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• W1979980294 abstract "Oxidative stress induced by reactive oxygen intermediates often causes cell death via apoptosis, which is regulated by many functional genes and their protein products. The evolutionarily conserved protein Bcl-2 blocks apoptosis induced by a wide array of death signals. Despite extensive research, the molecular milieu that characterizes the anti-apoptotic function of Bcl-2 has not been fully clarified. In this work, we have investigated the role of bcl-2 in protecting against oxidative death induced by H2O2 in cultured rat pheochromocytoma PC12 cells. Transfection with the bcl-2 gene rescued PC12 cells from apoptotic death caused by H2O2. Addition of NF-κB inhibitors such as pyrrolidine dithiocarbamate and N-tosyl-l-phenylalanine chloromethyl ketone to the medium aggravated oxidative cell death. PC12 cells overexpressing bcl-2 exhibited relatively high constitutive DNA binding and transcriptional activities of NF-κB compared with vector-transfected control cells. Western blot analysis and immunocytochemistry revealed that bcl-2-transfected PC12 cells retained a higher level of p65 (the functionally active subunit of NF-κB) in the nucleus compared with vector-transfected controls. In addition, sustained activation of ERK1/2 (upstream of NF-κB) was observed in bcl-2-overexpressing cells. In contrast, the cytoplasmic inhibitor IκBα was present in lower amounts in cells overexpressing bcl-2. The ectopic expression of bcl-2 increased the cellular glutathione level and γ-glutamylcysteine ligase expression, which were attenuated by NF-κB inhibitors. These results suggest that NF-κB plays a role in bcl-2-mediated protection against H2O2-induced apoptosis in PC12 cells through augmentation of antioxidant capacity. Oxidative stress induced by reactive oxygen intermediates often causes cell death via apoptosis, which is regulated by many functional genes and their protein products. The evolutionarily conserved protein Bcl-2 blocks apoptosis induced by a wide array of death signals. Despite extensive research, the molecular milieu that characterizes the anti-apoptotic function of Bcl-2 has not been fully clarified. In this work, we have investigated the role of bcl-2 in protecting against oxidative death induced by H2O2 in cultured rat pheochromocytoma PC12 cells. Transfection with the bcl-2 gene rescued PC12 cells from apoptotic death caused by H2O2. Addition of NF-κB inhibitors such as pyrrolidine dithiocarbamate and N-tosyl-l-phenylalanine chloromethyl ketone to the medium aggravated oxidative cell death. PC12 cells overexpressing bcl-2 exhibited relatively high constitutive DNA binding and transcriptional activities of NF-κB compared with vector-transfected control cells. Western blot analysis and immunocytochemistry revealed that bcl-2-transfected PC12 cells retained a higher level of p65 (the functionally active subunit of NF-κB) in the nucleus compared with vector-transfected controls. In addition, sustained activation of ERK1/2 (upstream of NF-κB) was observed in bcl-2-overexpressing cells. In contrast, the cytoplasmic inhibitor IκBα was present in lower amounts in cells overexpressing bcl-2. The ectopic expression of bcl-2 increased the cellular glutathione level and γ-glutamylcysteine ligase expression, which were attenuated by NF-κB inhibitors. These results suggest that NF-κB plays a role in bcl-2-mediated protection against H2O2-induced apoptosis in PC12 cells through augmentation of antioxidant capacity. Oxidative stress refers to the mismatched redox equilibrium between the production of reactive oxygen intermediates (ROIs) 1The abbreviations used are: ROIs, reactive oxygen intermediates; TPCK, N-tosyl-l-phenylalanine