TUMOR NECROSIS FACTOR-ALPHA-INDUCED APOPTOSIS IN HEPATOCYTES IN LONG-TERM CULTURE

Apoptosis occurs naturally in the liver and increases in specific path ogenic processes. We previously described the use of a chemically defi ned medium supplemented with epidermal growth factor and dimethylsulfo xide to maintain rat hepatocytes in a highly differentiated state for more than 30 days (long-term-culture). In this study, we showed that h epatocytes in long-term dimethylsulfoxide culture have definite advant ages over using cells in short-term culture (cells in culture for 2 to 4 days) to study apoptosis. We demonstrated that treatment with tumor necrosis factor (TNF)-alpha induced apoptosis (detected morphological ly and by formation of an oligonucleosomal DNA ladder) only in hepatoc ytes that had been subjected to dimethylsulfoxide removal. Neither tre atment with TNF-alpha alone or dimethylsulfoxide removal alone induced apoptosis. Apoptosis could be induced by concentrations as low as 500 U of TNF-alpha/ml. Although a DNA ladder was not detected by 12 hours after TNF-alpha treatment, it ws easily identified by 24 hours. We co nclude that this system can be used 1) to examine the underlying mecha nism by which TNF-alpha causes apoptosis in hepatocytes and 2) to stud y induction of apoptosis in hepatocytes by other agents.