Apoptosis has been the focus of intensive research efforts and is of central importance in normal development, homeostasis and pathogenic processes including cancer. Although little is known about apoptosis in liver, apoptosis most likely plays a role in liver development and regeneration. Apoptosis occurs naturally in the adult liver but at a sufficiently low rate that it cannot be easily detected. Apoptosis has been observed in acute hepatitis, chronic hepatitis, primary biliary cirrhosis, hepatocarcinogenesis, etc. An in-depth understanding of hepatocyte apoptosis and the factors that suppress or promote this process in hepatocytes may have clinical value in understanding and treating specific hepatic diseases. One agent that induces apoptosis is the cytokine tumor necrosis factor alpha (TNFalpha). It is conceivable that TNFalpha may be an important mediator of apoptosis in the liver because TNFalpha is produced by Kupffer cells and increased levels of TNFalpha production have been found in patients with alcoholic liver disease, chronic HBV infection, etc. Apoptosis can also be induced by activation of the Fas antigen, a member of the TNF receptor family. Activation of Fas in mice results in fulminate hepatic failure. The intracellular pathway for induction of apoptosis by TNFalpha or Fas in hepatocytes has not been studied in detail. Rat hepatocytes, plated on rat tail collagen-coated plates and fed serum free, chemically defined medium supplemented with 2% dimethylsulfoxide (DMSO) remain highly differentiated, at a biochemical, molecular and morphological level, for more than a year. The long-term DMSO hepatocyte culture model has been used previously to study molecular mechanisms of albumin expression, immortalization and transformation of hepatocytes, and DNA synthesis. Hepatocytes in long-term DMSO culture are also an excellent model for studying apoptosis; specifically, TNFalpha can induce apoptosis in hepatocytes in long-term DMSO culture but only after they have been sensitized by DMSO removal. This work provides the foundation for the studies proposed in this application. The long range goal is to define what factors in hepatocytes promote and suppress apoptosis and to understand the cellular changes that occur in hepatocytes when they undergo programmed cell death.
The specific aims of this proposal are: (1) To determine what cellular mechanisms mediate TNFalpha induced apoptosis in hepatocytes. (2) To determine whether Fas and/or """"""""death domain"""""""" proteins that associate with members of the TNF receptor family induce apoptosis in hepatocytes. (3) To determine how DMSO removal sensitizes hepatocytes to TNFalpha/Fas- induced apoptosis.