chloromethyl ketone; PDTC, pyrrolidine dithiocarbamate; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DCF-DA, dichlorofluorescein diacetate; TMRE, tetramethylrhodamine ethyl ester; GCL, γ-glutamylcysteine ligase; GCLC, GCL catalytic subunit; GCLM, GCL modulatory subunit; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling; PBS, phosphate-buffered saline; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/ERK kinase; MEKK, MEK kinase.1The abbreviations used are: ROIs, reactive oxygen intermediates; TPCK, N-tosyl-l-phenylalanine chloromethyl ketone; PDTC, pyrrolidine dithiocarbamate; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DCF-DA, dichlorofluorescein diacetate; TMRE, tetramethylrhodamine ethyl ester; GCL, γ-glutamylcysteine ligase; GCLC, GCL catalytic subunit; GCLM, GCL modulatory subunit; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling; PBS, phosphate-buffered saline; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/ERK kinase; MEKK, MEK kinase. and the ability of the cell to defend against them. Oxidative stress thus occurs when the production of ROIs increases or when the elimination of ROIs or the repair of oxidatively damaged macromolecules decreases or both. There is a growing body of data indicating that ROIs are a major cause of cellular injuries in a variety of clinical abnormalities, including cancer, diabetes, rheumatoid arthritis, and neurodegenerative disorders (1Markesbery W.R. Free Radic. Biol. Med. 1997; 23: 134-147Crossref PubMed Scopus (1949) Google Scholar, 2Droge W. Physiol. Rev. 2002; 82: 47-95Crossref PubMed Scopus (7276) Google Scholar). Multiple lines of evidence support that ROIs can cause cell death via apoptosis (3Wood K.A. Youle R.J. Ann. N. Y. Acad. Sci. 1994; 17: 400-407Google Scholar, 4Jacobson M.D. Trends Biochem. Sci. 1996; 21: 83-86Abstract Full Text PDF PubMed Scopus (727) Google Scholar, 5Slater A.F. Nobel C.S. Orrenius S. Biochim. Biophys. Acta. 1995; 1271: 59-62Crossref PubMed Scopus (281) Google Scholar). The concentration of ROIs and the microenvironment appear to be important in determining the mode of cell death (6Kim H.-J. So Y.J. Jang J.-H. Lee J.-S. Oh Y.J. Surh Y.-J. Mol. Pharmacol. 2001; 60: 440-449PubMed Google Scholar). Cells undergoing apoptosis exhibit shrinkage of the nucleus, blebbing of membranes, condensation or fragmentation of chromatin, and internucleosomal DNA degradation by endonucleases into fragments in multiples of 180-200 bp. Apoptosis is a tightly regulated process that involves changes in the expression of a distinct set of genes (7Burlacu A. J. Cell. Mol. Med. 2003; 7: 249-257Crossref PubMed Scopus (384) Google Scholar, 8McConkey D.J. Toxicol. Lett. 1998; 99: 157-168Crossref PubMed Scopus (208) Google Scholar). One of the major genes responsible for regulating apoptotic cell death is the proto-oncogene bcl-2, which encodes a 26-kDa protein found in the nuclear envelope, parts of the endoplasmic reticulum, and the outer mitochondrial membrane. The bcl-2 gene product has been shown to prolong cell survival by blocking apoptosis induced by a wide array of death signals (9Zhong L.T. Sarafian T. Kane D.J. Charles A.C. Mah S.P. Edwards R.H. Bredesen D.E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4533-4537Crossref PubMed Scopus (607) Google Scholar, 10Bruce-Keller A.J. Begley J.G. Fu W. Butterfield D.A. Bredesen D.E. Hutchins J.B. Hensley K. Mattson M.P. J. Neurochem. 1998; 70: 31-39Crossref PubMed Scopus (172) Google Scholar, 11Lee M. Hyun D.H. Halliwell B. Jenner P. J. Neurochem. 