Pok Sharon, Vohra Harpreet, Wehbe Charbel, Barn Vanessa A, Arfianti Evi, Dan Yock-Young, Farrell Geoffrey C, Teoh Narci C
Liver Research Group, Australian National University Medical School at The Canberra Hospital, Building 10 Level 5, Yamba Drive, Garran, Canberra, ACT 2605, Australia.
Imaging and Cytometry Facility, John Curtin School of Medical Research, Building 131, Garran Rd, Acton, Canberra, ACT 2601, Australia.
Exp Cell Res. 2017 Jul 1;356(1):48-56. doi: 10.1016/j.yexcr.2017.04.009. Epub 2017 Apr 10.
Dysplastic hepatocytes (DH) represent altered hepatocytes with potential for malignant transformation. To date, most research on pathways to hepatocarcinogenesis has focused on use of "hepatoma" cell lines derived from hepatocellular carcinoma (HCC). We describe a novel technique for deriving/culturing DH and demonstrate their utility for functional studies in vitro, compared to primary hepatocytes (PH) and HCC. PH and DH were prepared by portal vein collagenase perfusion from C57BL/6J mice. DH were subsequently subjected to FACS. HCC from diethylnitrosamine (DEN)-injected mice were mechanically isolated. Cell cycle analyses were performed by flow cytometry and PCNA immunohistochemistry. To establish utility of DH, we studied pathways of p53 turnover, apoptosis and cell proliferation using pfithrin-α (PFT) and nutlin-3. Like PH, DH were minimally proliferative compared to HCC. Only 30±0.03% of DH were in G/M phase versus 51±0.01% of HCC; this difference corroborated with PCNA-immunostaining of dysplastic nodules from DEN-injected mice. In DH and HCC, nutlin-3 suppressed p53 mRNA, induced p53 and mdm2 activation but paradoxically resulted in increased anti-apoptotic and proliferative activity. Primary murine DH display distinctive biological characteristics compared with PH and HCC. As an intermediate cell type to HCC, they offer a new pathobiologically relevant primary cell culture system with which to interrogate the molecular changes in hepatocarcinogenesis.
