Overexpression of Catalase in the Mitochondrial or Cytosolic Compartment Increases Sensitivity of HepG2 Cells to Tumor Necrosis Factor-α-induced Apoptosis
2000; Elsevier BV; Volume: 275; Issue: 25 Linguagem: Inglês
10.1074/jbc.m000438200
ISSN1083-351X
AutoresJingxiang Bai, Arthur I. Cederbaum,
Tópico(s)NF-κB Signaling Pathways
ResumoThe sensitivity of HepG2 cells overexpressing catalase in either the cytosolic or mitochondrial compartment to tumor necrosis factor-α (TNF-α) and cycloheximide was studied. Cells overexpressing catalase in the cytosol (C33 cells) and especially in mitochondria (mC5 cells) were more sensitive to TNF-α-induced apoptosis than were control cells (Hp cells). The activities of caspase-3 and -8 were increased by TNF-α, with the highest activities found in mC5 cells. Sodium azide, an inhibitor of catalase, reduced the increased sensitivity of mC5 and C33 cells to TNF-α to the level of toxicity found with control Hp cells. Azide also decreased the elevated caspase-3 activity of mC5 cells. A pan-caspase inhibitor prevented the TNF-α-induced apoptosis and toxicity produced by catalase overexpression. Addition of H2O2prevented TNF-α-induced apoptosis and caspase activation, an effect prevented by simultaneous addition of catalase. TNF-α plus cycloheximide increased ATP levels, with higher levels in C33 and mC5 cells compared with Hp cells. TNF-α did not produce apoptosis in mC5 cells maintained in a low energy state. TNF-α signaling was not altered by the overexpression of catalase, as activation of nuclear factor κB and AP-1 by TNF-α was similar in the three cell lines. These results suggest that catalase, overexpressed in the cytosolic or especially the mitochondrial compartment, potentiates TNF-α-induced apoptosis and activation of caspases by removal of H2O2. The sensitivity of HepG2 cells overexpressing catalase in either the cytosolic or mitochondrial compartment to tumor necrosis factor-α (TNF-α) and cycloheximide was studied. Cells overexpressing catalase in the cytosol (C33 cells) and especially in mitochondria (mC5 cells) were more sensitive to TNF-α-induced apoptosis than were control cells (Hp cells). The activities of caspase-3 and -8 were increased by TNF-α, with the highest activities found in mC5 cells. Sodium azide, an inhibitor of catalase, reduced the increased sensitivity of mC5 and C33 cells to TNF-α to the level of toxicity found with control Hp cells. Azide also decreased the elevated caspase-3 activity of mC5 cells. A pan-caspase inhibitor prevented the TNF-α-induced apoptosis and toxicity produced by catalase overexpression. Addition of H2O2prevented TNF-α-induced apoptosis and caspase activation, an effect prevented by simultaneous addition of catalase. TNF-α plus cycloheximide increased ATP levels, with higher levels in C33 and mC5 cells compared with Hp cells. TNF-α did not produce apoptosis in mC5 cells maintained in a low energy state. TNF-α signaling was not altered by the overexpression of catalase, as activation of nuclear factor κB and AP-1 by TNF-α was similar in the three cell lines. These results suggest that catalase, overexpressed in the cytosolic or especially the mitochondrial compartment, potentiates TNF-α-induced apoptosis and activation of caspases by removal of H2O2. reactive oxygen species tumor necrosis factor-α cycloheximide minimal essential medium phosphate-buffered saline nuclear factor κB benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone Apoptosis, a major form of cell death, is characterized by early and prominent condensation of nuclear chromatin, cell shrinkage, activation of proteases and endonucleases, enzymatic cleavage of the DNA into 180-base pair oligonucleosomal fragments, and segmentation of the cells into membrane-bound apoptotic bodies (1.Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar, 2.Mignotte B. Vayssiere J.L. Eur. J. Biochem. 1998; 252: 1-15Crossref PubMed Scopus (709) Google Scholar). The most critical protease families implicated in apoptosis are cysteine proteases known as caspases (3.Alnemri E.S. Livingston D.J. Nicholson D.W. Salvesen G. Thornberry N.A. Wong W.W. Yuan J. Cell. 1996; 87: 171Abstract Full Text Full Text PDF PubMed Scopus (2147) Google Scholar, 4.Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6182) Google Scholar). Caspases are constitutively present in cells as zymogens and require proteolytic cleavage into the catalytic active heterodimer. Inhibiting the activation of caspases suppresses the ability of cells to undergo apoptosis or causes a switch from apoptosis to necrosis (4.Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6182) Google Scholar, 5.Samali A. Nordgren H. Zhivotovsky B. Peterson E. Orrenius S. Biochem. Biophys. Res. Commun. 1999; 255: 6-11Crossref PubMed Scopus (179) Google Scholar). Reactive oxygen species (ROS)1 are thought to be involved in many forms of apoptosis. Increased levels of ROS have been detected in cells undergoing apoptosis (5.Samali A. Nordgren H. Zhivotovsky B. Peterson E. Orrenius S. Biochem. Biophys. Res. Commun. 1999; 255: 6-11Crossref PubMed Scopus (179) Google Scholar, 6.Tan S. Sagara Y. Liu Y. Maher P. Schubert D. J. Cell Biol. 1998; 141: 1423-1432Crossref PubMed Scopus (652) Google Scholar). Oxidative stress also affects the process of apoptosis. For example, treatment of cellsin vitro with H2O2 causes either apoptosis or necrosis depending on the concentration of H2O2 employed and the type of cells being studied (7.Lennon S.V. Martin S.J. Cotter T.G. Cell Proliferation. 1991; 24: 203-214Crossref PubMed Scopus (761) Google Scholar, 8.Hampton M.B. Orrenius S. FEBS Lett. 1997; 414: 552-556Crossref PubMed Scopus (593) Google Scholar). 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Pharmacol. 1995; 133: 43-52Crossref PubMed Scopus (299) Google Scholar, 13.Czaja M.J. Flanders K.C. Biempica L. Klein C. Zern M.A. Weiner F.R. Growth Factors. 1989; 1: 219-226Crossref PubMed Scopus (80) Google Scholar) and in humans during alcohol-induced liver disease (14.McClain C.J. Cohen D.A. Hepatology. 1989; 9: 349-351Crossref PubMed Scopus (489) Google Scholar, 15.Yoshioka K. Kakumu S. Arao M. Tsutsumi Y. Inoue M. Hepatology. 1989; 10: 769-773Crossref PubMed Scopus (122) Google Scholar). TNF-α kills cancer cells in intact animals and a variety of cell lines in vitro by inducing these cells to undergo either apoptosis or necrosis. However, the biochemical basis for the cytotoxic action of TNF-α is still largely unknown. TNF-α has to been shown to increase production of ROS, and this appears to be an important step in its cytotoxic mechanism (16.Beyaert R. Fiers W. FEBS Lett. 1994; 340: 9-16Crossref PubMed Scopus (244) Google Scholar, 17.Goossens V. Grooten J. De Vos K. Fiers W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8115-8119Crossref PubMed Scopus (558) Google Scholar). Although some studies reported that antioxidants could protect against TNF-α toxicity (18.Zimmerman R.J. Chan A. Leadon S.A. Cancer Res. 1989; 49: 1644-1648PubMed Google Scholar, 19.Wong G.H. Elwell J.H. Oberley L.W. Goeddel D.V. Cell. 1989; 58: 923-931Abstract Full Text PDF PubMed Scopus (767) Google Scholar), there are other reports that antioxidants including catalase did not prevent TNF-α-mediated cell death (20.O'Donnell V.B. Spycher S. Azzi A. Biochem. J. 1995; 310: 133-141Crossref PubMed Scopus (157) Google Scholar). In a previous study (21.Bai J. Rodriguez A.M. Melendez J.A. Cederbaum A.I. J. Biol. Chem. 1999; 274: 26217-26224Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar), stable HepG2 cell lines overexpressing catalase in the cytosol or mitochondria were established by transfection with catalase cDNA or with a catalase cDNA with a manganese-superoxide dismutase mitochondrial leader sequence that could conduct catalase into mitochondria. We found that the cells overexpressing catalase in either cellular compartment were more resistant to H2O2-, menadione-, or antimycin A-induced toxicity and apoptosis compared with cells transfected with the empty plasmid vector. In view of the conflicting reports concerning the ability of antioxidants to prevent TNF-α toxicity, and since one major locus of TNF-α-induced oxidative stress appears to be the mitochondrial compartment, studies were carried out to investigate the sensitivity of HepG2 cells