Disruption of Redox Homeostasis in Tumor Necrosis Factor-Induced Apoptosis in a Murine Hepatocyte Cell Line
2000; Elsevier BV; Volume: 157; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)64533-6
ISSN1525-2191
AutoresRobert H. Pierce, Jean S. Campbell, Alyssa B. Stephenson, Christopher C. Franklin, Michelle Chaisson, Martin Poot, Terrance J. Kavanagh, Peter S. Rabinovitch, Nelson Fausto,
Tópico(s)Macrophage Migration Inhibitory Factor
ResumoTumor necrosis factor (TNF) is a mediator of the acute phase response in the liver and can initiate proliferation and cause cell death in hepatocytes. We investigated the mechanisms by which TNF causes apoptosis in hepatocytes focusing on the role of oxidative stress, antioxidant defenses, and mitochondrial damage. The studies were conducted in cultured AML12 cells, a line of differentiated murine hepatocytes. As is the case for hepatocytesin vivo, AML12 cells were not sensitive to cell death by TNF alone, but died by apoptosis when exposed to TNF and a small dose of actinomycin D (Act D). Morphological signs of apoptosis were not detected until 6 hours after the treatment and by 18 hours ∼50% of the cells had died. Exposure of the cells to TNF+Act D did not block NFκB nuclear translocation, DNA binding, or its overall transactivation capacity. Induction of apoptosis was characterized by oxidative stress indicated by the loss of NAD(P)H and glutathione followed by mitochondrial damage that included loss of mitochondrial membrane potential, inner membrane structural damage, and mitochondrial condensation. These changes coincided with cytochrome C release and the activation of caspases-8, -9, and -3. TNF-induced apoptosis was dependent on glutathione levels. In cells with decreased levels of glutathione, TNF by itself in the absence of transcriptional blocking acted as an apoptotic agent. Conversely, the antioxidant α-lipoic acid, that protected against the loss of glutathione in cells exposed to TNF+Act D completely prevented mitochondrial damage, caspase activation, cytochrome C release, and apoptosis. The results demonstrate that apoptosis induced by TNF+Act D in AML12 cells involves oxidative injury and mitochondrial damage. As injury was regulated to a larger extent by the glutathione content of the cells, we suggest that the combination of TNF+Act D causes apoptosis because Act D blocks the transcription of genes required for antioxidant defenses. Tumor necrosis factor (TNF) is a mediator of the acute phase response in the liver and can initiate proliferation and cause cell death in hepatocytes. We investigated the mechanisms by which TNF causes apoptosis in hepatocytes focusing on the role of oxidative stress, antioxidant defenses, and mitochondrial damage. The studies were conducted in cultured AML12 cells, a line of differentiated murine hepatocytes. As is the case for hepatocytesin vivo, AML12 cells were not sensitive to cell death by TNF alone, but died by apoptosis when exposed to TNF and a small dose of actinomycin D (Act D). Morphological signs of apoptosis were not detected until 6 hours after the treatment and by 18 hours ∼50% of the cells had died. Exposure of the cells to TNF+Act D did not block NFκB nuclear translocation, DNA binding, or its overall transactivation capacity. Induction of apoptosis was characterized by oxidative stress indicated by the loss of NAD(P)H and glutathione followed by mitochondrial damage that included loss of mitochondrial membrane potential, inner membrane structural damage, and mitochondrial condensation. These changes coincided with cytochrome C release and the activation of caspases-8, -9, and -3. TNF-induced apoptosis was dependent on glutathione levels. In cells with decreased levels of glutathione, TNF by itself in the absence of