2001; 78: 209-220Crossref PubMed Scopus (70) Google Scholar). In addition, Bcl-2 heterodimerizes with Bax to form a high molecular mass complex. Bcl-2 may prevent apoptosis through regulation of an antioxidant pathway and is considered to act as a free radical scavenger. For instance, the protein was found to inhibit lipid peroxidation and oxidative DNA and/or protein damage induced by diverse pro-apoptotic stimuli capable of triggering apoptosis (9Zhong L.T. Sarafian T. Kane D.J. Charles A.C. Mah S.P. Edwards R.H. Bredesen D.E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4533-4537Crossref PubMed Scopus (607) Google Scholar, 10Bruce-Keller A.J. Begley J.G. Fu W. Butterfield D.A. Bredesen D.E. Hutchins J.B. Hensley K. Mattson M.P. J. Neurochem. 1998; 70: 31-39Crossref PubMed Scopus (172) Google Scholar, 11Lee M. Hyun D.H. Halliwell B. Jenner P. J. Neurochem. 2001; 78: 209-220Crossref PubMed Scopus (70) Google Scholar). Furthermore, induction or overexpression of bcl-2 is thought to confer resistance to oxidant injury (12Godley B.F. Jin G.F. Guo Y.S. Hurst J.S. Exp. Eye Res. 2002; 74: 663-669Crossref PubMed Scopus (47) Google Scholar, 13Chau Y.P. Shiah S.G. Don M.J. Kuo M.L. Free Radic. Biol. Med. 1998; 24: 660-670Crossref PubMed Scopus (109) Google Scholar, 14Amstad P.A. Liu H. Ichimiya M. Berezesky I.K. Trump B.F. Buhimschi I.A. Gutierrez P.L. Redox Rep. 2001; 6: 351-362Crossref PubMed Scopus (51) Google Scholar). The ubiquitous eukaryotic transcription factor NF-κB/Rel is known to regulate expression of numerous cellular genes that play important roles in mediating/regulating immune and stress responses, inflammation, apoptosis, and proliferation (15Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4563) Google Scholar, 16Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2910) Google Scholar). Recent studies have revealed that NF-κB is involved in regulating cell survival. Overexpression of NF-κB increases cell viability by suppressing induction of apoptosis in various cell types (17Giri D.K. Aggarwal B.B. J. Biol. Chem. 1998; 273: 14008-14014Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 18Bian X. Opipari Jr., A.W. Ratanaproeksa A.B. Boitano A.E. Lucas P.C. Castle V.P. J. Biol. Chem. 2002; 277: 42144-42150Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 19Poulaki V. Mitsiades C.S. Joussen A.M. Lappas A. Kirchhof B. Mitsiades N. Am. J. Pathol. 2002; 161: 2229-2240Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Cells that become resistant to oxidative cell death exhibit constitutive activation of NF-κB as an adaptive defense mechanism (20Lezoualc'h F. Sagara Y. Holsboer F. Behl C. J. Neurosci. 1998; 18: 3224-3232Crossref PubMed Google Scholar, 21Kim D.K. Cho E.S. Lee B.R. Um H.D. Free Radic. Biol. Med. 2001; 30: 563-571Crossref PubMed Scopus (32) Google Scholar). Conversely, inhibitors of NF-κB activity such as SN50, N-tosyl-l-phenylalanine chloromethyl ketone (TPCK), and pyrrolidine dithiocarbamate (PDTC) attenuate the survival of neurons, supporting that NF-κB is required for neuronal protection (18Bian X. Opipari Jr., A.W. Ratanaproeksa A.B. Boitano A.E. Lucas P.C. Castle V.P. J. Biol. Chem. 2002; 277: 42144-42150Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 20Lezoualc'h F. Sagara Y. Holsboer F. Behl C. J. Neurosci. 1998; 18: 3224-3232Crossref PubMed Google Scholar, 22Taglialatela G. Robinson R. Perez-Polo J.R. J. Neurosci. Res. 1997; 47: 155-162Crossref PubMed Scopus (156) Google Scholar, 23Koulich E. Nguyen T. Johnson K. Giardina C. D'mello S. J. Neurochem. 