that overexpress catalase in the cytosol or mitochondria to apoptosis induced by TNF-α. To our surprise and in contrast to the decreased sensitivity to H2O2, menadione, or antimycin A, cells overexpressing catalase, especially in the mitochondrial compartment, displayed an increased sensitivity to TNF-α-induced apoptosis. Recombinant human TNF-α, cycloheximide (CHX), sodium azide, hydrogen peroxide (H2O2), horseradish peroxidase-conjugated goat anti-rabbit IgG, MEM, and fetal bovine serum were purchased from Sigma. Propidium iodide was purchased from Molecular Probes, Inc. (Eugene, OR). Bovine catalase, caspase inhibitor I, substrates of caspase-3 and -8, and polyclonal antibodies raised in rabbit against human caspase-3 or cytochrome cwere obtained from Calbiochem. Zeocin for selection was from Invitrogen (Carlsbad, CA). HepG2 cells overexpressing cytosolic catalase (C33 cells) and mitochondrial catalase (mC5 cells) as well as control cells (Hp cells) were established in our laboratory previously (21.Bai J. Rodriguez A.M. Melendez J.A. Cederbaum A.I. J. Biol. Chem. 1999; 274: 26217-26224Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar) by transfection with plasmid vector pZeoSV2(+) containing human catalase cDNA (pZeoSV-CAT), plasmid vector pZeoSV2(+) containing human catalase cDNA with a manganese-superoxide dismutase mitochondrial leader sequence (pZeoSV/MSP-CAT), and empty vector pZeoSV2(+) into HepG2 cells. Cells were cultured in MEM containing 10% fetal calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 300 μg/ml Zeocin, and 2 mm glutamine in a humidified atmosphere in 5% CO2 at 37 °C. The DNA fragmentation pattern (DNA ladder) was carried out by agarose gel electrophoresis. Cells (1× 106) treated with various reagents were scraped and centrifuged at 1200 rpm for 10 min. The cell pellet was resuspended in 1 ml of lysis buffer consisting of 10 mm Tris-HCl, pH 7.4, 10 mm NaCl, 10 mm EDTA, 100 μg/ml proteinase K, and 0.5% SDS and incubated for 2 h at 50 °C. DNA was extracted with 1 ml of phenol, pH 8.0, followed by extraction with 1 ml of phenol/chloroform (1:1) and chloroform. The aqueous phase was precipitated with 2.5 volumes of ice-cold ethanol and 0.1 volume of 3m sodium acetate, pH 5.2, at −20 °C overnight. The precipitates were collected by centrifugation at 13,000 ×g for 10 min. The pellets were air-dried and resuspended in 50 μl of Tris/EDTA buffer supplemented with 100 μg/ml RNase A. DNA was loaded onto a 1.5% agarose gel containing ethidium bromide, electrophoresed in Tris acetate/EDTA buffer for 2 h at 50 V, and photographed under UV illumination. Flow cytometry DNA analysis was used to quantify the percentage of apoptotic cells. Cells (5 × 105) were seeded onto six-well plates and incubated with various reagents. At different time points, cells were harvested by trypsinization and washed with PBS, followed by centrifugation at 2000 rpm for 10 min. The cell pellet was resuspended in 80% ethanol and stored at 4 °C for 24 h. Cells were washed twice with PBS. The pellet was resuspended in PBS containing 100 μg/ml RNase A, incubated at 37 °C for 30 min, stained with propidium iodide (50 μg/ml), and analyzed by flow cytometry DNA analysis as described previously (21.Bai J. Rodriguez A.M. Melendez J.A. Cederbaum A.I. J. Biol. Chem. 1999; 274: 26217-26224Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). Hp, C33, and mC5 cells treated as described in the figure legends were harvested by scraping from the dishes, washed with ice-cold PBS, and resuspended in lysis buffer containing 50 mm Hepes, pH 7.5, 10% sucrose, and 0.1% Triton X-100 on ice for 30 min. After centrifugation at 16,000 ×g for 20 min at 4 °C, supernatants were transferred to new tubes. Protein concentration was measured with the DC protein assay reagent (Bio-Rad). Caspase-3 activity was measured in the supernatant using the CaspACETM assay system kit (Promega) by following the cleavage of 50 μm acetyl-Asp-Glu-Val-Asp 7-amino-4-methylcoumarin. The fluorescence of the cleaved 7-amino-4-methylcoumarin substrate was determined using a fluorescence spectrophotometer (Perkin-Elmer 650-10S, Hitachi, Ltd.) set at