Tumor necrosis factor-alpha-induced apoptosis in hepatocytes in long-term culture.
Apoptosis occurs naturally in the liver and increases in specific pathogenic processes. We previously described the use of a chemically defined medium supplemented with epidermal growth factor and dimethylsulfoxide to maintain rat hepatocytes in a highly differentiated state for more than 30 days (long-term culture). In this study, we showed that hepatocytes in long-term dimethylsulfoxide culture have definite advantages 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 morphologically and by formation of an oligonucleosomal DNA ladder) only in hepatocytes that had been subjected to dimethylsulfoxide removal. Neither treatment 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 was easily identified by 24 hours. We conclude that this system can be used 1) to examine the underlying mechanism by which TNF-alpha causes apoptosis in hepatocytes and 2) to study induction of apoptosis in hepatocytes by other agents.
Bour
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Department of Microbiology and Immunology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey 17033, USA.
Ward
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LK
Cornman
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GA
Isom
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HC
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D018929:Cell Culture Techniques; D003470:Culture Media; D004247:DNA / Q000096:biosynthesis; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004587:Electrophoresis, Agar Gel; D008099:Liver / Q000166:cytology* / Q000187:drug effects; D008297:Male; D009336:Necrosis; D016133:Polymerase Chain Reaction; D051381:Rats; D011916:Rats, Inbred F344; D012402:Rotenone / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D003470:Culture Media; D014409:Tumor Necrosis Factor-alpha; D012402:Rotenone; D004247:DNA; D004121:Dimethyl Sulfoxide