2001; 76: 1188-1198Crossref PubMed Scopus (98) Google Scholar). However, the molecular mechanisms underlying the anti-apoptotic effect of NF-κB have not been clarified. In this work, we investigated whether Bcl-2 can protect against H2O2-induced apoptosis through activation of NF-κBin cultured rat pheochromocytoma PC12 cells. For this purpose, we compared the extent of NF-κB activation and levels of antioxidative defense capacity, especially glutathione metabolism, in bcl-2-transfected and vector-treated control cells to link Bcl-2 and the NF-κB signaling pathways in the context of their commitment to cellular protection against oxidative insults. Chemical and Biochemical Reagents—Poly-d-lysine, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), PDTC, and TPCK were purchased from Sigma. H2O2 was a product of Junsei Chemical Co., Ltd. (Tokyo, Japan). Dulbecco's modified Eagle's medium, Hanks' balanced salt solution, fetal bovine serum, horse serum, Geneticin (G418), nutrient mixture F-12, and N-2 supplement were provided by Invitrogen. Dichlorofluorescein diacetate (DCF-DA), 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide (JC-1), and tetramethylrhodamine ethyl ester (TMRE) were obtained from Molecular Probes, Inc. (Eugene, OR). The in situ cell death detection kit was supplied by Roche Diagnostics (Mannheim, Germany). The NF-κB consensus oligonucleotide and the luciferase assay kit with reporter lysis buffer were purchased from Promega (Madison, WI). [γ-32P]ATP was the product of PerkinElmer Life Sciences. The NF-κB and γ-glutamylcysteine ligase (GCL) promoter-luciferase constructs were kindly provided by Dr. Young Mi Kim (University of Ulsan Medical School, Seoul, Korea) and Dr. Shelly C. Lu (University of Southern California School of Medicine), respectively. Cell Culture—PC12 cells transfected with a eukaryotic expression vector containing the human cytomegalovirus major immediate-early enhancer/promoter followed by a full-length human Bcl-2 cDNA sequence were kindly provided by Dr. Young J. Oh (Yonsei University, Seoul) and maintained in our laboratory. Briefly, DNA transfection was performed with 1 × 105 PC12 cells cultivated on poly-d-lysine-coated 100-mm Petri dishes by adding a transfection mixture of 2 μg of plasmid/10 μl of LipofectAMINE (Invitrogen) in Dulbecco's modified Eagle's medium. Subsequently, single neomycin-resistant colonies were selected and expanded in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 5% horse serum, and 500 μg/ml G418. Stable PC12 cell lines overexpressing bcl-2 were characterized by immunoblot analysis. PC12 cells transfected with a eukaryotic expression vector without a human bcl-2 cDNA sequence were utilized as a control cell line. The subsequent cultures were conducted as reported previously (6Kim H.-J. So Y.J. Jang J.-H. Lee J.-S. Oh Y.J. Surh Y.-J. Mol. Pharmacol. 2001; 60: 440-449PubMed Google Scholar). Determination of Cell Viability—PC12 cells were plated at a density of 4 × 104 cells/300 μl in 48-well plates, and cell viability was determined using the conventional MTT reduction assay. After incubation, cells were treated with MTT solution (1 mg/ml final concentration) for 2 h. The dark blue formazan crystals formed in intact cells were solubilized with lysis buffer (20% SDS in 50% aqueous N,N-dimethylformamide), and absorbance at 540-595 nm was measured with a microplate reader (Molecular Devices, Inc., Sunnyvale, CA). Results are expressed as percent MTT reduction. Terminal Deoxynucleotidyltransferase-mediated dUTP Nick End Labeling (TUNEL)—The commercially available in situ death detection kit was utilized to assess DNA fragmentation. PC12 cells (5 × 105 cells/3 ml on a chamber slide) were fixed for 30 min in 10% neutral buffered formalin solution at room temperature. Endogenous peroxidase was inactivated by incubation with 0.3% hydrogen peroxide in methanol for 30 min at room temperature and further incubated in a permeabilizing solution (0.1% sodium citrate and 0.1% Triton X-100) for 2 min at 4 °C. The cells were incubated with the TUNEL reaction mixture for 60 min at 37 °C, followed by labeling with peroxidaseconjugated