Involvement of ICE-like protease(s) in TGF-beta 1 induced apoptosis in hepatocytes

Internucleosomal DNA cleavage is often regarded as the biochemical hallmark of apoptosis and can be reproduced in vitro in rat liver nuclei. This chromatin cleavage in rat liver nuclei was further characterised and studies described in this thesis showed that the DNA was initially cleaved into 700kbp, 200-250kbp and 30-50kbp fragments via a Mg2+ dependent process which was potentiated by Ca2+. Further investigations of the DNA cleavage processes were carried out in primary hepatocytes using TGF-1 to induce apoptosis. The studies showed for the first time that treatment of hepatocytes with TGF-1 resulted in multi-step DNA cleavage as seen in rat nuclei. Cycloheximide and an interleukin-1-converting enzyme (ICE)-like inhibitor, benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone (Z-VAD.FMK) and Z-Asp-Glu-Val-Asp-(OMe) fluoromethyl ketone (Z-DEVD.FMK), a potent inhibitor of CPP32, blocked DNA cleavage and apoptosis. During apoptosis there was a time dependent increase in CPP32-like proteolytic activity in lysates isolated from TGF-1 treated hepatocytes, which was detected with the fluorogenic assay using Z-DEVD-amino-trifluoromethyl-coumarin (Z-DEVD.AFC). This activity was abolished when hepatocytes were pre-treated with either Z-VAD.FMK, Z-DEVD.FMK or cycloheximide. Unlike cycloheximide, Z-VAD.FMK and Z-DEVD.FMK were potent inhibitors of activated lysate from TGF-1 treated hepatocytes. Immunoblotting showed the processing of pro-CPP32 to its active form. In conclusion, this study shows for the first time that TGFF-1 mediated apoptosis involves the activation of ICE-like proteases and that cycloheximide inhibits apoptosis by blocking upstream of this ICE-like activity.