Liver Pathophysiology: Cellular and Molecular Analyses of Growth Control, Differentiation, Apoptosis, Cell Injury, Iron Metabolism, and Hepatitis Virus Infection in Hepatic Cell Systems

The research in my laboratory focuses on utilizing in vitro cell culture systems to understand liver function and pathogenesis. The cell systems include primary rat hepatocytes in long-term dimethylsulfoxide (DMSO) culture, SV40 immortalized hepatocyte cell lines and cell lines of human hepatic origin. Our laboratory established an in vitro model in which hepatocytes are plated on collagen-coated plates and maintained in serum free medium supplemented with DMSO. These cells remain highly differentiated at a biochemical, molecular and morphological level for more than a year. Current studies can be divided into four related areas of research.

Regulation of Growth Control in Hepatocytes: A series of immortalized, transformed and tumor derived rat hepatocyte cell lines have been generated and characterized in our laboratory. The combination of hepatocytes in long-term DMSO culture and these hepatic cell lines can be used to examine progression to malignancy. We have previously determined that α1 integrin expression present in immortalized rat hepatocyte cell lines is lost when these cells are transformed by activated H-ras and short term expression of activated H-ras downregulates α1 integrin expression. The current aims are to determine whether α1 integrin expression plays a role in normal growth control in hepatocytes and how α1 integrin expression is regulated, in particular, by ras, in primary hepatocytes and hepatocyte cell lines. We have previously shown that TGFβ1 treatment reverses the transformed phenotype of ras-transformed hepatic cell lines and also increases α1 integrin expression. The current aims are to elucidate the key players in the TGFβ1 signaling pathway involved in suppressing the transformed phenotype, and/or inducing α1 integrin expression.

Apoptosis in Hepatocytes: We have recently shown that hepatocytes in long-term DMSO culture are an excellent model for studying apoptosis in hepatocytes. One advantage of this system over short term primary hepatocyte cultures is that the background level of spontaneous apoptosis is minimal. We have used this system to demonstrate that tumor necrosis factor α (TNFα can induce apoptosis in hepatocytes in long term culture, but only after the cells have been sensitized by removal of DMSO or treatment with cycloheximide. We have also demonstrated that hepatocytes in long-term DMSO culture can be used to study apoptosis induced by treatment with transforming growth factor β or activation of Fas. The current aims are to dissect the cellular mechanisms that mediate TNFα-induced apoptosis in hepatocytes concentrating on identifying the proteins that interact with the TNF receptors and the aspects of the signaling pathway that are unique to hepatocytes. Recent technical advances that will facilitate our ability to achieve our goals are the development of a highly sensitive radioactive assay for oligonucleosomal DNA ladder formation and the use of baculoviruses to mediate gene delivery into primary hepatocytes. The latter will facilitate our ability to examine the effects of gain or loss of function of a specific gene on apoptosis.

Iron Metabolism and Metal-Induced Cellular Injury: Hemochromatosis is the term used to describe a state of iron overload in an individual. Eventually, after chronic iron overload, fibrosis occurs, the liver becomes cirrhotic and hepatocellular carcinomas may arise. Our goal is to use hepatocytes in culture to address the isolated issue of how iron-overload alters the function of well-differentiated hepatocytes in the absence of the other liver cell types. Recently, we have demonstrated that hepatocytes in long-term DMSO culture can be iron-loaded and can be used to study the effects of chronic iron loading on hepatocytes. Iron loading was accompanied by an increase in ferritin cores within lysosomes and an increase in total cellular ferritin. Our current aim is to test the hypothesis that iron loading of hepatocytes in long-term DMSO culture induces specific types of cellular damage which are potentiated if the cells are treated with cytokines. We will also characterize the molecular mechanism underlying ferritin induction caused by chronic iron overload.

Hepatitis B virus (HBV) Replication: We recently developed a novel transient mechanism for studying HBV gene expression and replication. Recombinant HBV baculovirus Autographa californica is used to deliver the HBV genome to human hepatoblast HepG2 cells. In HBV baculovirus infected HepG2 cells, HBV transcripts, intracellular and secreted HBV antigens are produced, enveloped HBV virions are secreted and replication occurs as evidenced by the presence of high levels of intracellular replicative intermediates and protected HBV DNA in the medium. Covalently closed circular (CCC) DNA is present indicating that, in this system, HBV core particles are capable of delivering newly synthesized HBV genomes back into the nuclei of infected cells. The recombinant HBV baculovirus system has numerous advantages over existing in vitro HBV expressing cell cultures systems. HBV CCC DNA can be rapidly detected from low numbers of HepG2 cells and the system lends itself readily to experimental manipulation. Our current aims are to use the system (1) to evaluate the effects of antivirals and/or cytokines on HBV replication, (2) to dissect specific aspects of HBV replication, and (3) to study the interaction of HBV with the host cell.