anti-goat antibody (Fab fragment) for an additional 30 min. The cells were rinsed with phosphate-buffered saline (PBS) and examined under a confocal microscope (Leica Microsystems Heidelberg GmbH, Heidelberg, Germany) with excitation at 488 nm and emission at 525 nm. Western Blot Analysis—Treated cells (1 × 107 cells/7 ml in a 100-mm dish) were collected and washed with PBS. After centrifugation, cell lysis was carried out at 4 °C by vigorous shaking for 15 min in radio-immune precipitation assay buffer (150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mm Tris-HCl (pH 7.4), 50 mm glycerophosphate, 20 mm NaF, 20 mm EGTA, 1 mm dithiothreitol, 1 mm Na3VO4, and protease inhibitors). After centrifugation at 23,000 × g for 15 min, the supernatant was separated and stored at -70 °C until used. The protein concentration was determined using the BCA protein assay kit (Pierce). After addition of sample loading buffer, protein samples were electrophoresed on a 12.5% SDS-polyacrylamide gel. Proteins were transferred to polyvinylidene difluoride blots at 300 mA for 3 h. The blots were blocked for 1 h at room temperature in fresh blocking buffer (0.1% Tween 20 in Tris-buffered saline (pH 7.4) containing 5% nonfat dry milk). Dilutions (1:1000) of anti-Bcl-2, anti-Bax, anti-poly-(ADP-ribose) polymerase, anti-IκBα, anti-p65, anti-phospho-ERK, anti-ERK, anti-phospho-JNK, anti-JNK, anti-phospho-p38, and anti-p38 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); anti-cleaved caspase-3 and anti-phospho-IκBα (Cell Signaling Technology, Inc., Beverly, MA); and anti-actin (Sigma) primary antibodies were made in PBS with 3% nonfat dry milk. The blots were incubated overnight at 4 °C. Following three washes with 0.1% Tween 20 in PBS (PBST), the blots were incubated with horseradish peroxidase-conjugated secondary antibodies in PBS with 3% nonfat dry milk for 1 h at room temperature. The blots were washed again three times with 0.1% PBST, and transferred proteins were incubated with ECL substrate solution (Amersham Biosciences) for 1 min according to the manufacturer's instructions and visualized with x-ray film. Measurement of the Mitochondrial Transmembrane Potential—To measure the mitochondrial membrane potential (Δψm), the lipophilic cationic probes JC-1 and TMRE were used. The green fluorescent JC-1 probe exists as a monomer at low membrane potential. However, at higher potential, JC-1 forms red fluorescent J-aggregates that exhibit a broad excitation spectrum. Following treatment with H2O2 for 6 h, cells (5 × 105 cells/3 ml on a chamber slide) were rinsed with PBS, and JC-1 (10 μg/ml) was loaded. After a 20-min incubation at 37 °C, cells were examined under a confocal microscope with excitation at 488 nm and emission at 530/590 nm. Determination of Δψm was also carried out using TMRE, which rapidly equilibrates between cellular compartments due to potential differences. Thus, a decrease in fluorescence is indicative of reduced Δψm. Following the incubation, the cells were treated with TMRE (150 nm) for 30 min, rinsed, and examined by confocal microscopy in the same manner as done for JC-1, except that the TMRE fluorescence was measured at 590 nm. Measurement of Intracellular ROI Accumulation—To monitor net intracellular accumulation of ROIs, the fluorescent probe DCF-DA was used. After treatment with H2O2 for 30 min, cells (1 × 106 cells/3 ml in 6-well plates) were rinsed with Krebs-Ringer phosphate solution, and 10 μm DCF-DA was loaded. Following an additional incubation for 15 min at 37 °C, cells were examined under a confocal microscope equipped with an argon laser (488 nm, 200 milliwatts). Preparation of Nuclear Proteins—After treatment with H2O2, cells (1 × 107 cells/7 ml in a 100-mm dish) were washed