-transparent.png){kind=logo}
The mechanism of Fas antigen-induced hepatocyte apoptosis was investigated. Using a monoclonal antibody directed against the Fas antigen, apoptosis was induced in freshly isolated murine hepatocytes within 90 minutes of antibody addition as assessed by plasma membrane bleb formation, chromatin condensation, and DNA fragmentation. Pretreatment of the cells with the caspase inhibitors, N-acetyl-Asp-Glu-Val-Asp aldehyde (Ac-DEVD-CHO), benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD-FMK), or Z-Asp-2,6-dichlorobenzoyloxymethylketone inhibited anti-Fas-mediated apoptosis. Likewise, the serine protease inhibitors, N-tosyl-L-phenyl chloromethyl ketone (TPCK) and 3,4-dichloroisocoumarin (DCI), prevented apoptosis, whereas N-tosyl-L-lysine chloromethyl ketone (TLCK), Ac-Leu-Leu-L-norleucinal, Ac-Leu-Leu-L-methional, and trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane were without effect. Examination of CED-3/caspase-3-related caspases revealed that pro-caspases-3 (CPP32) and -7 (Mch-3alpha) were rapidly processed after Fas antigen stimulation. Caspase-7 was further cleaved to form the catalytically active subunits. In contrast, the p17 subunit of caspase-3 was not detected, indicating slow formation or rapid degradation. The activation of CED-3-related caspases was further confirmed by an increase in the rate of Z-DEVD-7-amino-4-trifluoromethylcoumarin (Z-DEVD-AFC) hydrolysis that was sensitive to Ac-DEVD-CHO and was inhibited by pretreatment of the cells with TPCK but not by DCI. In contrast, no increase in the rates of hydrolysis of Z-YVAD-AFC, a substrate for caspase-1, was detected. Investigation of the in situ proteolytic cleavage of the CED-3 related caspases substrate, poly(ADP-ribose) polymerase, revealed that this protein was not degraded in hepatocytes undergoing Fas-mediated apoptosis. Taken together, our results show that processing of caspases, in particular, caspases-7 and -3, occurs during Fas-induced apoptosis of mouse hepatocytes and suggest a role of these proteases as well as serine protease(s) in the apoptotic response.
R A Jones, and
V L Johnson, and
N R Buck, and
M Dobrota, and
R H Hinton, and
S C Chow, and
G E Kass
December 1994,
Experimental cell research,
R A Jones, and
V L Johnson, and
N R Buck, and
M Dobrota, and
R H Hinton, and
S C Chow, and
G E Kass
April 1999,
Clinical and experimental immunology,
R A Jones, and
V L Johnson, and
N R Buck, and
M Dobrota, and
R H Hinton, and
S C Chow, and
G E Kass
January 2004,
Cell transplantation,
R A Jones, and
V L Johnson, and
N R Buck, and
M Dobrota, and
R H Hinton, and
S C Chow, and
G E Kass
December 2001,
American journal of physiology. Gastrointestinal and liver physiology,
R A Jones, and
V L Johnson, and
N R Buck, and
M Dobrota, and
R H Hinton, and
S C Chow, and
G E Kass
August 2000,
American journal of physiology. Cell physiology,
R A Jones, and
V L Johnson, and
N R Buck, and
M Dobrota, and
R H Hinton, and
S C Chow, and
G E Kass
September 1999,
Gastroenterology,
R A Jones, and
V L Johnson, and
N R Buck, and
M Dobrota, and
R H Hinton, and
S C Chow, and
G E Kass
May 2012,
Cell stem cell,
R A Jones, and
V L Johnson, and
N R Buck, and
M Dobrota, and
R H Hinton, and
S C Chow, and
G E Kass
March 2005,
Molecular pharmacology,
R A Jones, and
V L Johnson, and
N R Buck, and
M Dobrota, and
R H Hinton, and
S C Chow, and
G E Kass
April 2000,
The Journal of biological chemistry,
R A Jones, and
V L Johnson, and
N R Buck, and
M Dobrota, and
R H Hinton, and
S C Chow, and
G E Kass
July 2002,
Hepatology (Baltimore, Md.),
