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• W1979173881 abstract "Acrolein is a highly electrophilic α,β-unsaturated aldehyde to which humans are exposed in various situations. In the present study, the effects of sublethal doses of acrolein on nuclear factor κB (NF-κB) activation in A549 human lung adenocarcinoma cells were investigated. Immediately following a 30-min exposure to 45 fmol of acrolein/cell, glutathione (GSH) and DNA synthesis and NF-κB binding were reduced by more than 80%. All parameters returned to normal or supranormal levels by 8 h post-treatment. Pretreatment with acrolein completely blocked 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced activation of NF-κB. Cells treated for 1 h with 1 mmdiethyl maleate (DEM) showed a 34 and 53% decrease in GSH and DNA synthesis, respectively. DEM also reduced NF-κB activation by 64% at 2 h post-treatment, with recovery to within 22% of control at 8 h. Both acrolein and DEM decreased NF-κB function ∼50% at 2 h after treatment with TPA, as shown by a secreted alkaline phosphatase reporter assay. GSH returned to control levels by 8 h after DEM treatment, but proliferation remained significantly depressed for 24 h. Interestingly, DEM caused a profound decrease in NF-κB binding, even at doses as low as 0.125 mm that had little effect on GSH. Neither acrolein nor DEM had any effect on the levels of phosphorylated or nonphosphorylated inhibitor κB-α (IκB-α). Furthermore, acrolein decreased NF-κB activation in cells depleted of IκB-α by TPA stimulation in the presence of cycloheximide, demonstrating that the decrease in NF-κB activation was not the result of increased binding by the inhibitory protein. This conclusion was further supported by the finding that acrolein modified NF-κB in the cytosol prior to chemical dissociation from IκB with detergent. Together, these data support the conclusion that the inhibition of NF-κB activation by acrolein and DEM is IκB-independent. The mechanism appears to be related to direct modification of thiol groups in the NF-κB subunits. Acrolein is a highly electrophilic α,β-unsaturated aldehyde to which humans are exposed in various situations. In the present study, the effects of sublethal doses of acrolein on nuclear factor κB (NF-κB) activation in A549 human lung adenocarcinoma cells were investigated. Immediately following a 30-min exposure to 45 fmol of acrolein/cell, glutathione (GSH) and DNA synthesis and NF-κB binding were reduced by more than 80%. All parameters returned to normal or supranormal levels by 8 h post-treatment. Pretreatment with acrolein completely blocked 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced activation of NF-κB. Cells treated for 1 h with 1 mmdiethyl maleate (DEM) showed a 34 and 53% decrease in GSH and DNA synthesis, respectively. DEM also reduced NF-κB activation by 64% at 2 h post-treatment, with recovery to within 22% of control at 8 h. Both acrolein and DEM decreased NF-κB function ∼50% at 2 h after treatment with TPA, as shown by a secreted alkaline phosphatase reporter assay. GSH returned to control levels by 8 h after DEM treatment, but proliferation remained significantly depressed for 24 h. Interestingly, DEM caused a profound decrease in NF-κB binding, even at doses as low as 0.125 mm that had little effect on GSH. Neither acrolein nor DEM had any effect on the levels of phosphorylated or nonphosphorylated inhibitor κB-α (IκB-α). Furthermore, acrolein decreased NF-κB activation in cells depleted of IκB-α by TPA stimulation in the presence of cycloheximide, demonstrating that the decrease in NF-κB activation was not the result of increased binding by the inhibitory protein. This conclusion was further supported by the finding that acrolein modified NF-κB in the