with PBS, centrifuged, and resuspended in ice-cold isotonic buffer (10 mm HEPES (pH 7.9), 1.5 mm MgCl2, 10 mm KCl, 0.5 mm dithiothreitol, and 0.2 mm phenylmethylsulfonyl fluoride). After incubation in an ice bath for 10 min, cells were centrifuged again and resuspended in ice-cold buffer containing 20 mm HEPES (pH 7.9), 20% glycerol, 420 mm NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 0.5 mm dithiothreitol, and 0.2 mm phenylmethylsulfonyl fluoride, followed by incubation at 0 °C for 20 min. After Vortex mixing, the resulting suspension was centrifuged, and the supernatant was stored at -70 °C for the NF-κB DNA binding assay. The protein concentration was determined using the BCA protein assay kit. Electrophoretic Mobility Shift Assay—A synthetic double-strand oligonucleotide harboring the NF-κB-binding domain was labeled with [γ-32P]ATP using T4 polynucleotide kinase and separated from unincorporated [γ-32P]ATP by gel filtration using a nick spin column (Amersham Biosciences). Prior to addition of the γ-32P-labeled oligonucleotide (100,000 cpm), 10 μg of the nuclear extract was kept on ice for 15 min in gel shift binding buffer (4% glycerol, 1 mm EDTA, 1 mm dithiothreitol, 100 mm NaCl, 10 mm Tris-HCl (pH 7.5), and 0.1 mg/ml sonicated salmon sperm DNA). DNA-protein complexes were resolved by 6% nondenaturing PAGE at 200 V for 2 h, followed by autoradiography. Immunocytochemical Staining for p65 and Phospho-ERK—To detect the activation of p65 and ERK, we have adopted the immunocytochemical method with a monoclonal antibody recognizing p65 or phospho-ERK. Cells (105 cells/1 ml on a chamber slide) were fixed in 10% neutral buffered formalin solution for 30 min at room temperature. After rinsing with PBS, cells were blocked for 1 h at room temperature in fresh blocking buffer (0.5% PBST, pH 7.4) containing 10% normal goat serum). Dilutions (1:100) of primary antibodies were made in 0.1% PBST with 1% bovine serum albumin, and cells were incubated overnight at 4 °C. Following two washes with 0.1% PBST, the cells were incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG secondary antibody in 0.1% PBST with 3% bovine serum albumin for 1 h at room temperature. Cells were rinsed with PBS, and stained cells were analyzed under a confocal microscope and photographed. Transient Transfection and Luciferase Assay—One day before transfection, PC12 cells were subcultured at a density of 1 × 106 cells/60-mm dish to maintain ∼60-80% confluency. They were transiently transfected with the NF-κB or GCL promoter-luciferase construct using the transfection reagent N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium (Roche Diagnostics) according to the instructions supplied by the manufacturer. After overnight transfection, the treated cells were harvested and lysed with reporter lysis buffer (Promega luciferase assay system). The cell extract (20 μl) was mixed with 100 μl of the luciferase assay reagent and analyzed with a luminometer (AutoLumat LB 953, EG&G Berthold, Bad Widbad, Germany). The β-galactosidase assay (Promega β-galactosidase enzyme assay system) was done according to the supplier's instructions to normalize the luciferase activity. Assessment of Intracellular GSH Levels—The intracellular GSH levels were assessed using the commercially available colorimetric assay kit BIOXYTECH GSH-400 (OXIS Research, Portland, OR). Cells were harvested and homogenized in meta-phosphoric acid working solution. After centrifugation, 50 μl of R1 solution (solution of the chromogenic reagent in HCl) was added to the 700-μl supernatant, followed by gentle Vortex mixing. Following addition of 50 μl of R2 solution (30% NaOH), the mixtures were incubated at 25 ± 3 °C for 10 min. After centrifugation, the