Activation of multiple interleukin-1beta converting enzyme homologues in cytosol and nuclei of HL-60 cells during etoposide-induced apoptosis.
Recent genetic and biochemical studies have implicated cysteine-dependent aspartate-directed proteases (caspases) in the active phase of apoptosis. In the present study, three complementary techniques were utilized to follow caspase activation during the course of etoposide-induced apoptosis in HL-60 human leukemia cells. Immunoblotting revealed that levels of procaspase-2 did not change during etoposide-induced apoptosis, whereas levels of procaspase-3 diminished markedly 2-3 h after etoposide addition. At the same time, cytosolic peptidase activities that cleaved DEVD-aminotrifluoromethylcoumarin and VEID-aminomethylcoumarin increased 100- and 20-fold, respectively; but there was only a 1. 5-fold increase in YVAD-aminotrifluoromethylcoumarin cleavage activity. Affinity labeling with N-(Nalpha-benzyloxycarbonylglutamyl-Nepsilon-biotin yllysyl)aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone indicated that multiple active caspase species sequentially appeared in the cytosol during the first 6 h after the addition of etoposide. Analysis on one- and two-dimensional gels revealed that two species comigrated with caspase-6 and three comigrated with active caspase-3 species, suggesting that several splice or modification variants of these enzymes are active during apoptosis. Polypeptides that comigrate with the cytosolic caspases were also labeled in nuclei of apoptotic HL-60 cells. These results not only indicate that etoposide-induced apoptosis in HL-60 cells is accompanied by the selective activation of multiple caspases in cytosol and nuclei, but also suggest that other caspase precursors such as procaspase-2 are present but not activated during apoptosis.
Martins
L M
LM
Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh, EH9 3JR Scotland, United Kingdom.
Kottke
T
T
Mesner
P W
PW
Basi
G S
GS
Sinha
S
S
Frigon
N
N
Tatar
E
E
Tung
J S
JS
Bryant
K
K
Takahashi
A
A
Svingen
P A
PA
Madden
B J
BJ
McCormick
D J
DJ
Earnshaw
W C
WC
Kaufmann
S H
SH
D000972:Antineoplastic Agents, Phytogenic / Q000633:toxicity*; D017209:Apoptosis / Q000187:drug effects*; D020170:Caspase 1; D002467:Cell Nucleus / Q000201:enzymology*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology*; D004789:Enzyme Activation; D005047:Etoposide / Q000633:toxicity*; D018922:HL-60 Cells; D006801:Humans
D000972:Antineoplastic Agents, Phytogenic; D005047:Etoposide; D003546:Cysteine Endopeptidases; D020170:Caspase 1