cytosol prior to chemical dissociation from IκB with detergent. Together, these data support the conclusion that the inhibition of NF-κB activation by acrolein and DEM is IκB-independent. The mechanism appears to be related to direct modification of thiol groups in the NF-κB subunits. nuclear factor κB inhibitor κB 12-O-tetradecanoylphorbol-13-acetate Dulbecco's modified Eagle's medium diethyl maleate Earl's balanced salt solution high pressure liquid chromatography secreted alkaline phosphatase cycloheximide β-mercaptoethanol Acrolein, an α,β-unsaturated aldehyde, is a highly electrophilic (1Witz G. Free Radical Biol. Med. 1989; 7: 333-349Crossref PubMed Scopus (327) Google Scholar), volatile liquid with a pungent and irritating odor. It is produced by a wide variety of both natural and synthetic processes including incomplete combustion or pyrolysis of organic materials such as fuels, wood, synthetic polymers, food, and tobacco. In addition, patients treated with the cytostatic agent cyclophosphamide are exposed to acrolein as a metabolite of the parent drug (2Sladek N.E. Pharmacol. Ther. 1988; 37: 301-355Crossref PubMed Scopus (310) Google Scholar). Extensive research has been done on the acute biochemical effects of acrolein. However, the effects of subacute exposures have been little studied, particularly at the molecular level. Recent work has shown that acrolein can inhibit cell proliferation at doses that do not cause lethality (3Horton N.D. Mamiya B.M. Kehrer J.P. Toxicology. 1997; 122: 111-122Crossref PubMed Scopus (52) Google Scholar), and such information may have major significance in terms of signal transduction pathways as well as, perhaps, in the control of cell division and apoptosis. As a metabolite of cyclophosphamide, acrolein may also play a role in the unique antineoplastic efficacy of this drug through molecular effects associated with low acrolein doses. Myriad adverse cellular effects are seen following exposure to acrolein, including growth inhibition, alterations in the levels of glutathione (GSH), protein sulfhydryls, and thiol-containing enzymes, and increased cell membrane permeability (4Francelyne M. Puiseux-Dao S. Toxicol. Lett. 1982; 14: 143-149Crossref PubMed Scopus (7) Google Scholar, 5Perry C.S. Liu X. Lund L.G. Whitman C.P. Kehrer J.P. Toxicol. In Vitro. 1995; 9: 21-26Crossref PubMed Scopus (15) Google Scholar, 6Cooper K.O. Witz G. Witmer C. Fundam. Appl. Toxicol. 1992; 19: 343-349Crossref PubMed Scopus (27) Google Scholar, 7Grafstrom R.C. Dypbukt J.M. Willey J.C. Sundqvist K. Edman C. Atzori L. Harris C.C. Cancer Res. 1988; 48: 1717-1721PubMed Google Scholar, 8Patel J.M. Block E.R. Toxicol. Appl. Pharmacol. 1993; 122: 46-53Crossref PubMed Scopus (54) Google Scholar). The primary source of acrolein's reactivity is its α,β-unsaturated carbon–carbon bond. This molecule will react via a Michael addition in the presence of a nucleophile to form an alkylated adduct. Acrolein's potential role as a carcinogen is based on the observation that it binds GSH (9Ohno Y. Ormstad K. Arch. Toxicol. 1985; 57: 99-103Crossref PubMed Scopus (43) Google Scholar) and nuclear chromatin (10Marano F. Demestere M. Experientia (Basel). 1976; 32: 501-502Crossref PubMed Scopus (7) Google Scholar) and can form a number of adducts with DNA (11Marinelli E.R. Hohnson F. Iden C.R. Yu P.L. Chem. Res. Toxicol. 