an excitation wavelength of 360 nm and an emission wavelength of 460 nm. The same method was used to measure the activity of caspase-8 using 50 μm benzyloxycarbonyl-Ile-Glu-Thr-Asp 7-amino-4-trifluoromethylcoumarin as a substrate and 400 nm as the excitation wavelength and 505 nm as the emission wavelength. Activities are expressed as arbitrary units of fluorescence. To detect procaspase-3, cells treated with or without TNF-α plus CHX were washed twice with PBS, harvested by scraping, and subsequently sonicated at duty cycle 50% and output control 4 for 10 s (Heat Systems-Ultrasonics, Inc.). The sonicated suspensions were centrifuged at 10,000 × g for 10 min at 4 °C. The supernatant was transferred to a new tube, and the protein concentration was measured. To detect cytochrome c, a post-mitochondrial supernatant fraction was prepared by homogenization and differential centrifugation as described previously (21.Bai J. Rodriguez A.M. Melendez J.A. Cederbaum A.I. J. Biol. Chem. 1999; 274: 26217-26224Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). 50 μg (for procaspase-3) or 20 μg (for cytochromec) of denatured protein was resolved on 15% SDS-polyacrylamide gel and electroblotted onto nitrocellulose membranes (Bio-Rad). The membrane was incubated with rabbit anti-human caspase-3 (1:300) or rabbit anti-human cytochrome c (1:1000) polyclonal antibody as the primary antibody, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG as the secondary antibody (1:5000). Detection by the chemiluminescence reaction was carried out for 1 min using the ECL kit (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom). Cells treated with medium or with medium containing 15 ng/ml TNF-α for 15 or 30 min were harvested by scraping and centrifugation. The cell pellets were resuspended in 800 μl of buffer containing 10 mm Hepes, 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 1 mmdithiothreitol, and 0.5 mm phenylmethylsulfonyl fluoride. After incubation on ice for 15 min, 50 μl of 10% Nonidet P-40 was added; the lysed cells were mixed in a Vortex mixer for 10 s and then spun down (13,000 × g, 1 min, 4 °C); and the supernatant was removed. The nuclear pellet was quickly resuspended in 400 μl of buffer containing 20 mm Hepes, 400 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, and 1 mmphenylmethylsulfonyl fluoride. The nuclei were extracted on ice for 15 min with Vortex mixing every minute. The nuclei were then pelleted by centrifugation (13,000 × g, 5 min, 4 °C); the supernatant, considered as the nuclear extract, was removed; and the protein concentration was measured. The DNA binding reaction was performed using the gel shift assay system from Promega according to the instructions of the manufacturer. 2–5 μg of nuclear extract was incubated in gel shift binding buffer for 10 min at room temperature. NF-κB and AP-1 DNA probes (5′-AGT TGA GGG GAC TTT CCC AGG C-3′ and 5′-CGC TTG ATG AGT CAG CCG GAA-3′, respectively) were labeled with [γ-32P]ATP by T4 polynucleotide kinase. Approximately 10,000 cpm of32P-labeled DNA probe was then added to the nuclear extract in binding buffer and allowed to bind for 30 min. The reaction was then loaded onto a 4% nondenaturing acrylamide gel. After gel electrophoresis, the gels were dried and exposed to Kodak XAR5 film. Intracellular ATP levels were determined using the ENLITENTM ATP assay kit (Promega). Hp, C33, and mC5 cells (∼5 × 105 cells) were treated with or without TNF-α/CHX for 3 h. Cells were washed twice with ice-cold PBS, and 250 μl of ice-cold 2.5% (w/v) trichloroacetic acid was added to the six-well dishes. After scrapping from the dish, the cell extract was immediately centrifuged at 10,000 × g for 5 min at 4 °C. The supernatant was diluted 10 times and neutralized with Tris acetate buffer, pH 7.75. 