Macrophage Colony-Stimulating Factor: Possible Existence of Caspase 3-Like Pathway E Okuma1, K Saeki1, M Shimura2, Y Ishizaka2, E Yasugi1 and a Yuo1
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Leukemia (2000) 14, 612–619 2000 Macmillan Publishers Ltd All rights reserved 0887-6924/00 $15.00 www.nature.com/leu Induction of apoptosis in human hematopoietic U937 cells by granulocyte– macrophage colony-stimulating factor: possible existence of caspase 3-like pathway E Okuma1, K Saeki1, M Shimura2, Y Ishizaka2, E Yasugi1 and A Yuo1 Departments of 1Hematology and 2Intractable Diseases, Research Institute, International Medical Center of Japan, Tokyo, Japan Granulocyte–macrophage colony-stimulating factor (GM-CSF) U937 cells probably via unknown pathways distinct from induced apoptosis in human hematopoietic U937 cells by itself conventional caspase 3 cascade. and in a synergistic manner with tumor necrosis factor (TNF). GM-CSF-induced apoptosis was not inhibited by caspase inhibitors YVAD-CMK, DEVD-CHO and z-VAD-FMK, under the condition that these inhibitors potently suppressed TNF- Materials and methods induced apoptosis. Both GM-CSF and TNF induced caspase 3- like activity in this cell line though the time course was distinct Reagents between two cytokines, and combined stimulation of cells with GM-CSF plus TNF induced additive or synergistic activation of caspase 3-like activity. Amount of immunoreactive cleaved Highly purified recombinant human GM-CSF and TNF pro- forms of caspase 3 recognized by specific antibody was com- duced by Escherichia coli were provided by Schering Plough, pletely dissociated with its enzymatic activity when the cells Osaka, Japan and Dainippon Pharmaceutical, Osaka, Japan, were stimulated with GM-CSF, but not with TNF. These results respectively. Contamination of lipopolysaccharide was less indicate that GM-CSF induces apoptosis of U937 cells via than 100 pg/mg protein, as determined by limulus amebocyte unknown pathway, which seems to be mediated by caspase 3- lysate assay. Sodium dodecyl sulfate (SDS), dimethyl sulfoxide like activity, yet not caspase 3 itself, resistant to the caspase inhibitors, and synergistically interacts with conventional cas- (DMSO), RNase, ethidium bromide, Marker 4 and the tetra- pase 3 pathway of TNF. Possible involvement of caspases 1 peptide caspase 1 inhibitor, acetyl-Tyr-Val-Ala-Asp-chloro- and 8 (-like activity) but not caspase 7 in this pathway was also methylketone (YVAD-CMK) were purchased from Wako Pure suggested. Leukemia (2000) 14, 612–619. Chemical, Tokyo, Japan. RPMI 1640 medium was from Gibco Keywords: GM-CSF; apoptosis; U937 cells; caspase 3 Laboratories, Grand Island, NY, USA; fetal calf serum from JRH Bioscience, Lenexa, KS, USA; Proteinase K from Boehr- inger Mannheim, Mannheim, Germany; BCA protein assay Introduction reagent from Pierce, Rockford, IL, USA; a fluorogenic sub- strate for caspase 7, amino-methylcumarin-Val-Asp-Gln-Val- Apoptosis is a way of cell death that is programmed geneti- Asp-Gly-Trp-[Lys-DNP] (AMC-VDQVDGW[K-DNP]), from cally and occurs actively. Detailed analyses for this phenom- Calbiochem-Novabiochem, La Jolla, CA, USA; fluorogenic enon have been performed and its important roles in a wide substrates for caspase 1, acetyl-Tyr-Val-Ala-Asp-amino- variety of biological systems have been determined.1 In methylcumarin (YVAD-AMC), caspase 3, acetyl-Asp-Glu-Val- addition to these physiological significances, intracellular sig- Asp-amino-methylcumarin (DEVD-AMC), and caspase 8, ace- naling mechanisms of apoptosis have also been intensively tyl-Ile-Glu-Thr-Asp-amino-methylcumarin (IETD-AMC), and studied. Among the signaling molecules for apoptosis, caspase the tetrapeptide caspase 3 inhibitor, acetyl-Asp-Glu-Val-Asp 3 (CPP32) has been reported to play critical roles2,3 and its (aldehyde) (DEVD-CHO) and the tripeptide caspase inhibitor, direct downstream target seems to be DNase which induces carbobenzoxy Val-Ala-Asp-fluoromethylketone (z-VAD-FMK) the internucleosomal fragmentation of DNA.4 were from Peptide Institute, Osaka, Japan. The monoclonal Granulocyte–macrophage colony-stimulating factor (GM- anti-caspase 3 antibody was purchased from Transduction CSF) is one member of hematopoietic growth factors that sup- Laboratories, Lexington, KY, USA; monoclonal anti-caspase 1 ports the proliferation and/or differentiation of hematopoietic antibody from Pharmingen, San Diego, CA, USA; polyclonal cell.5 GM-CSF is known to activate signaling molecules such anti-caspase 7 and caspase 8 antibodies were from Santa Cruz as Stat5 and extracellular-regulated kinase (ERK) to exert these Biotechnology, Santa Cruz, CA, USA; alkaline phosphatase- biological effects.6,7 Although this factor is well known to conjugated anti-mouse IgG and alkaline phosphatase-conju- induce the proliferation and inhibit apoptosis,5–7 its possible gated anti-rabbit IgG antibodies from Promega, Madison, WI, apoptosis-inducing effects on hematopoietic cells have not USA; and nitrocellulose