1990; 3: 49-58Crossref PubMed Scopus (51) Google Scholar, 12Nath R.G. Ocando J.E. Chung F.L. Cancer Res. 1996; 56: 452-456PubMed Google Scholar, 13Smith R.A. Williamson D.S. Cerny R.L. Cohen S.M. Cancer Res. 1990; 50: 3005-3012PubMed Google Scholar). Some researchers have suggested that acrolein's antiproliferative effects may be the result of its binding to RNA polymerase, thereby serving as a transcriptional restraint (14Moulé Y. Frayssinet C. FEBS Lett. 1971; 16: 216-218Crossref PubMed Scopus (16) Google Scholar). However, the fact that GSH appears to play some role in cell division (15Atzori L. Dypbukt J.M. Hybbinette S.S. Moldeus P. Grafstrom R.C. Exp. Cell Res. 1994; 211: 115-120Crossref PubMed Scopus (22) Google Scholar, 16Kang Y.J. Enger M.D. Exp. Cell Res. 1990; 187: 177-179Crossref PubMed Scopus (39) Google Scholar) raises the possibility that acrolein-mediated alterations in this tripeptide may also be an important factor. Our previous data (3Horton N.D. Mamiya B.M. Kehrer J.P. Toxicology. 1997; 122: 111-122Crossref PubMed Scopus (52) Google Scholar, 17Ramu K. Perry C.S. Ahmed T. Pakenham G. Kehrer J.P. Toxicol. Appl. Pharmacol. 1996; 140: 487-498Crossref PubMed Scopus (43) Google Scholar) demonstrated that inhibiting the proliferation of human lung adenocarcinoma A549 cells with acrolein correlated with acrolein-induced changes in GSH. Although a cause-and-effect relationship between acrolein-induced changes in GSH and proliferation has not been shown, it is apparent that acrolein can alter redox-regulated cellular pathways. Nuclear factor-κB (NF-κB)1 is one of the most widely studied molecules affected by cellular redox status. It was first identified as a factor that activated the Ig κ-light chain intron enhancer during B-lymphocyte development (18Sen R. Baltimore D. Cell. 1986; 46: 705-716Abstract Full Text PDF PubMed Scopus (1901) Google Scholar). High levels of interest in this transcription factor are based on its broad role in coordinately controlling a number of genes including those encoding inflammatory cytokines, chemokines, interferons, proteins of the major histocompatibility complex, growth factors, cell adhesion molecules, and viruses (19Baeuerle P.A. Henkel T. Annu. Rev. Immunol. 1995; 12: 141-179Crossref Scopus (4563) Google Scholar). NF-κB, which comprises a 50- and 65-kDa heterodimer complex, is the prototype of a family of dimeric transcription factors consisting of monomers that have approximately 300-amino acid Rel regions that bind to DNA and interact with each other (20Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2910) Google Scholar). These factors are normally bound to a member of a family of inhibitory proteins known as inhibitor κB (IκB). The inhibitors all have 5–7 ankyrin repeat domains, each with approximately 30 amino acids, that form a unit able to interact with Rel regions. IκB-α, the best characterized member of this family, binds the p50/p65 heterodimer of NF-κB and retains it in the cytoplasm. The exposure of cells to NF-κB activators, including oxidants, cytokines (such as tumor necrosis factor-α or interleukin-1), or the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), causes phosphorylation of two serine residues of IκB-α (Ser32and Ser36). This phosphorylation is the signal for ubiquitination and degradation of IκB-α by the 26 S proteasome. NF-κB is then released and translocated to the nucleus where it can bind to κB sites on the DNA, thereby activating transcription of target genes (21Thanos D. Maniatis T. Cell. 1995; 80: 529-532Abstract Full Text PDF PubMed Scopus (1216) Google Scholar, 22Maniatis T. Science. 1997; 278: 818-819Crossref PubMed Scopus (233) Google Scholar). NF-κB is thought to be under redox control at two distinct levels. The activation and nuclear translocation of NF-κB involve reactive oxygen intermediates and can be blocked by reducing agents such asN-acetylcysteine and GSH (23Staal F.J.T. Roederer M. Herzenberg L.A. Herzenberg L.