title: Activation of multiple interleukin-1beta converting enzyme homologues in cytosol and nuclei of HL-60 cells during etoposide-induced apoptosis.
abstract: 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.
authors.affiliation: Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh, EH9 3JR Scotland, United Kingdom.
authors.forename: L M
authors.initials: LM
authors.lastname: Martins
authors.forename: T
authors.initials: T
authors.lastname: Kottke
authors.forename: P W
authors.initials: PW
authors.lastname: Mesner
authors.forename: G S
authors.initials: GS
authors.lastname: Basi
authors.forename: S
authors.initials: S
authors.lastname: Sinha
authors.forename: N
authors.initials: N
authors.lastname: Frigon
authors.forename: E
authors.initials: E
authors.lastname: Tatar
authors.forename: J S
authors.initials: JS
authors.lastname: Tung
authors.forename: K
authors.initials: K
authors.lastname: Bryant
authors.forename: A
authors.initials: A
authors.lastname: Takahashi
authors.forename: P A
authors.initials: PA
authors.lastname: Svingen
authors.forename: B J
authors.initials: BJ
authors.lastname: Madden
authors.forename: D J
authors.initials: DJ
authors.lastname: McCormick
authors.forename: W C
authors.initials: WC
authors.lastname: Earnshaw
authors.forename: S H
authors.initials: SH
authors.lastname: Kaufmann
mesh_terms: 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
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D005047:Etoposide; D003546:Cysteine Endopeptidases; D020170:Caspase 1