membrane and protein A-Sepharose been well characterized. In addition, its effects on caspases from BioRad, Richmond, CA, USA. Other reagents for the cell are largely unknown. culture, FACS analysis, DNA fragmentation assay, determi- In the present study, we investigated the apoptosis-inducing nation of caspase activity, Western blotting and immunopreci- effects of GM-CSF on human hematopoietic cell line in pitation were purchased from Sigma Chemical, St Louis, comparison with those of TNF, and extended the study to the MO, USA. intracellular signaling pathways linking to apoptosis, caspase cascade. Results indicate that GM-CSF induces apoptosis of Culture and preparation of cells U937 cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, penicillin Correspondence: A Yuo, Department of Hematology, Research µ Institute, International Medical Center of Japan, 1–21–1, Toyama, (100 U/ml) and streptomycin (100 g/ml). For the induction of 4 Shinjuku-ku, Tokyo 162–8655, Japan; Fax: 81 3 3204 5422 apoptosis, U937 cells were seeded at 4–8 × 10 cells/ml and Received 8 February 1999; accepted 29 July 1999 grown in the presence or absence of cytokines and other GM-CSF-induced apoptosis in U937 cells E Okuma et al 613 agents for up to 4 days. Cultured cells were harvested after as substrates for caspase 3-, 1-, 8- and 7-like activities, the cultivation with these agents, washed one to three times respectively. An aliquot of each sample was incubated with with phosphate-buffered saline (PBS), and then suspended in the substrate for 1 h at 37°C. The release of AMC was meas- appropriate buffer for each experiment. ured by a fluorescence microplate reader, model MTP-100F (Corona Electric, Hitachi, Ibaragi, Japan) or Hitachi F-4010 fluorescence spectrophotometer (Hitachi, Tokyo, Japan). The FACS analysis excitation and emission wavelengths were set at 360 and 450 nm for caspase 3-like activity, 380 and 460 nm for cas- FACS analysis for determination of cell cycle distribution was pase 1- and 8-like activities, and 316 and 394 nm for caspase performed as described.8 After exposure to appropriate stim- 7-like activity, respectively. The sample composed of substrate uli, cells were washed in PBS once, and then resuspended in and lysis buffer was used as blank. Caspase activity was 80% ice-cold ethanol for fixing. After treatment with RNase measured as fluorescence units normalized by the protein (100 µg/ml) for 30 min at 37°C, DNA was stained with concentration in the samples. 50 µg/ml of propidium iodide. Cell cycle analysis was perfor- med by FACScan (Nippon Becton Dickinson, Tokyo, Japan), and the number of cells in the area corresponding to the sub- Preparation of cell lysate for Western blotting and G1 region was calculated using the Lysis II program (Nippon immunoprecipitation Becton Dickinson). For Western blotting, the cell pellets were resuspended in ice- cold solution containing 50 mM Hepes (pH 7.4), 2 mM sodium Morphological evaluation of cells orthovanadate, 100 mM sodium fluoride, 1 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aproti- Light microscopic examination of cell viability and mor- nin and 10 µg/ml leupeptin. The cell suspension was mixed phology was done to determine the apoptosis of U937 cells. with 1:1 with 2 × sample buffer (4% SDS, 20% glycerol, 10% For the determination of cell viability, a cell count was made mercaptoethanol and a trace amount of bromophenol blue on the hemocytometer using the trypan blue dye exclusion dye in 125 mM Tris-HCl, pH 6.8), heated for 5 min at 100°C test. For the morphological evaluation of apoptosis, cells sus- and then frozen at −80°C until use. For immunoprecipitation, pended in Hanks’ balanced salt solution (HBSS) were caused the cell pellets were resuspended in ice-cold solution contain- to adhere to slide glasses by centrifugation using Cytospin 2 ing 50 mM Hepes (pH 7.4), 1% Triton X-100, 2 mM sodium (Shandon, Pittsburgh, PA, USA) and cytospin slides were then orthovanadate, 100 mM sodium fluoride, 1 mM EDTA, 1 mM subjected to Wright–Giemsa staining for light microscopic EGTA, 1 mM phenymethylsufonyl fluoride, 10 µg/ml aprotinin examination of the cells. and 10 µg/ml leupeptin, and lysed for 20 min at 4°C. Insol- uble materials were removed by centrifugation for 15 min at 4°C. DNA fragmentation assay Small molecular weight chromosomal DNA from the nucleus Western blotting and cytoplasm was prepared as described.9 Briefly, 3 × 106 cells were lysed with 350 µl of lysis buffer containing 0.6% Samples were subjected to 10% or 14% SDS polyacrylamide SDS and 1 mM EDTA (pH 8.0), and after 5 min incubation at gel electrophoresis. After electrophoresis, proteins were elec- room temperature, 95 µlof5M NaCl was added. This was trophoretically transferred from the gel on to a nitrocellulose mixed gently and incubated for more than 8 h at 4°C. It was membrane in a buffer containing 25 mM Tris, 192 mM glycine then centrifuged at 20 000 g for 20 min at 4°C, and the super- and 20% methanol at 2 mA/cm2 for 2 h at 4°C.