transcriptional blocking acted as an apoptotic agent. Conversely, the antioxidant α-lipoic acid, that protected against the loss of glutathione in cells exposed to TNF+Act D completely prevented mitochondrial damage, caspase activation, cytochrome C release, and apoptosis. The results demonstrate that apoptosis induced by TNF+Act D in AML12 cells involves oxidative injury and mitochondrial damage. As injury was regulated to a larger extent by the glutathione content of the cells, we suggest that the combination of TNF+Act D causes apoptosis because Act D blocks the transcription of genes required for antioxidant defenses. Tumor necrosis factor (TNF) is a multifunctional cytokine that binds to two cell surface receptors,1Ashkenazi A Dixit VM Death receptors: signaling and modulation.Science. 1998; 281: 1305-1308Crossref PubMed Scopus (5201) Google Scholar tumor necrosis factor receptor type 1 and type 2 (TNFR1 and TNFR2, respectively). Most of the biological effects of TNF, including cell death,2Wallach D Boldin M Varfolomeev E Beyaert R Vandenabeele P Fiers W Cell death induction by receptors of the TNF family: towards a molecular understanding.FEBS Lett. 1997; 410: 96-106Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar are transduced through TNFR1, a receptor that contains a death domain near the intracellular C-terminus, similar to Fas (CD95 or Apo1). Clustering of the death domain of trimerized TNFR1 after ligand binding provides a docking site for TRADD (TNFR-associated death domain), an adapter protein3Arch RH Gedrich RW Thompson CB Tumor necrosis factor receptor-associated factors (TRAFs): a family of adapter proteins that regulates life and death.Genes Dev. 1998; 12: 2821-2830Crossref PubMed Scopus (525) Google Scholar that can bind signaling molecules such as TRAF2 (TNFR-associated factor 2), RIP (receptor interacting protein), and FADD (Fas-associated death domain). Association of TRAF2 and RIP to the receptor results in the downstream activation of NFκB and AP-1, whereas FADD signaling initiates caspase activation.4Ghosh S May MJ Kopp EB NFκB and Rel proteins: evolutionarily conserved mediators of immune responses.Annu Rev Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4652) Google Scholar, 5Hsu H Xiong J Goeddel D The TNF receptor 1-associated protein TRADD signals cell death and NFκB activation.Cell. 1995; 81: 495-504Abstract Full Text PDF PubMed Scopus (1763) Google ScholarThus, TNFR1 signaling is bifurcated into two opposing pathways; one activating pro-inflammatory and mitogenic or survival responses while the other initiates programmed cell death. In the liver, endotoxin (lipopolysaccharide)-mediated release of TNF triggers the up-regulation as well as down-regulation of acute phase response genes.6Heinrich P Behrmann I Graeve L Grotzinger J Haan S Horn F Horsten U Kerr I May P Muller-Newen G Schaper F Terstegen L Thiel S The acute-phase response of the liver: molecular mechanism of IL-6 signalling from the plasma membrane to the nucleus.in: Haussinger D Heinrich P Signalling in the Liver. Kluwer Academic Publishers, Dordrecht1998: 55-71Google Scholar In addition to its role in inflammatory responses, TNF has other important effects in the liver. Recent studies from this and other laboratories7Akerman P Cote P Yang SQ McClain C Nelson S Bagby GJ Diehl AM Antibodies to tumor necrosis factor-alpha inhibit liver regeneration after partial hepatectomy.Am J Physiol. 1992; 263: G579-G585PubMed Google Scholar, 8Yamada Y Kirillova I Peschon JJ Fausto N Initiation of liver growth by tumor necrosis factor: deficient liver regeneration in mice lacking type I tumor necrosis factor receptor.Proc Natl Acad Sci USA. 1997; 94: 1441-1446Crossref PubMed Scopus (845) Google Scholar established that TNF is involved in the initiation of liver regeneration after partial hepatectomy or chemical injury through TNFR1 signaling.9Yamada Y Fausto N Deficient liver regeneration after carbon tetrachloride injury in mice lacking type 1 but not type 2 tumor necrosis factor receptor.Am J Pathol. 