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9943-9947Crossref PubMed Scopus (884) Google Scholar, 24Pinkus R. Weiner L.M. Daniel V. J. Biol. Chem. 1996; 271: 13422-13429Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar). In contrast, the DNA-binding activity of NF-κB is inhibited by oxidative agents and potentiated by reducing thiols (25Mihm S. Galter D. Dröge W. FASEB J. 1995; 9: 246-252Crossref PubMed Scopus (152) Google Scholar, 26Kumar S. Rabson A.B. Gélinas C. Mol. Cell. Biol. 1992; 12: 3094-3106Crossref PubMed Google Scholar). These are likely the results of the requirement that cysteine residues present in the DNA-binding domain of all members of the Rel protein family be reduced to bind DNA (26Kumar S. Rabson A.B. Gélinas C. Mol. Cell. Biol. 1992; 12: 3094-3106Crossref PubMed Google Scholar). Acrolein's reactivity with nucleophiles suggests that it may interfere with NF-κB binding either by altering the redox balance of the nucleus or by forming adducts with NF-κB. In this study, we describe acrolein's attenuation of NF-κB activation in A549 human lung adenocarcinoma cells in a manner independent of IκB-α and consistent with the formation of acrolein-NF-κB conjugates. Dulbecco's modified Eagle's medium (DMEM), acrolein (90%; water and dimers make up the other 10%), diethyl maleate (DEM), and o-phthalaldehyde were obtained from Sigma. Fetal bovine serum was purchased from Summit Biotechnology (Fort Collins, CO). [3H]Thymidine (55 Ci/mmol) was obtained from ICN (Costa Mesa, CA). All antibodies were secured from Santa Cruz Biotechnology (Santa Cruz, CA) or New England Biolabs (Beverly, MA). The double-stranded NF-κB consensus oligonucleotide was purchased from Promega Corp. (Madison, WI). Phosphorylated and nonphosphorylated IκB control cell extracts were obtained from New England Biolabs. A549 human lung adenocarcinoma cells, obtained originally from the American Type Culture Collection (Manassas, VA), were cultured in DMEM (pH 7.4) supplemented with 10% (v/v) fetal bovine serum, 3.7 g/liter sodium bicarbonate, and 100 mg/liter gentamicin. Cells were maintained at 37 °C with 5% CO2. Cultures were passaged at confluency (approximately every 3 days) and were removed from monolayer stock cultures with trypsin-EDTA. Cells were counted with a T-890 Coulter counter (Miami, FL) and plated in either Falcon 6-well dishes (9.6 cm2/well) or Corning 10-cm tissue culture plates (55 cm2) with a medium volume of 2 ml/well and 10 ml/plate, respectively. Changes in cell growth were monitored by the uptake of [3H]thymidine. Cells were seeded 48 h before treatment. DEM was dissolved in 100% ethanol and added to culture dishes at an amount equivalent to 0.1% (v/v) of the medium. For treatment with acrolein, cells were washed twice in one volume/wash of Earl's balanced salt solution (EBSS). Cells were then incubated for 30 min at 37 °C with 5% CO2 in sterile EBSS containing the desired dose of acrolein. Incubation in EBSS was essential because of the reactivity of acrolein with components of DMEM (27Grafstrom R.C. Mutat. Res. 1990; 238: 175-184Crossref PubMed Scopus (54) Google Scholar). Following treatment, the cells were replenished with fresh DMEM + 10% fetal bovine serum. Additional washes to remove acrolein were not incorporated because any residual acrolein would rapidly react with nucleophiles present in the complete medium. The uptake of exogenous 3H-labeled thymidine was measured in cells treated with acrolein, DEM, or vehicle. Cells were pulsed for 2 h with 2.5 μCi/ml [3H]thymidine before isolating the DNA (28$$Google Scholar). DNA was quantitated by fluorescence after treatment with ethidium bromide (29Karsten U. Wollenberger A. Anal. Biochem. 