10-μl samples and 100 μl of luciferin-luciferase reagent were used for ATP measurements according to the instructions of the manufacturer (Promega). Luminescence units were measured using a Model 1251 luminometer (LKB Wallac). The relative luminescence units were used as an index of the intracellular ATP levels. Results are expressed as means ± S.E. The numbers of experiments are indicated in the figure legends. Statistical evaluation was carried out using Student's t test. HepG2 cells were transfected with empty plasmid or plasmid containing human catalase cDNA or human catalase cDNA with a 80-base pair manganese-superoxide dismutase mitochondrial leader sequence, and stable cells were generated (21.Bai J. Rodriguez A.M. Melendez J.A. Cederbaum A.I. J. Biol. Chem. 1999; 274: 26217-26224Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). Catalase activity was increased from values of ∼40 units/mg of total cell protein in the cells transfected with empty vector (Hp cells) to values of 100–120 units/mg of cell protein in the cells transfected with catalase cDNA (C33 cells) or catalase cDNA with the mitochondrial leader sequence (mC5 cells). Isolated mitochondria from Hp or C33 cells displayed low catalase activity ( 2-fold higher in mC5 cells than in Hp cells. The only apparent difference between Hp cells and C33 or mC5 cells is the increased activity and content of catalase in the cytosol or mitochondria of the latter compared with the former. To validate that the increased sensitivity of C33 and mC5 cells to TNF-α is indeed due to overexpression of catalase, the effect of sodium azide on TNF-α/CHX-induced apoptosis and caspase activation was determined. Addition of sodium azide to a final concentration of 1 mmresulted in a 70% decrease in catalase activity in C33 and mC5 cells. As shown in Fig. 3 A, pretreating the cells with 1 mm sodium azide for 6 h, followed by incubation with 15 ng/ml TNF-α and 40 μmCHX for 6 h, resulted in an inhibition of the TNF-α-induced DNA ladder formation in all three cell lines (compare lanes 4,7, and 10 with lanes 3, 6, and 9). Similarly, sodium azide lowered the percentage of cells undergoing apoptosis in the presence of TNF-α/CHX (Fig.3 B). This prevention of apoptosis by sodium azide was observed in all three cell lines, and much of the increase in apoptosis found in C33 and mC5 cells was prevented by sodium azide (percent apoptosis induced by TNF-α/CHX was 16, 33, and 41% in Hp, C33, and mC5 cells in the absence of sodium azide and 8, 12, and 15% in its presence). The striking increase in caspase-3 activity induced by TNF-α/CHX in mC5 cells was also reduced by sodium azide (Fig.3 C). The increased sensitivity of C33 and mC5 cells to TNF-α/CHX and the prevention of TNF-α toxicity by sodium azide suggest that H2O2 may be a key modulator of sensitivity to TNF-α. To evaluate this, Hp, C33, and mC5 cells were incubated with 15 ng/ml TNF-α and 40 μm CHX in the absence or presence of 200 μmH2O2; and after 6 h, apoptosis was determined by the DNA fragmentation assay. H2O2completely inhibited apoptosis in Hp cells (Fig.4 A, lanes 3 and4) and partially inhibited apoptosis in C33 and mC5 cells (lanes 8 and 9 and lanes 13 and14, respectively). Adding catalase to the medium abolished this inhibitory effect of H2O2 and reestablished TNF-α-induced apoptosis (lanes 5,10, and 15). At this dosage and time, cells did not show significant necrosis caused by H2O2. The increase in caspase-3 activity produced by TNF-α/CHX in all three cell lines was largely prevented by 200 μmH2O2 (Fig. 4 B). H2O2 also produced inhibition of caspase-8 activity, which was slightly increased by addition of TNF-α/CHX; the inhibition of caspase-8 activity by H2O2 was less than the inhibition of caspase-3 activity (Fig. 4 B). The inhibition of TNF-α-induced caspase activation by H2O2 was confirmed by Western blot analysis of the levels of procaspase-3. Addition of TNF-α/CHX to Hp cells and especially to mC5 cells decreased the levels of procaspase-3 (Fig.4 C, compare lanes 2 and 6 withlanes 1 and 5), consistent with the cleavage of the procaspase form to the active caspase-3 fragments. H2O2 prevented the TNF-α-induced cleavage of procaspase-3 especially in Hp cells, which show the least sensitivity to TNF-α-induced apoptosis (lane 3). The activation of caspases such as caspase-3 in all three cell lines upon addition of TNF-α/CHX, the increased activation of caspase-3 in mC5 cells, and the inhibition by H2O2 of TNF-α-induced apoptosis and caspase-3 activation