1998; 152: 1577-1589PubMed Google Scholar, 10Yamada Y Webber EM Kirillova I Peschon JJ Fausto N Analysis of liver regeneration in mice lacking type 1 or type 2 tumor necrosis factor receptor: requirement for type 1 but not type 2 receptor.Hepatology. 1998; 28: 959-970Crossref PubMed Scopus (216) Google Scholar In contrast, this cytokine acts as a cytotoxic agent in different types of liver injury such as in transplantation rejection and alcohol-induced hepatic disease,11Colell A Garcia-Ruiz C Miranda M Ardite E Mari M Morales A Corrales F Kaplowitz N Fernandez-Checa JC Selective glutathione depletion of mitochondria by ethanol sensitizes hepatocytes to tumor necrosis factor.Gastroenterology. 1998; 115: 1541-1551Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar although the precise mechanisms of TNF effects in these complex conditions are not entirely understood.12Faubion WA Gores GJ Death receptors in liver biology and pathobiology.Hepatology. 1999; 29: 1-4Crossref PubMed Scopus (162) Google Scholar It has been reported that TNF by itself does not cause hepatocyte cell death.13Leist M Gantner F Bohlinger I Germann PG Tiegs G Wendel A Murine hepatocyte apoptosis induced in vitro and in vivo by TNF-alpha requires transcriptional arrest.J Immunol. 1994; 153: 1778-1788PubMed Google Scholar However, sensitization to TNF cytotoxicity can be caused by drugs that directly block gene transcription or RNA translation or by less specific drugs that also inhibit gene transcription such as galactosamine and α-amanitine.14Leist M Gantner F Kunstle G Wendel A Cytokine-mediated hepatic apoptosis.Rev Physiol Biochem Pharmacol. 1998; 133: 109-155PubMed Google Scholar Despite the extensive studies of TNF-mediated apoptosis in lymphocytes and other cells, there is relatively little specific information regarding the cell-death pathways activated by TNFR1 in hepatocytes. TNF causes cell death by necrosis or apoptosis depending on the intensity and duration of the stimulus and the overall metabolic status.15Lemasters JJ Nieminen AL Qian T Trost LC Elmore SP Nishimura Y Crowe RA Cascio WE Bradham CA Brenner DA Herman B The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy.Biochim Biophys Acta. 1998; 1366: 177-196Crossref PubMed Scopus (1237) Google Scholar In cultured hepatocytes infected with an adenovirus expressing a dominant NFκB repressor, TNF-induced apoptosis and necrosis was preceded by the opening of high-conductance mitochondrial pores.16Bradham CA Qian T Streetz K Trautwein C Brenner DA Lemasters JJ The mitochondrial permeability transition is required for tumor necrosis factor alpha-mediated apoptosis and cytochrome c release.Mol Cell Biol. 1998; 18: 6353-6364Crossref PubMed Scopus (369) Google Scholar Although mitochondrial alterations may have been produced by reactive oxygen species (ROS), it remains controversial what role alterations in redox potential and cell antioxidant defenses may play in TNF-induced cell death. In L929 cells,17Goossens V Grooten J De Vos K Fiers W Direct evidence for tumor necrosis factor-induced mitochondrial reactive oxygen intermediates and their involvement in cytotoxicity.Proc Natl Acad Sci USA. 1995; 92: 8115-8119Crossref PubMed Scopus (558) Google Scholar, 18Schulze-Osthoff K Bakker AC Vanhaesebroeck B Beyaert R Jacob WA Fiers W Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial functions. Evidence for the involvement of mitochondrial radical generation.J Biol Chem. 1992; 267: 5317-5323Abstract Full Text PDF PubMed Google Scholar TNF toxicity was associated with ROS production while glutathione (γ-glutamylcysteinylglycine, GSH) acted as the main agent capable of reducing ROS levels. In contrast with these findings, Leist et al13Leist M Gantner F Bohlinger I Germann PG Tiegs G Wendel A Murine hepatocyte apoptosis induced in vitro and in vivo by TNF-alpha requires transcriptional arrest.J Immunol. 