1976; 77: 464-470Crossref Scopus (252) Google Scholar). Cell counts at the time of treatment (48 h post-seeding) were obtained using the CyQuant cell proliferation assay. This assay has a linear detection range of 50–50,000 cells/200 μl and is dependent on a green dye (CyQuant-GR) that fluoresces when bound to cellular nucleic acids. Cell monolayers were washed twice with phosphate-buffered saline, trypsinized, suspended in phosphate-buffered saline, and pelleted at 200 × g. The supernatant was carefully removed and the cells frozen at −80 °C. At the time of the assay, cells were thawed at room temperature and lysed in buffer containing the CyQuant-GR dye prepared according to manufacturer's instructions. Fluorescence was measured (excitation, 480 nm; emission, 520 nm) and compared with a standard curve for cell number determination. Previous work in our laboratory showed that acrolein treatment does not significantly alter the level of glutathione disulfide (17Ramu K. Perry C.S. Ahmed T. Pakenham G. Kehrer J.P. Toxicol. Appl. Pharmacol. 1996; 140: 487-498Crossref PubMed Scopus (43) Google Scholar). Therefore, only total glutathione (GSH + glutathione disulfide) was measured by HPLC (30Neuschwander-Tetri B.A. Roll F.J. Anal. Biochem. 1989; 179: 236-241Crossref PubMed Scopus (206) Google Scholar) or enzymatically (31Tietze F. Anal. Biochem. 1969; 27: 502-522Crossref PubMed Scopus (5488) Google Scholar). Briefly, cells were seeded at 5000 cells/cm2 in six-well plates and treated with acrolein or DEM. Cell monolayers were washed twice with PBS and lysed with 1 ml of 20 mm EDTA followed by sonication for 1 min. For HPLC analyses, 250 μl of the lysate were combined with 83 μl of 25 mm NaH2PO4, pH 7.0. Samples were then processed, derivatized with o-phthalaldehyde, and analyzed as described previously (17Ramu K. Perry C.S. Ahmed T. Pakenham G. Kehrer J.P. Toxicol. Appl. Pharmacol. 1996; 140: 487-498Crossref PubMed Scopus (43) Google Scholar). For the enzymatic assay, 100 μl of cell lysate were combined with 600 μl of 0.2 mKH2PO4 and 5 mm EDTA (pH 7.4) and analyzed. Total protein in the lysates was determined (32Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar) and compared with a bovine serum albumin standard curve. Electrophoretic mobility shift assays were carried out after the method of Denisonet al. (33Denison M.S. Fisher J.M. Whitlock J.P.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2528-2532Crossref PubMed Scopus (236) Google Scholar) as modified by Bowes et al. (34Bowes R.C. Weber T.J. Ramos K.S. Arch. Biochem. Biophys. 1995; 323: 243-250Crossref PubMed Scopus (13) Google Scholar). Briefly, cells were rinsed twice and lysed in ice-cold HEGD (25 mm HEPES, pH 7.6, 1.5 mm EDTA, 10% glycerol, 1 mm dithiothreitol, 0.1 mg/ml phenylmethylsulfonyl fluoride, 0.75 mm spermidine, 0.15 mm spermine) by homogenization. The homogenate was centrifuged at 12,000 ×g for 10 min at 4 °C. Experiments examining the activation of cytosolic latent NF-κB by detergents used the 12,000 × g supernatant fraction. Cytosol (4 μg of protein) was pretreated with 0.8% (w/v) sodium deoxycholate and 1.1% Nonidet P-40 for 10 min on ice before incubation with the labeled oligonucleotide probe (35Mahon T.M. O'Neill L.A.J. J. Biol. Chem. 1995; 270: 28557-28564Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). NF-κB was assessed using the 12,000 × g pellet extracted with 20 μl of HEGDK (HEGD + 0.5m KCl) for 1 h on ice. Extracted pellets were centrifuged at 16,000 × g for 10 min at 4 °C, and the supernatant containing the nuclear extracts was collected and assayed for protein content (32Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar). Extracts were frozen using liquid nitrogen and stored at −80 °C until analyzed. 