Comparison of Caspase Activation and Subcellular Localization in HL-60 and K562 Cells Undergoing Etoposide-Induced Apoptosis by Luis M. Martins, Peter.

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Comparison of Caspase Activation and Subcellular Localization in HL-60 and K562 Cells Undergoing Etoposide-Induced Apoptosis by Luis M. Martins, Peter W. Mesner, Timothy J. Kottke, Guriqbal S. Basi, Sukanto Sinha, Jay S. Tung, Phyllis A. Svingen, Benjamin J. Madden, Atsushi Takahashi, Daniel J. McCormick, William C. Earnshaw, and Scott H. Kaufmann Blood Volume 90(11):4283-4296 December 1, 1997 ©1997 by American Society of Hematology

Luis M. Martins et al. Blood 1997;90:4283-4296 ©1997 by American Society of Hematology

Luis M. Martins et al. Blood 1997;90:4283-4296 ©1997 by American Society of Hematology

Luis M. Martins et al. Blood 1997;90:4283-4296 ©1997 by American Society of Hematology

Luis M. Martins et al. Blood 1997;90:4283-4296 ©1997 by American Society of Hematology

Luis M. Martins et al. Blood 1997;90:4283-4296 ©1997 by American Society of Hematology

Luis M. Martins et al. Blood 1997;90:4283-4296 ©1997 by American Society of Hematology

Luis M. Martins et al. Blood 1997;90:4283-4296 ©1997 by American Society of Hematology

RT-PCR analysis of procaspase transcripts in untreated K562 cells. RT-PCR analysis of procaspase transcripts in untreated K562 cells. Each lane contains 10% of the product from the PCR reaction for the indicated target mRNA. The lane marked β-actin (−RT) is a control PCR reaction using poly A+ RNA as the template. The lack of a product in this lane confirms that the products observed in the other lanes are not derived from contaminating genomic DNA. Luis M. Martins et al. Blood 1997;90:4283-4296 ©1997 by American Society of Hematology

Luis M. Martins et al. Blood 1997;90:4283-4296 ©1997 by American Society of Hematology

Luis M. Martins et al. Blood 1997;90:4283-4296 ©1997 by American Society of Hematology

Luis M. Martins et al. Blood 1997;90:4283-4296 ©1997 by American Society of Hematology

Affinity labeling of etoposide-treated HL-60 and K562 cytosol and nuclei. Affinity labeling of etoposide-treated HL-60 and K562 cytosol and nuclei. (A) Aliquots containing 37 μg of cytosolic protein prepared from HL-60 (lanes 2 through 7) or K562 (lanes 8 through 11) cells treated with 68 μmol/L etoposide for the indicated length of time were reacted with z-EK(bio)D-aomk, subjected to SDS-PAGE, and reacted with peroxidase-coupled streptavidin. Additional experiments (see B) indicate that bands detected in 0-hour samples bind streptavidin in the absence of reaction with z-EK(bio)D-aomk. Lane 1, apoptosis-inducing S/M extract prepared from DU249 chicken hepatoma cells.24 53 (B) z-EK(bio)D-aomk labeling of cytosol and nuclei from HL-60 cells and K562 cells treated with 68 μmol/L etoposide for the indicated length of time. Samples containing cytosol or nuclei from 3 × 106 cells were incubated with 1 μmol/L z-EK(bio)D-aomk for 1 hour, then subjected to SDS-PAGE and blotting as described in the legend for (A). Lanes marked 0* contain nuclei that were subjected to SDS-PAGE without z-EK(bio)D-aomk treatment, thereby revealing the streptavidin-binding polypeptides that are normal constituents of these nuclei. Luis M. Martins et al. Blood 1997;90:4283-4296 ©1997 by American Society of Hematology

Analysis of z-EK(bio)D-aomk-labeled caspases by two-dimensional gel electrophoresis. Analysis of z-EK(bio)D-aomk-labeled caspases by two-dimensional gel electrophoresis. (A) Analysis of active caspases in HL-60 cytosol (left) and nuclei (middle). The spots are indexed as recently described.38 In these gels, the pH decreases from left to right. This analysis showed a novel active species (D) in HL-60 nuclei. The merged picture (right) was obtained by co-electophoresing mixtures of, respectively, HL-60 cytoplasm + active caspase-2 and HL-60 nuclei + active caspase-2 (data not shown). Caspase-2, which was previously shown not to comigrate with any of the caspases detected in HL-60 cytosol,38 provided a reference point for alignment of the cytosolic and nuclear images. Arrows in the merged image point to species observed in the nuclear fractions. (B) Analysis of active caspases in K562 cytosol (left) and nuclei (middle). Images were merged (right) as described for (A). (C) Comparison of the pattern of active caspases in cytosol and nuclei of HL-60 and K562 cells. Crosses correspond to the exact positions of the active species in HL-60, and circles correspond to the exact positions of the active species in K562. Luis M. Martins et al. Blood 1997;90:4283-4296 ©1997 by American Society of Hematology