1994; 153: 1778-1788PubMed Google Scholar reported that both oxidative stress and decreases in GSH occurred after morphological changes of apoptosis that were evident in hepatocytes exposed to TNF. Furthermore, Xu et al19Xu Y Bialik S Jones BE Iimuro Y Kitsis RN Srinivasan A Brenner DA Czaja MJ NF-kappaB inactivation converts a hepatocyte cell line TNF-alpha response from proliferation to apoptosis.Am J Physiol. 1998; 275: C1058-C1066PubMed Google Scholar also concluded that oxidative stress played no role in TNF-induced hepatocyte apoptosis because prevention of the up-regulation of antioxidant responses did not sensitize hepatocytes to TNF-induced cell death. The interpretations of experiments to test the role of oxidant stress in TNF-mediated hepatocyte apoptosis are complicated by several factors. First, TNF may be capable of triggering apoptosis by more than one signaling pathway depending on variables such as the metabolic state of the cell and oxidative phosphorylation coupling.20Qian T Herman B Lemasters JJ The mitochondrial permeability transition mediates both necrotic and apoptotic death of hepatocytes exposed to Br-A23187.Toxicol Appl Pharmacol. 1999; 154: 117-125Crossref PubMed Scopus (135) Google Scholar For instance in HeLa cells, TNF given in combination with the protein synthesis inhibitor, emeline, activated two distinct apoptotic pathways, only one of which depended on the early release of mitochondrial ROS.21Sidoti-de Fraisse C Rincheval V Risler Y Mignotte B Vayssiere JL TNF-alpha activates at least two apoptotic signaling cascades.Oncogene. 1998; 17: 1639-1651Crossref PubMed Scopus (132) Google Scholar In addition, the intensity of the stimulus may determine the nature of the cellular response as excessive oxidative stress can block caspase activity by a direct effect on these proteases. Nonhepatic cells exposed to high concentrations of H2O2, as well as hepatocytes depleted of GSH, were resistant to apoptosis and had no detectable caspase-3 activity after treatment with agonist Fas antibodies.22Hampton MB Orrenius S Dual regulation of caspase activity by hydrogen peroxide: implications for apoptosis.FEBS Lett. 1997; 414: 552-556Abstract Full Text Full Text PDF PubMed Scopus (599) Google Scholar Lastly, hepatocytes may differ from other cell types in their response to oxidative injury because they contain very high GSH levels.23Lu SC Regulation of hepatic glutathione synthesis.Semin Liver Dis. 1998; 18: 331-343Crossref PubMed Scopus (82) Google Scholar, 24Fernandez-Checa JC Kaplowitz N Garcia-Ruiz C Colell A Mitochondrial glutathione: importance and transport.Semin Liver Dis. 1998; 18: 389-401Crossref PubMed Scopus (191) Google Scholar It is likely that modulations of the cellular GSH content would have major effects on cell survival. All of these observations are consistent with a general model for mammalian cell apoptosis25Scaffidi C Fulda S Srinivasan A Friesen C Li F Tomaselli KJ Debatin KM Krammer PH Peter ME Two CD95 (APO-1/Fas) signaling pathways.Embo J. 1998; 17: 1675-1687Crossref PubMed Scopus (2645) Google Scholar, 26Gross G McDonnell JM Korsmeyer SJ BCL-2 family members and the mitochondria in apoptosis.Genes Dev. 1999; 13: 1899-1911Crossref PubMed Scopus (3280) Google Scholar that proposes that there are two main execution pathways: one involving mitochondrial damage (type II cells) and the other, a