5–20 μg of extracted protein were incubated in a reaction mixture consisting of 18.8 mm HEPES, 1.1 mm EDTA, 7.5% glycerol, 0.75 mm dithiothreitol, and 62.5 ng/ml poly(dI-dC) for 15 min at room temperature to reduce interference by nonspecific DNA-binding proteins. To determine NF-κB binding activity, 0.1 ng of NF-κB labeled with [γ-32P]ATP (3000–5000 Ci/mmol; NEN Life Science Products, Boston, MA) was added to the nuclear or cytosolic extracts for 15 min. The specificity of the binding reaction was assessed using unlabeled NF-κB, which competitively eliminated the induced band, or with an excess of a non-NF-κB competitor oligonucleotide, which was without effect. Bound NF-κB was separated from the free probe on a 4% polyacrylamide nondenaturing gel for 2 h at 120 V. Gels were dried under vacuum and exposed to Kodak XAR-5 film (Sigma) for 1–4 h at −80 °C with intensifying screens. Gels were also evaluated with a Packard Instant Imager and Packard imaging software (version 2.02, Packard Instrument Co.). A549 cells were transfected with the pNF-κB secreted alkaline phosphatase (SEAP) vector (CLONTECH Laboratories, Palo Alto, CA). Induction of the NF-κB pathway enables it to bind to the κ enhancer element located in the promoter region of the vector, thus activating transcription of the reporter gene and leading to increases in alkaline phosphatase activity in the culture medium. The alkaline phosphatase assay was done using the Great EscAPe SEAP fluorescence detection kit (CLONTECH) per the manufacturer's instructions. Conditions for transfection of A549 cells were optimized using FuGENE transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN). 2 μg of DNA were used for each transfection. 24 h post-transfection, the cells were washed with EBSS and treated with acrolein or DEM for 30 min as described previously. After treatment, cells were washed with EBSS, and fresh DMEM medium with fetal bovine serum containing 100 ng/ml TPA was added. 100 μl of media were collected after 2 h for the alkaline phosphatase assay and stored at −20 °C. Monolayer cells (106) were lysed in 300 μl of lysis buffer (10 mm Tris-HCl (pH 7.4), 10 mm NaCl, 3 mm MgCl2, 1 mm EDTA, 0.1% (v/v) Nonidet P-40, 100 μg/ml phenylmethylsulfonyl fluoride, 30 μl/ml aprotinin, and 1 mm sodium orthovanadate). The lysate was collected and incubated on ice for 15 min. Samples were centrifuged at 16,000 ×g for 10 min, and the supernatant was collected, assayed for protein (36Lowry O.H. Rosenbrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar), and stored at −20 °C. Thawed supernatants were mixed 1:3 with loading dye (4% (w/v) SDS, 20% (w/v) glycerol, 4% (w/v) β-mercaptoethanol, 0.2 m Tris-HCl (pH 6.8), and 0.02% (w/v) bromphenol blue) and separated on SDS-polyacrylamide gels (8–15%). Protein was transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA) and blocked from 1 h to overnight in 5% (w/v) nonfat dry milk (Bio-Rad) in TBS-T (25 mm Tris-HCl (pH 7.6), 0.2 m NaCl, and 0.15% Tween 20 (v/v)). Membranes were incubated with a polyclonal antibody specific for the protein of interest (1:1500 dilution in TBS-T) for 1 h. After washing in TBS-T, the membranes were rinsed and incubated with a horseradish peroxidase-conjugated secondary antibody (1:3000 dilution in TBS-T; Amersham Pharmacia Biotech) for 1 h. After the secondary antibody incubation, the membranes were rinsed with TBS-T, and bound antibodies were detected using enhanced chemiluminescence (ECL) with a kit from Amersham Pharmacia Biotech. Developed film was scanned, and individual band densities were integrated using NIH Image public domain software. Immunoblots following the various treatments were run a minimum of two times. Representative blots are shown in the figures. Data are expressed as means ± S.E. Comparisons between groups were done with a one-way analysis of variance followed by the Student-Newman-Keul's test. A pvalue of less than 0.05 was considered significant. Six-well plates were seeded at 5000 cells/cm2 (48,000 cells/well) and incubated 48 h before treatment for 30 min with 45 