title: Comparison of caspase activation and subcellular localization in HL-60 and K562 cells undergoing etoposide-induced apoptosis.
abstract: Previous studies have shown that K562 chronic myelogenous leukemia cells are resistant to induction of apoptosis by a variety of agents, including the topoisomerase II (topo II) poison etoposide, when examined 4 to 24 hours after treatment with an initiating stimulus. In the present study, the responses of K562 cells and apoptosis-proficient HL-60 acute myelomonocytic leukemia cells to etoposide were compared, with particular emphasis on determining the long-term fate of the cells. When cells were treated with varying concentrations of etoposide for 1 hour and subsequently plated in soft agar, the two cell lines displayed similar sensitivities, with a 90% reduction in colony formation at 5 to 10 mu mol/L etoposide. After treatment with 17 mu mol/L etoposide for 1 hour, cleavage of the caspase substrate poly(ADP-ribose) polymerase (PARP), DNA fragmentation, and apoptotic morphological changes were evident in HL-60 cells in less than 6 hours. After the same treatment, K562 cells arrested in G2 phase of the cell cycle but otherwise appeared normal for 3 to 4 days before developing similar apoptotic changes. When the etoposide dose was increased to 68 mu mol/L, apoptotic changes were evident in HL-60 cells after 2 to 3 hours, whereas the same changes were observed in K562 cells after 24 to 48 hours. This delay in the development of apoptotic changes in K562 cells was accompanied by delayed release of cytochrome c to the cytosol and delayed appearance of peptidase activity that cleaved the fluorogenic substrates Asp-Glu-Val-Asp-aminotrifluoromethylcoumarin (DEVD-AFC) and Val-Glu-Ile-Asp-aminomethylcoumarin (VEID-AMC) as well as an altered spectrum of active caspases that were affinity labeled with N-(Nalpha-benzyloxycarbonylglutamyl-Nepsilon-biotin yllysyl) aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone [z-EK(bio)D-aomk]. On the other hand, the activation of caspase-3 under cell-free conditions occurred with indistinguishable kinetics in cytosol prepared from the two cell lines. Collectively, these results suggest that a delay in the signaling cascade upstream of cytochrome c release and caspase activation leads to a long latent period before the active phase of apoptosis is initiated in etoposide-treated K562 cells. Once the active phase of apoptosis is initiated, the spectrum and subcellular distribution of active caspase species differ between HL-60 and K562 cells, but a similar proportion of cells are ultimately killed in both cell lines.
authors.affiliation: Institute of Cell & Molecular Biology, University of Edinburgh, Edinburgh, UK.
authors.forename: L M
authors.initials: LM
authors.lastname: Martins
authors.forename: P W
authors.initials: PW
authors.lastname: Mesner
authors.forename: T J
authors.initials: TJ
authors.lastname: Kottke
authors.forename: G S
authors.initials: GS
authors.lastname: Basi
authors.forename: S
authors.initials: S
authors.lastname: Sinha
authors.forename: J S
authors.initials: JS
authors.lastname: Tung
authors.forename: P A
authors.initials: PA
authors.lastname: Svingen
authors.forename: B J
authors.initials: BJ
authors.lastname: Madden
authors.forename: A
authors.initials: A
authors.lastname: Takahashi
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 / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism; D005047:Etoposide / Q000494:pharmacology*; D018922:HL-60 Cells; D006801:Humans; D007700:Kinetics; D016315:Proto-Oncogene Proteins c-abl / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D004792:Enzyme Precursors; D005047:Etoposide; D016315:Proto-Oncogene Proteins c-abl; D003546:Cysteine Endopeptidases