mitochondrial-independent pathway in which early caspase-8 activation is a major initiating event (type I cells). We hypothesized that because of the abundance of mitochondria, high oxidative phosphorylation rates, and the high levels of GSH in hepatocytes, TNF-induced apoptosis in these cells may be highly dependent on mitochondrial functional and structural integrity. In this report, we focused our analysis of the mechanisms mediating TNF-induced apoptosis in hepatocytes on the role of oxidative stress, antioxidant defenses, and mitochondrial damage. The studies were conducted in cultured AML12 hepatocytes, which, as is the case for murine hepatocytes in vivo,13Leist M Gantner F Bohlinger I Germann PG Tiegs G Wendel A Murine hepatocyte apoptosis induced in vitro and in vivo by TNF-alpha requires transcriptional arrest.J Immunol. 1994; 153: 1778-1788PubMed Google Scholar are not sensitive to cell killing by TNF, but die by apoptosis when exposed to TNF and in the presence of a small dose of actinomycin D (Act D). The results obtained indicate that the balance between mitochondrial oxidant stress and endogenous antioxidant defense mechanisms involving GSH plays a determinant role in TNF-induced apoptosis in hepatocytes. AML12 hepatocytes, a well-differentiated, nontransformed murine hepatocyte cell line derived from transforming growth factor-α (TGF-α) transgenic mice were used for all experiments.27Wu JC Merlino G Fausto N Establishment and characterization of differentiated, nontransformed hepatocyte cell lines derived from mice transgenic for transforming growth factor alpha.Proc Natl Acad Sci USA. 1994; 91: 674-678Crossref PubMed Scopus (253) Google Scholar In brief, AML12 cells were maintained in Dulbecco's minimal essential medium/F12 (Life Technologies Inc., Grand Island, NY) with 10% fetal bovine serum (Hyclone, Logan, UT), 5 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml selenium (ITS Premix, Collaborative Biomedical Products (Bedford, MA)), 50 μg/ml gentamicin, and 0.1 μmol/L dexamethasone. Cultures were grown at 37°C in a humidified 6% CO2 atmosphere, fed approximately every 72 hours, and passaged at ∼80 to 90% confluence. Act D, buthionine sulfoximine (BSO), diethyl maleate (DEM), dexamethasome, and menadione (MEN) were purchased through Sigma Chemical Co. (St. Louis, MO). Acetyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-aminomethylcoumarin (DEVD-AMC), acetyl-Ilu(OMe)-Glu(OMe)-Thr-Asp(OMe)-aminomethylcoumarin (IETD-AMC), and acetyl-Leu(OMe)-Glu(OMe)-His-Asp(OMe)-aminomethylcoumarin (LEHD-AMC) were purchased from Biomol (Plymouth Meeting, PA) whereas benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-FMK) and α-lipoic (α-LA) acid were obtained from Bachem (Torrance, CA) or Calbiochem (La Jolla, CA), respectively. Mitotracker green (MTG), chloromethyl-x-rosamine (CMX), nonyl acridine orange (NAO), monochlorobimane (MCB), and monobromobimane were purchased from Molecular Probes (Eugene, OR). Recombinant murine TNF was purchased from R&D Systems (Minneapolis, MN). Antibodies to p65 (sc-372-G), and caspase-8 (Mch 5, sc-6134) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), whereas anti-caspase-3 antibodies (C76920) were purchased from Pharmingen (San Diego, CA). Horseradish peroxidase-conjugated anti-mouse secondary antibody was purchased from Amersham Pharmacia Biotech (NA 931; Piscataway, NJ) and the horseradish peroxidase-conjugated anti-goat secondary antibody was purchased from Santa Cruz (sc-2020). AML12 cells were trypsinized, plated, and allowed to adhere and grow overnight. At 90 to 95% confluence, the cells were pretreated with either 200 nmol/L (250 ng/ml) Act D or phosphate-buffered saline (PBS) in fresh medium for 30 minutes followed by either 20 ng/ml TNF (dissolved in PBS containing 1.0% bovine serum albumin, fraction V, to a stock concentration of 2.0 μg/ml) or PBS control. Treatment of cells plated at lower densities resulted in much more rapid kinetics of apoptosis than cultures plated at 90 to 95% confluency. In vivo induction of hepatocyte apoptosis was done as described by Leist et al.13Leist M Gantner F Bohlinger I Germann PG Tiegs G Wendel A Murine hepatocyte apoptosis induced in vitro and in vivo by TNF-alpha requires transcriptional arrest.J Immunol. 