifithrin-α administration results in decreased p53 mRNA in HCC cells but elicits no changes in DHs. puma mRNA is decreased in both cell types. Inhibition of p53 exhibits increased mdm2 and downregulation of usp18 mRNA expression in both cell types. Nutlin-3 treatment suppresses mdm2 mRNA only in DHs but not HCCs. p53 and usp18 mRNA are downregulated in both cell types. Conclusion: Isolated murine dysplastic hepatocytes exhibit distinct molecular mechanisms/properties compared to HCC cells and may prove to be an appropriate in vitro resource to elucidate the molecular changes early in hepatocarcinogenesis. Show more