Toxicological Sciences 59, 75-81 (2001)
Copyright © 2001 by the Society of Toxicology
Carcinogenicity |
The Activity of NF-
B in Swiss 3T3 Cells Exposed to Aqueous Extracts of Cigarette Smoke Is Dependent on Thioredoxin
INBIFO Institut für biologische Forschung, Fuggerstr.3, D-51149 Köln, Germany
Received August 17, 2000; accepted September 21, 2000
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ABSTRACT |
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Multiple studies in vitro have demonstrated that aqueous extracts of mainstream cigarette smoke (CS) [smoke-bubbled phosphate-buffered saline (PBS)] induce a distinct pattern of stress response in cultured cells, which may be related to the reported pro-inflammatory activities of CS in vitro and in vivo. Nuclear factor
B (NF-B) is a transcription factor involved in both inflammatory and stress-dependent cell-signaling processes. Here we report on the activity of NF-B in cells exposed to subcytotoxic concentrations of smoke-bubbled PBS. Using electrophoretic mobility shift assay (EMSA) techniques, we observed a decreased DNA binding of NF-B during the first 2 h of exposure, which was followed by a more than 2-fold increase over controls after 4 to 6 h of exposure. This type of kinetics is not regulated by IB-
, as evidenced by the lack of phosphorylation and degradation of IB- in CS-treated cells. However, as demonstrated in immuno-coprecipitation experiments, the kinetics of NF-B DNA binding is strictly paralleled by decreased and increased complex formation between NF-B and thioredoxin (Trx), the reducing catalyst of Cys-62 of NF-B subunit p50, the reduced thiol function of which is essential for efficient NF-B DNA binding. Monitoring the expression of the gene encoding thioredoxin reductase (TrxR), which is required to keep Trx in a functional reduced state, we observed a significant increase in TrxR mRNA after 2 to 6 h of exposure. Based on the correspondence between the kinetics of NF-B DNA binding, NF-B/Trx complex formation, and TrxR expression, along with a lack of IB- phosphorylation and degradation, these results suggest that the activity of NF-B in CS-treated cells is subject mainly to a redox-controlled mechanism dependent on the availability of reduced Trx rather than being controlled by its normal regulator, IB-.
Key Words: cigarette smoke; NF-B; thioredoxin; thiol oxidation; glutathione.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It is widely accepted that cigarette smoke (CS) is a risk factor in cancer development (Doll and Peto, 1981
; Peto et al., 1994). However, less is known about the underlying mechanisms, particularly in terms of the contribution of water-soluble compounds in CS. Several studies have demonstrated that aqueous extracts of CS [smoke-bubbled phosphate-buffered saline (PBS)] induce a distinct pattern of (oxidative) stress-related effects in exposed cells (Cosgrove et al., 1985; Müller, 1995; Müller and Gebel, 1994; Nakayama et al., 1985), e.g., as reflected by the formation of DNA strand breaks and by the expression of stress response genes such as heme oxygenase and the immediate early gene c-fos. In addition to this stress-inducing potential, CS has been shown to exert inflammatory activities in vitro and in vivo, e.g., as indicated by the release of the chemotactic cytokine interleukin (IL)-8 from bronchial epithelial cells in response to CS (Mio et al., 1997; Nishikawa et al., 1999).
Hydroxyl radicals formed by Fenton chemistry (Imlay et al., 1988) are associated mainly with CS-dependent genotoxicity (Cosgrove et al., 1985; Müller and Gebel, 1994; Nakayama et al., 1985). However, they were found not to be the driving force of stress gene expression (Müller, 1995; Müller and Gebel, 1994). Instead, evidence has been provided that c-fos mRNA accumulation in smoke-exposed cells is dependent mainly on the formation of peroxynitrite (Müller et al., 1997). Although the potential peroxynitrite concentration in smoke-bubbled PBS is not sufficient to exert this effect by itself, CS-related aldehydes such as formaldehyde, acetaldehyde, and acrolein might allow this powerful oxidant to interfere with critical components that feed into stress signal transduction, most probably by significantly decreasing the intracellular glutathione (GSH) content (Müller and Gebel, 1998). Peroxynitrite is thought to be formed in aqueous extracts of CS in the presence of nitric oxide, which is abundant in CS, and superoxide, which in turn is generated by quinone/hydroquinone-like redox systems, e.g., provided by polyphenols such as catechol (Pryor and Stone, 1993)
A prominent proinflammatory transcription factor implicated in stress signal transduction is NF-B, which has also been linked to the cellular response to environmental stresses (for review, see Mercurio and Manning, 1999). Activation of NF-B in response to extracellular stress stimuli appears to be controlled by the redox status of the cell (for review, see Meyer et al., 1994; Sun and Oberley, 1996), since NF-B is significantly inhibited by a broad range of chemically unrelated antioxidants and becomes strongly activated by physical and chemical stresses that tend to trigger the formation of reactive oxygen species (for review, see Flohé et al., 1997). Mechanistically, the signaling pathway(s) leading to NF-B activation function mainly through the release of NF-B from its physical interaction with a member of the family of NF-B-inhibiting proteins known as inhibitor Bs (IBs), of which IB- is the best characterized. Complex formation with IB- retains NF-B in the cytoplasm, whereas phosphorylation of specific serine residues and subsequent degradation of IB- primed by ubiquitination results in the translocation of NF-B to the nucleus, followed by transcriptional activation of NF-B responsive genes (for review, see May and Ghosh, 1998). Alternatively, active NF-B may also be released from IB- by the phosphorylation of a specific tyrosine residue in the N-terminal region of IB-. This mechanism, in contrast to the regular path, does not include the proteasome-dependent degradation of IB- (Imbert et al., 1996). Finally, DNA binding of and consequently transactivation by NF-B requires a thioredoxin (Trx)-dependent reduced status of cysteine 62 (Cys-62) in the DNA-binding domain of the p50 subunit of NF-B, which renders the activity of NF-B subject to redox regulation (Matthews et al., 1992; Okamoto et al., 1992; Hayashi et al., 1993).
