Oval cells, bipotent cells of ductal origin, are activated in human liver by hepatotoxins, carcinogens, viral agents or chronic disease states, all which greatly increase the risk of liver cancer. A better understanding of oval cells as progenitors of hepatocellular carcinoma (HCC) could lead to valuable insights, particularly if oval cell-derived HCC differ in behavior from their diploid hepatocyte-derived counterparts. The proposed research is structured around the hypothesis that HCC originate, at least in part, from hepatocytes derived de novo from initiated oval cells. These initiated oval cells have newly acquired genetic change(s) that arrest differentiation and produce oval-cell-antigen positive hepatocytes capable of progression to HCC. This hypothesis will be tested by assessing the fate of carcinogen-induced Fischer rat oval cells transplanted via the spleen into the liver of syngeneic hosts with mutated, inactive dipeptidylpeptidase IV (DPPIV). Donor cells will be distinguished histochemically by their expression of active DPPIV or exogenous beta-galactosidase (B-gal). Experiments in Specific Aim I will determine whether established cultures of oval cells induced by ethionine in a choline deficient (CD) diet (CDE) can integrate, differentiate and progress to HCC when promoted by CD diet. To encourage donor cell expansion, host rats will be treated with retrorsine/partial hepatectomy (PH) prior to transplantation. Changes in gene expression following integration will be monitored by phenotyping donor cells at various timepoints after transplantation.
In Specific Aim 2, a similar series of experiments will be performed using primary oval cell isolates from DPPIV+ rats maintained on CDE diet or treated with diethylnitrosamine, 2-AAF and PH (resistant hepatocyte regimen). Oval cells will be isolated by high speed FACS using anti Thy-1 antibodies and a unique panel of monoclonal antibodies defining surface lineage markers. HCC will be quantitated by measuring serum DPPIV activity, DPPIV/B-gal activity in liver extracts or by direct quantitation of HCC transections stained for DPPIV, B-gal or known neoplastic markers.
In Specific Aim 3, the focus will be on determining if the failure of oval cell and high passage BDEC cultures to upregulate the BDEC antigen, BD.1, following G1/S arrest represents a preneoplastic event. Comparative studies will examine growth characteristics, ductal morphogenesis, adhesion phenotypes, apoptosis and expression of genes activated in the early stages of cholangiocarcinogenesis (c-neu, COX-2, c-met). By elucidating the role of oval cells in liver carcinogenesis, the results from these studies will provide valuable information that can be translated into new strategies for the diagnosis, treatment and prognosis of HCC.