fmol of acrolein/cell (6.7 μm) in EBSS or for 1 h with 6.7 pmol of DEM/cell (1 mm). DNA synthesis was reduced to 30 and 63% of vehicle-treated cells 2 h after acrolein or DEM exposure, respectively (Table I). DNA synthesis in acrolein-treated cells recovered to supranormal levels by 8 h post-treatment, whereas growth in DEM-treated cells remained significantly suppressed, reaching only 54% of the level of growth in vehicle-treated cells (64% of control cells) at 24 h.Table I[3H]Thymidine incorporationTime45 fmol of acrolein/cellaAll values are significantly different from vehicle control (p < 0.05).1 mm DEMaAll values are significantly different from vehicle control (p < 0.05).h230 ± 1663 ± 2447 ± 169 ± 38130 ± 278 ± 22489 ± 154 ± 2Cells were seeded at 5000 cells/cm2 in six-well plates and incubated for 48 h at 37 °C in 5% CO2 before treatment. Following treatment with 45 fmol of acrolein/cell (30 min) or 1 mm DEM (1 h), cells were incubated for 2 h with medium containing 2.5 μCi/ml [3H]thymidine before harvesting. Data are expressed as the mean percent of [3H]thymidine incorporation relative to vehicle-treated cells ± S.E. (n = 3).a All values are significantly different from vehicle control (p < 0.05). Open table in a new tab Cells were seeded at 5000 cells/cm2 in six-well plates and incubated for 48 h at 37 °C in 5% CO2 before treatment. Following treatment with 45 fmol of acrolein/cell (30 min) or 1 mm DEM (1 h), cells were incubated for 2 h with medium containing 2.5 μCi/ml [3H]thymidine before harvesting. Data are expressed as the mean percent of [3H]thymidine incorporation relative to vehicle-treated cells ± S.E. (n = 3). Under slightly different conditions, we have shown previously that acrolein rapidly decreases total cellular GSH (3Horton N.D. Mamiya B.M. Kehrer J.P. Toxicology. 1997; 122: 111-122Crossref PubMed Scopus (52) Google Scholar). In the current study, cells were treated with 45 fmol of acrolein/cell or 6.7 pmol of DEM/cell. The level of GSH in acrolein-treated cells declined to 13% of that in vehicle-treated cells immediately following treatment and recovered to normal or supranormal levels by 8 h post-treatment (Fig.1). DEM-treated cells showed a smaller decline in GSH to 63% of the level in vehicle-treated cells with recovery again occurring by 8 h post-treatment (Fig. 1). Treating A549 cells with 35 fmol of acrolein/cell caused a significant decrease in NF-κB activation relative to TPA-treated or serum-deprived controls after as little as a 5-min exposure. This binding inhibition increased with the time of exposure. However, NF-κB activation in serum-deprived, vehicle-treated cells also began to decline at 2 h post-treatment (Fig. 2). To minimize the effects of serum deprivation, 30-min acrolein treatments were selected for further studies. With this length of treatment, both constitutive and TPA-stimulated NF-κB activation were inhibited by acrolein (Fig.2). Nonspecific binding was evident in this gel but did not correlate with either time or acrolein treatment. A time-response study of NF-κB activation in which cells were treated with 45 fmol of acrolein/cell for 30 min (Fig.3) showed inhibition and recovery patterns very much like those seen when examining changes in DNA synthesis (Table I) and total GSH (Fig. 1). Acrolein caused a dramatic decline in NF-κB binding at 30 min and at 2 h post-treatment. Some recovery of NF-κB activation was evident at 4 h, and the inhibitory effect of 30 min of acrolein treatment was fully reversed by 8 h post-treatment (Fig. 3). DEM also caused a decrease in NF-κB activation. There was a clear dose-response relationship with NF-κB binding increasingly reduced after 1-h exposures to DEM doses from 0.125 to 2 mm (Fig.4). Interestingly, the inhibition seen in NF-κB