absorbance of the clear supernatant was read at 400 nm. The protein concentration was determined using the BCA protein assay kit. Reverse Transcription-PCR—Total RNA was lysed, extracted with TRIzol (Invitrogen), and converted to cDNA by Moloney murine leukemia virus reverse transcriptase (Promega) according to the manufacturer's instructions. Reverse transcription-PCR was performed following standard procedures. Specific DNA sequences were amplified with a PCR mixture (HyMed, Seoul). Each PCR primer used in this study was as follows: GCL catalytic subunit (GCLC), 5′-GCC AAG GTC ATCCAT GAC AAC-3′ (sense) and 5′-AGT GTA GCC CAG GAT GCC CTT-3′ (antisense); GCL modulatory subunit (GCLM), 5′-AGA CCG GGA ACC TGC TCA AC-3′ (sense) and 5′-CAT CAC CCT GAT GCC TAA GC-3′ (antisense); glyceraldehyde-3-phosphate dehydrogenase, 5′-AGT GTA GCC CAG GAT GCC CTT-3′ (sense) and 5′-GCC AAG GTC ATC CAT GAC AAC-3′ (antisense). The reaction conditions were 25 cycles at 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 45 s. After amplification, the products were resolved by electrophoresis on 1.0% agarose gel, stained with ethidium bromide, and photographed under ultraviolet light. bcl-2-Overexpressing PC12 Cells Are Less Susceptible to the Cytotoxic Effects of H2O2—To address whether ectopic expression of Bcl-2 can protect against H2O2-induced cell death, PC12 cells were stably transfected with a plasmid harboring the bcl-2 gene. The effects of bcl-2 overexpression on cell survival following exposure to H2O2 were then examined by the MTT reduction assay. As illustrated in Fig. 1A, bcl-2 overexpression rescued PC12 cells from H2O2-induced cytotoxicity. Bcl-2 Overexpression Attenuates Apoptotic Cell Death Induced by H2O2—One of the distinctive biochemical hallmarks of apoptotic cell death is the unique occurrence of internucleosomal DNA fragmentation. Apoptotic cells were confirmed by TUNEL staining, which is widely used to detect DNA fragmentation in situ. Bcl-2 overexpression reduced H2O2-induced DNA fragmentation as revealed by the decreased proportion of TUNEL-positive cells (Fig. 1B). H2O2 treatment also led to the increased expression of Bax, activation of caspase-3, and cleavage of poly(ADP-ribose) polymerase, which were attenuated in bcl-2-overexpressing cells (Fig. 1C). bcl-2 Mitigates the Dissipation of the Mitochondrial Transmembrane Potential and Intracellular Accumulation of Hydroperoxide in H2O2-treated PC12 Cells—When PC12 cells were exposed to H2O2 (250 μm), the mitochondrial membrane became rapidly depolarized, as shown by an increase in green fluorescence and the concomitant disappearance of red fluorescence derived from the JC-1 dye (Fig. 2A, panels a and c). Bcl-2 overexpression reduced the changes in mitochondrial membrane transition (Δψm) as indicated by repression of green fluorescence and restoration of red fluorescence (Fig. 2A, panels b and d). These findings were further supported by use of another voltage-sensitive dye, TMRE. Again, H2O2-induced dissipation of Δψm (Fig. 2A, panel e) was found to be blocked by bcl-2 overexpression (panel f). Accumulation of intracellular hydroperoxide was detected by use of DCF-DA, which is freely permeable to cell membranes. Once inside cells, the compound is hydrolyzed by an esterase activity to DCF and trapped intracellularly. DCF is then able to interact with peroxides to form fluorescent 2′,7′-dichlorofluorescin, which is readily detectable by confocal microscopy. The activation of DCF is relatively specific for the detection of H2O2 and secondary or tertiary peroxides such as lipid peroxides. H2O2-derived intracellular peroxide accumulation decreased in PC12 cells overexpressing bcl-2 (Fig. 2B). NF-κB Inhibitors Render PC12 Cells More Vulnerable to