1994; 153: 1778-1788PubMed Google Scholar For these experiments C57BL/6 mice received either TNF (3.3 μg/kg) or Act D (800 μg/kg) alone or in combination. The Act D injection was given 15 minutes before the TNF injection and the mice were killed 3 to 6 hours after the TNF injection. Livers were harvested, fixed in 10% buffered formalin, processed, and stained with hematoxylin and eosin. Apoptosis was assessed morphologically. AML12 cells were pretreated with 100 μmol/L zVAD-FMK (3 hours pretreatment) or 1 mmol/L α-LA acid (60 minutes pretreatment) followed by treatment with Act D and then TNF as described above. To alter intracellular GSH content, AML12 cells were pretreated with BSO (1 mmol/L dissolved in AML12 media) for 60 minutes to block de novo GSH synthesis then 0.8 mmol/L DEM (made fresh in AML12 media) to acutely deplete GSH. For experiments with menadione, AML12 cells were pretreated for 3 hours at a concentration of 100 μmol/L, followed by treatment with Act D or Act D with α-LA (1 mmol/L) for 9 hours. AML12 cells were lysed and nuclei were extracted as reported previously.28FitzGerald MJ Webber EM Donovan JR Fausto N Rapid DNA binding by nuclear factor kappa B in hepatocytes at the start of liver regeneration.Cell Growth Differ. 1995; 6: 417-427PubMed Google Scholar In brief, 5 μg of nuclear protein were incubated at room temperature for 30 minutes with 0.2 ng of32P-end-labeled double-stranded oligonucleotide (NFκB binding site from the class 1 major histocompatibility enhancer element H2K), followed by electrophoresis through a 5% polyacrylamide Tris-glycine-ethylenediaminetetraacetic acid (EDTA) gel. Gels were dried under a vacuum and exposed overnight to Kodak X-AR film (Eastman Kodak Co., Rochester, NY) at −80°C with an intensifying screen. AML12 cells were rinsed in PBS and lysed in a 1% Triton X-100 buffer containing 50 mmol/L Tris-HCl, pH 7.4, 50 mmol/L β-glycerophosphate, 150 mmol/L NaCl, 2 mmol/L EDTA, 1 mmol/L Na3VO4, 1 mmol/L benzamidine, 1 mmol/L dithiothreitol, 10 μg/ml leupeptin, 10 μg/ml pepstatin A, 10 μg/ml aprotinin, 0.5 mmol/L 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochlorine (ICN Biomedicals, Irvine, CA), and 10% glycerol. Protein quantitation was performed using Bradford reagent (Bio-Rad, Hercules, CA) and 50 μg of total protein lysate was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (Millipore, Bedford, MA). Immunoblot analysis of nuclear protein was performed using 10 μg of protein isolated as described above. Release of cytochrome C into the cytosol was determined by immunoblot as described by Ghibelli et al29Ghibelli L Coppola S Fanelli C Rotilio G Civitareale P Scovassi AI Ciriolo MR Glutathione depletion causes cytochrome c release even in the absence of cell commitment to apoptosis.FASEB J. 1999; 13: 2031-2036Crossref PubMed Scopus (134) Google Scholar using a mouse monoclonal anti-cytochrome C antibody (PharMingen, La Jolla, CA). Membranes were blocked in Tris-buffered saline with 0.1% Tween 20 containing 5% milk (blotting grade; BioRad, Hercules, CA) at 4°C and incubated with primary antibodies at the following dilutions: p65, 1:2,000; Mch5, 1:1,000; caspase-3, 1:1,000; and cytochrome C, 1:1,000, in 0.5% milk in Tris-buffered saline with 0.1% Tween 20 for 1 to 2 hours. The appropriate secondary antibodies were added for 2 to 3 hours in 0.5% milk in Tris-buffered saline with 0.1% Tween 20 and antigen-antibody complexes were detected with enhanced chemiluminescent reagents purchased from either Dupont-New England Nuclear (Boston, MA) or Pierce (Rockford, IL). AML12 cells were co-transfected with a 4× NFκB luciferase reporter gene30Berberich I Shu GL Clark EA Cross-linking CD40 on B cells rapidly activates nuclear factor-kappa B.J Immunol. 