activation with 0.125 mm DEM was profound, yet little or no change in total GSH was evident at this dose (data not shown). Treating cells with 3.33 pmol of DEM/cell (1 mm) for 1 h resulted in a NF-κB activation time response (Fig.5) that was almost identical to that obtained following acrolein exposure (Fig. 3), and again paralleled changes in GSH (Fig. 1). NF-κB binding decreased dramatically at 1–2 h post-treatment and showed a recovery to near normal binding by 8–12 h. Nonspecific binding was more intense in this gel, but again did not correlate with either time or DEM treatment.Figure 5Time course of NF-κB binding after treatment with DEM. Cells were seeded at 10,000 cells/cm2 in 10-cm plates and incubated for 48 h at 37 °C in 5% CO2 before treatment. Nuclear fractions were harvested at the indicated times as measured from the beginning of treatment. 10 μg of total protein were loaded per lane. C, control; −, vehicle (0.1% (v/v) ethanol)-treated cells; +, DEM-treated cells (1 mm for 1 h).View Large Image Figure ViewerDownload (PPT) The SEAP reporter assay confirmed that both acrolein and DEM diminished the transcriptional activity of NF-κB. Two h after adding TPA to cells pretreated for 30 min with either 45 fmol/cell acrolein or 1 mm DEM, SEAP activity was decreased by 51 and 45%, respectively. Changes in NF-κB activation are generally controlled by IκB. In a number of different experiments, there were no consistent changes in the levels of IκB-α up to 2 h after cells were treated for 30 min with 45 fmol of acrolein/cell or for 1 h with 3.33 pmol of DEM/cell (Fig.6, A and B). To further examine this phenomenon, changes in the level of phosphorylated IκB-α were examined following treatment with acrolein. Once again, no changes in the levels of this protein were observed (Fig.6 C), suggesting that acrolein blocks NF-κB activation by an IκB-independent mechanism. A more thorough analysis of the possibility of IκB-independent changes in NF-κB activation involved examining the degradation of IκB following stimulation with TPA, a tumor promoter that up-regulates protein kinase C. Treatment with 100 ng/ml TPA caused a temporary decrease in the levels of IκB (Fig.7 A). By stimulating cells with TPA in the presence of the protein synthesis inhibitor cycloheximide (CHX), IκB levels were almost completely abrogated at 2 h post-treatment (Fig. 7 B). NF-κB activation was also checked at this time to ensure that the treatment with CHX had not affected NF-κB binding. Stimulating cells with TPA in the presence of CHX resulted in maximum NF-κB activation at 2 h post-treatment (Fig. 8), the same time that IκB levels were at their nadir. Finally, cells that had been stimulated with TPA for 1.5 h in the presence of CHX were treated with 45 fmol of acrolein/cell for 30 min. Under these conditions, acrolein still caused a 63% decline in NF-κB activation (Fig.9), indicating that the effect of acrolein was independent of IκB.Figure 8NF-κB binding in the presence of TPA and CHX. Cells were seeded at 10,000 cells/cm2 in 10-cm plates and incubated for 48 h at 37 °C in 5% CO2 before treatment. Cells were treated with 100 ng/ml TPA in the presence of 100 μg/ml CHX (2 h). Nuclear" @default.
• W1979173881 created "2016-06-24" @default.
• W1979173881 creator A5014341579 @default.
• W1979173881 creator A5041033681 @default.
• W1979173881 creator A5048650670 @default.
• W1979173881 creator A5069551927 @default.
• W1979173881 creator A5091803236 @default.
• W1979173881 date "1999-04-01" @default.
• W1979173881 modified "2023-10-18" @default.
• W1979173881 title "Acrolein Causes Inhibitor κB-independent Decreases in Nuclear Factor κB Activation in Human Lung Adenocarcinoma (A549) Cells" @default.
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