H2O2-induced Cell Death—NF-κB activation in PC12 cells was assessed by electrophoretic mobility shift assay with an oligonucleotide harboring a consensus NF-κB-binding element. Treatment of PC12 cells with H2O2 (100, 250, and 500 μm) caused a concentration-dependent increase in NF-κB DNA binding activity in these cells (Fig. 3A). PDTC, an antioxidant reported to effectively block the IκB degradation pathway (24Schreck R. Meier B. Mannel D.N. Droge W. Baeuerle P.A. J. Exp. Med. 1992; 175: 1181-1194Crossref PubMed Scopus (1439) Google Scholar, 25Chung K.C. Park J.H. Kim C.H. Lee H.W. Sato N. Uchiyama Y. Ahn Y.S. J. Neurosci. Res. 2000; 59: 117-125Crossref PubMed Scopus (76) Google Scholar), reduced the DNA binding activity of NF-κB in a concentration-dependent manner (Fig. 3B). To examine the possible role of NF-κB in protecting against H2O2-induced cytotoxicity, cells were exposed to H2O2 for 9 h in the absence and presence of PDTC (10 μm) or another NF-κB inhibitor, TPCK (5 μm), and cell viability was assessed by the MTT reduction assay. Both PDTC (Fig. 3C) and TPCK (Fig. 3D) exacerbated the H2O2-induced cytotoxicity. Neither of these NF-κB inhibitors exhibited apparent toxicity to PC12 cells at the concentrations used in this experiment. Ectopic Expression of Bcl-2 Leads to Constitutive Activation of NF-κB through Stimulating the Degradation of Cytoplasmic IκBα—In PC12 cells, the DNA binding activity of NF-κB was transiently enhanced by H2O2 treatment (Fig. 4A). PC12 cells overexpressing bcl-2 exhibited relatively high levels of constitutively activated NF-κB compared with vector-transfected control cells (Fig. 4A). Since the NF-κB DNA binding activity is largely regulated by IκBα, which sequesters NF-κB in the cytoplasm, we determined whether the observed increase in nuclear NF-κB binding activity in bcl-2-overexpressing PC12 cells is due to increased IκBα degradation. Protein extracts of both the nucleus and cytoplasm were subjected to Western blot analysis to measure p65, IκBα, or phospho-IκBα. Cytoplasmic IκBα levels were profoundly reduced, whereas phospho-IκB and nuclear p65 levels were constitutively increased in PC12 cells overexpressing bcl-2 compared with control cells (Fig. 4B). We also verified the nuclear accumulation of p65 by immunocytochemistry using anti-p65 antibody (Fig. 4C). The transcriptional activity of NF-κB was also constitutively increased in bcl-2-transfected cells as assessed using an NF-κB-reporter plasmid containing the consensus NF-κB-binding DNA site linked to a luciferase reporter gene (pELAM-Luc). As illustrated in Fig. 4D, the base-line transcriptional activity of NF-κB was found to be approximately six times higher in bcl-2-transfected cells than in vector-transfected control cells. To elucidate a molecular target for NF-κB-mediated potentiation of cellular defense against oxidative insult in bcl-2-overexpressing cells, we examined the effect of GSH on H2O2-induced cell death. GSH, a ubiquitous tripeptide thiol, is a vital intra- and extracellular antioxidant against oxidative stress. N-Acetyl-l-cysteine undergoes a rapid deacetylation in cells and provides a rate-limiting amino acid (cysteine) needed for the intracellular synthesis of GSH, thereby replenishing the depleted levels of GSH. Pretreatment with GSH o" @default.
• W1979980294 created "2016-06-24" @default.
• W1979980294 creator A5051630057 @default.
• W1979980294 creator A5061532156 @default.
• W1979980294 date "2004-09-01" @default.
• W1979980294 modified "2023-10-17" @default.
• W1979980294 title "Bcl-2 Attenuation of Oxidative Cell Death Is Associated with Up-regulation of γ-Glutamylcysteine Ligase via Constitutive NF-κB Activation" @default.
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