1994; 153: 4357-4366Crossref PubMed Google Scholar and a CMV β-galactosidase gene using lipofectamine (Life Technologies, Inc.) following the manufacturer's protocol in a 2:1 ratio (1.5 μg total DNA). The transfection medium containing DNA/lipofectamine was removed after 5 hours and cells were treated immediately. Cells were harvested 14 hours after transfection and processed for luciferase and β-galactosidase assays using the Dual Light System (Tropix, Bedford, MA), according to the manufacturer's instructions. This system allows for sequential determinations of luciferase and β-galactosidase on the same experimental sample. Flow cytometry was performed on a Coulter Epics Elite (Coulter Electronics, Hialeah, FL) using time-resolved dual laser excitation: 15 mW 488 argon (nondelayed) and 20 mW UV argon (delayed, 40 μS). Fluorescence measurements were done with the following dyes as previously described:31Poot M Pierce RH Detection of changes in mitochondrial function during apoptosis by simultaneous staining with multiple fluorescent dyes and correlated multiparameter flow cytometry.Cytometry. 1999; 35: 311-317Crossref PubMed Scopus (98) Google Scholar, 32Poot M Verkerk A Koster JF Jongkind JF De novo synthesis of glutathione in human fibroblasts during in vitro ageing and in some metabolic diseases as measured by a flow cytometric method.Biochim Biophys Acta. 1986; 883: 580-584Crossref PubMed Scopus (58) Google Scholar, 33Hedley DW Chow S Evaluation of methods for measuring cellular glutathione content using flow cytometry.Cytometry. 1994; 15: 349-358Crossref PubMed Scopus (226) Google Scholar MCB and monobromobimane to measure GSH and reduced cellular thiols, respectively; CMX to measure mitochondrial membrane potential; MTG to measure mitochondrial mass; NAO to measure cardiolipin content; and dichloromethyl-x-rosamine (H2-CMXROS), a mitochondrial selective dye that becomes fluorescent when oxidized, to measure mitochondrial ROS. UV excited blue autofluorescence was used to measure reduced NAD, designated NADPH to represent contributions by both NADH and NADPH. For each sample, 488-nm forward light scatter to determine cell size and UV laser right-angle light scatter were measured. Fluorescent intensity was displayed on a logarithmic scale. AML12 cells were cultured in 6-well tissue culture plates. The culture supernatant was pooled with the attached cells, which were harvested with trypsin/EDTA (Life Technologies, Inc.). An aliquot of cells was added to a tube containing an equal volume of 2× solution of the indicated dyes in cell culture medium and incubated with occasional mixing for 30 minutes in the tissue culture incubator. The cells were then placed on ice in the dark and run on the flow cytometer. At least 15,000 events were recorded per sample. Analysis of flow cytometric experiments was performed using the software package MPLUS (Phoenix Flow Systems, San Diego, CA). To help eliminate debris, a UV-side scatter versus 488-forward scatter gate determined by analysis of the untreated control samples was applied. Analytic (nonexclusionary) gates drawn around the entire population or distinct subpopulations were used to determine the median fluorescence values and percentage of cells associated with those groups. Identical gates were used for all comparisons. Trypsinized cells were fixed in 70% ETOH, centrifuged at 50 to 80 × g for 5 minutes and resuspended in a solution containing 0.5% Nonidet P-40 and 10 μg/ml DAPI. Apoptotic nuclear morphology was assessed as described by Hotz et al34Hotz MA Gong J Traganos F Darzynkiewicz Z Flow cytometric detection of apoptosis: comparison of the assays of in situ DNA degradation and chromatin changes.Cytometry. 