suggest that caspases play an important role in the developing apoptosis and in the different sensitivity of Hp, C33, and mC5 cells to TNF-α. This was validated by studying the effect of Z-VAD-fmk, an inhibitor of caspase-3 as well as several other caspases, on the TNF-α toxicity. At a final concentration of 50 μm, Z-VAD-fmk prevented the induction of apoptosis by TNF-α/CHX in all three cell lines (Fig. 4 A, comparelanes 6, 11, and 16 with lanes 3, 8, and 13), prevented the activation of caspase-3 by TNF-α/CHX in all three cell lines (Fig. 4 B), and prevented the TNF-α/CHX-induced cleavage of procaspase-3 (Fig.4 C, compare lane 4 with lane 2 andlane 8 with lane 6). Caspase-8 activity was not inhibited by Z-VAD-fmk at a concentration that strongly inhibited caspase-3 activity (Fig. 4 B). In summary, the results of Fig. 4 show that the TNF-α/CHX-induced apoptosis and activation of caspase-3 and the increased sensitivity of C33 cells and especially mC5 cells to TNF-α/CHX can be prevented by H2O2and by the caspase inhibitor Z-VAD-fmk. Release of cytochrome cfrom the mitochondria to the cytosol occurs in certain systems undergoing apoptosis (23.Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T.I. Jones D.P. Wang X. 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Increased release of cytochrome c from the mitochondria does not appear to be responsible for the increased caspase-3 activity and sensitivity to apoptosis of mC5 cells treated with TNF-α/CHX. Activation of oxidant-sensitive transcription factors such as NF-κB by TNF-α has been observed in many experimental models (26.Hu W.H. Johnson H. Shu H.B. J. Biol. Chem. 1999; 274: 30603-30610Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 27.Kohler H.B. Knop J. Martin M. de Bruin A. Huchzermeyer B. Lehmann H. Kietzmann M. Meier B. Nolte I. Vet. Immunol. Immunopathol. 1999; 71: 125-142Crossref PubMed Scopus (15) Google Scholar, 28.Xu Y. Kiningham K.K. Devalaraja M.N. Yeh C.C. Majima H. Kasarskis E.J. St. Clair D.K. DNA Cell Biol. 1999; 18: 709-722Crossref PubMed Scopus (203) Google Scholar). It is generally believed that activation of NF-κB and the subsequent activation of NF-κB-responsive genes are a cellular response to minimize the toxicity of TNF-α (29.Wang C.Y. Guttridge D.C. Mayo M.W. Baldwin Jr., A.S. Mol. Cell. Biol. 1999; 19: 5923-5929Crossref PubMed Scopus (543) Google Scholar, 30.Kitamura M. Eur. J. Immunol. 1999; 29: 1647-1655Crossref PubMed Scopus (38) Google Scholar, 31.Sumitomo M. Tachibana M. Nakashima J. Murai M. Miyajima A. Kimura F. Hayakawa M. Nakamura H. J. Urol. 1999; 161: 674-679Crossref PubMed Google Scholar). Could the increased sensitivity of C33 and mC5 cells to TNF-α reflect a failure to activate NF-κB in these cells (i.e. overexpression of catalase in C33 or mC5 cells minimizes TNF-α production of ROS and ROS activation of NF-κB (32.Schmidt K.N. Amstad P. Cerutti P. Baeuerle P.A. Chem. Biol. 1995; 2: 13-22Abstract Full Text PDF PubMed Scopus (431) Google Scholar))? We initially discounted differences in protective responses by NF-κB activation in the three cell lines because the presence of CHX, which was required for the TNF-α toxicity, would prevent synthesis of short-lived death antagonists; indeed, the requirement for CHX to observe TNF-α toxicity is likely due to the failure to synthesize protective factors. To study this further, the ability of TNF-α to activate NF-κB and AP-1 was evaluated by electrophoretic mobility gel shift assays. As shown in Fig.6 A, all three cell lines showed a very early response to TNF-α addition with respect to activation of NF-κB; increased binding to a NF-κB consensus sequence could be observed 15 min after addition of TNF-α. Similarly, all three cell lines showed a low activation of AP-1 binding, which increased 15 and 30 min after addition of TNF-α (Fig. 6 B). In general, activation of NF-κB and AP-1 by TNF-α appeared to be similar in the three cell lines. Importantly, this suggests that TNF-α binding and signal transduction are not significantly altered by the overexpression of catalase. ATP is necessary for cells to
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