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Apoptosis induced in rat hepatocytes by transforming growth factor beta1 (TGF-beta1) was accompanied by the activation of interleukin-1beta converting enzyme (ICE)-like proteases. Cell lysates were isolated at various times after TGF-beta1 treatment and analyzed for ICE and CPP32-like activity, using N-acetyl-Tyr-Val-Ala-Asp-7-amino-4-methylcoumarin (Ac-YVAD.AMC) and benzyloxycarbonyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin (Z-DEVD.AFC), respectively. CPP32-like but not ICE protease activity increased in a time dependent manner and preceded the onset of apoptosis. Kinetic studies in cell lysates indicated that more than one CPP32-like protease was being activated. This was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/Western blotting of TGF-beta1-treated cells, which showed limited processing of CPP32 as shown by the appearance of the catalytically active p17 subunit. Loss of pro-Mch3alpha was also observed but the catalytically active p19 subunit was not detected. Staurosporine, which induced a much greater level of hepatocyte apoptosis, produced a concomitant increase in CPP32/Mch3alpha processing as shown by the appearance of the p17/p19 subunits and the corresponding increase in CPP32-like protease activity. Apoptosis, CPP32/Mch3alpha processing and the increase in CPP32-like protease activity induced by TGF-beta1 and staurosporine were abolished in hepatocytes pretreated with Z-Asp-Glu-Val-Asp (OMe) fluoromethylketone (Z-DEVD.FMK) or Z-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK). These peptide analogues were potent inhibitors of CPP32-like protease activity in lysates. Pretreatment of hepatocytes with cycloheximide also blocked TGF-beta1-induced apoptosis and the increase in CPP32-like activity. Unlike Z-VAD.FMK and Z-DEVD.FMK, cycloheximide did not inhibit CPP32-like protease activity in cell lysates. Thus, cycloheximide may block apoptosis by inhibiting the synthesis of a protein, which is involved in the upstream events responsible for the activation of the CPP32-like protease activity. Our studies have identified two of the CPP32-like proteases, namely CPP32 and Mch3alpha, which are activated during the execution phase of hepatocyte apoptosis.
S H Inayat-Hussain, and
C Couet, and
G M Cohen, and
K Cain
November 1997,
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S H Inayat-Hussain, and
C Couet, and
G M Cohen, and
K Cain
February 1998,
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S H Inayat-Hussain, and
C Couet, and
G M Cohen, and
K Cain
July 1998,
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S H Inayat-Hussain, and
C Couet, and
G M Cohen, and
K Cain
June 2001,
Cell research,
S H Inayat-Hussain, and
C Couet, and
G M Cohen, and
K Cain
June 1999,
European journal of endocrinology,
S H Inayat-Hussain, and
C Couet, and
G M Cohen, and
K Cain
September 1998,
Toxicology and applied pharmacology,
S H Inayat-Hussain, and
C Couet, and
G M Cohen, and
K Cain
January 2000,
Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society,
S H Inayat-Hussain, and
C Couet, and
G M Cohen, and
K Cain
October 2008,
BMB reports,
S H Inayat-Hussain, and
C Couet, and
G M Cohen, and
K Cain
May 2003,
Journal of cellular biochemistry,
S H Inayat-Hussain, and
C Couet, and
G M Cohen, and
K Cain
October 1997,
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