1994; 15: 237-244Crossref PubMed Scopus (226) Google Scholar on 200 cells per experimental point and each point was determined in triplicate. AML12 cells were cultured on 2-well coverslip chambers (Nunc Inc., Bountiful, UT) and after treatment, the cultures were stained with CMXROS and MTG for 15 minutes at 37°C. Cells were scanned with an ACAS Ultima Confocal Laser Cytometer (Meridian Instruments, Okemos, MI) and confocal images were acquired using a 60× oil objective (NA = 1.3). Fields were first scanned for fluorescence from the mitochondrial stains, and then the NAD(P)H fluorescence images were collected from the same field. Images are displayed as composite images (red, green, blue) using Adobe Photoshop (Adobe Systems, Inc., San Jose, CA). Cells harvested by trypsinization were fixed at room temperature overnight in one-half strength Karnovsky's fixative. Cells were then rinsed in 0.1 mol/L phosphate buffer, postfixed in a 2% buffered osmium tetroxide solution, followed by dehydration through a graded series of ethanol solutions. Cells were rinsed with propylene oxide and infiltrated with 1:1 mix of PolyBed (Polysciences, Warrington, PA)/propylene oxide followed by 100% PolyBed. Cells were then embedded in PolyBed and polymerized at 60°C overnight. Sections (70 to 90 nm) were stained with saturated solutions of uranyl acetate and lead tartrate and micrographs were taken on a Philips 410 transmission electron microscope (FEI Co., Hillsboro, OR). Caspase-8, caspase-9, and caspase-3 activities were measured using the substrates, IETD-AMC, LEHD-AMC, and DEVD-AMC (Biomol), respectively. Cells were harvested and lysed in the Triton X-100 lysis buffer described above. Protein lysate (50 μg) was incubated for 30 minutes at 37°C in a caspase assay buffer (50 mmol/L Hepes, pH 7.4, 100 mmol/L NaCl, 2 mmol/L EDTA, 10% sucrose, 0.1% CHAPS, (3-[(3-cholamidopropyl)dimethyl ammonio]-1-propanesulfonate (Meriden, CN) 10 mmol/L dithiothreitol) containing 10 μmol/L of each peptide-aminomethylcoumarin substrate. Enzymatic assays and aminomethylcoumarin standard curves were carried out in duplicates using a fluorescent plate reader (Packard Instruments, Meriden, CT) with excitation and emission wavelengths of 360 nm and 460 nm, respectively. Fluorescence of the substrate blank was subtracted as background in each assay. Data analysis was performed using Packard's I-Smart software. Statview 4.5 (Abacus Concepts, Berkeley, CA) was used to perform the statistical analyses. For comparisons between means of multiple groups multifactorial analysis of variance with a Fischer's least significant difference was used. To assess the interaction between variables, 2-factor analysis of variance was used. The degree of correlation between flow cytometric parameters was assessed using a simple regression analysis. For all tests, P < 0.05 was accepted as significant. To study TNF-induced apoptosis in murine hepatocytes, we first determined whether TNF alone or in conjunction with Act D would cause apoptosis of mouse hepatocytes in vivo. In agreement with the data of Leist et al,13Leist M Gantner F Bohlinger I Germann PG Tiegs G Wendel A Murine hepatocyte apoptosis induced in vitro and in vivo by TNF-alpha requires transcriptional arrest.J Immunol. 1994; 153: 1778-1788PubMed Google Scholar histological analysis of
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