Based on the implication of NF-B in inflammatory and stress-signaling processes, the aim of this study was to evaluate the role of NF-B in the CS-evoked cellular stress response. We found that the DNA-binding activity of NF-B dropped significantly during the first 2 h of exposure but was subsequently elevated more than 2-fold. This pattern of deactivation and reactivation was not the result of IB- regulation, but appears to depend on the availability of reduced Trx, as can be concluded from the kinetics of both the complex formation between Trx and NF-B and the expression of thioredoxin reductase (TrxR) in smoke-bubbled PBS-treated cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Materials.
All chemicals, radiochemicals, and enzymes were obtained from Sigma Chemical Co., Deisenhofen, Amersham, Freiburg, and Roche, Mannheim, respectively, at the highest purity available. Generation of CS with the Reference Cigarette 2R1 (University of Kentucky) and preparation of smoke-bubbled PBS were performed as described (Müller, 1995
; Müller and Gebel, 1994).
Cell culture and treatment.
Swiss albino 3T3 mouse fibroblast cells (ATCC CCL 92) were cultured in 15-cm tissue culture dishes (Greiner, Frickenhausen), in 20 ml of Dulbecco's modified Eagle medium (DMEM) supplemented with 4% sodium hydrogen carbonate, L-glutamine, streptomycin, penicillin, and 10% fetal calf serum (FCS) (GIBCO-BRL, Karlsruhe).
Growth-arrested (0.5% FCS; 48 h) 3T3 cells were used in these experiments. Prior to treatment, the cells were washed with serum-free DMEM and immediately exposed for the indicated incubation times to serum-free culture medium containing smoke-bubbled PBS at the concentrations indicated.
Trx/NF-B coprecipitation.
Cells were lysed on ice (10 min) in 20 mM Tris pH 7.5, 150 mM NaCl, 10 mM EDTA, 0.5% Triton X-100, 1 mM Pefabloc, 15 µg/ml aprotinin, and centrifuged at 18000 x g for 15 min. Immunoprecipitations of clarified extracts were performed with protein A sepharose and 2.5 µl of a Trx-specific antibody (American Diagnostica, Pfungstadt). The purified precipitates were dissolved in sample buffer, separated on a polyacrylamide gel, and blotted on nitrocellulose. Western analysis was performed with an NF-B (p65)-specific antibody (1:200) (H286, Santa Cruz, Heidelberg). In parallel, Western analysis of whole cell extract proteins was performed with a Trx- or NF-B (p65)-specific antibody.
Western analysis.
Whole-cell extracts were prepared by lysing the cells in RIPA buffer [50 mM Tris pH 8, 125 mM NaCl, 0.5% NP40, 0.5% NaDOC, 0.1% SDS, 100 µM Na3VO3, 1 mM Pefabloc, leupeptin, aprotinin, and pepstatin (10 µg/ml each)]. Nuclear extracts were generated from cells lysed in 10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2, and 0.5% NP40. Purified nuclei were kept in RIPA buffer. Both types of extracts were clarified by centrifugation at 35000 x g for 30 min. Equal amounts of proteins were separated by polyacrylamide gel electrophoresis and blotted on nitrocellulose using a Trans-Blot Cell (BioRad, München). Detection of IB- and IB- phosphorylated on Ser-32 (BioLabs, Frankfurt am Main) or NF-B by Western analysis was performed with commercially available kit systems.
Northern hybridization.
Total RNA was isolated from quiescent 3T3 cells (48 h, 0.5% FCS) by standard methods with a commercially available RNA isolation kit (Wak-Chemie, Bad Soden). Fifteen micrograms of RNA/sample was analyzed by routine methods including denaturing RNA gel electrophoresis, blotting, hybridization, and autoradiography (Sambrook et al., 1989). 32P-labeled fragments of the murine c-myc and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (as internal control) served as hybridization probes.
Reverse transcriptase-PCR.
Quantitative reverse transcriptase-polymerase chain reaction (PCR) analysis of TrxR expression was performed from 5 µg of total RNA according to standard procedures. After reverse transcription, a 326-bp fragment internal of the TrxR coding sequence was amplified by 25 cycles of PCR with the following primer pair: 5`-TCCCGCAGAGCTACTCGGTA-3` ("cDNA primer") and 5`-CCTTATCATCATTGGAGGTG-3` ("amplimer"). Amplification of a 369-bp fragment internal of the GAPDH gene by quantitative reverse transcriptase-PCR was performed as an internal control.
Electrophoretic mobility shift assay (EMSA).
Cells were lysed in 20 mM Tris/HCl, pH 7.5, 250 mM NaCl, 20% glycerol, 0.25% NP40, 5 mM MgCl2, 2 mM EDTA, 2.5 mM DTT, 100 µM Na3VO3, 1 mM Pefabloc, and leupeptin, aprotinin, and pepstatin (10 µg/ml each) by freezing and thawing (three times). Extracts were clarified by centrifugation at 18,000 x g for 15 min. Gelshift reactions (10 µl) containing the cellular extracts (5–10 µg protein) in 15 mM Tris/HCl (pH 7.5), 100 mM NaCl, 5% glycerol, 5% Ficoll, 0.05% NP40, 2 mM MgCl2, 1 mM EDTA, 1 mM DTT, 0.05 mg/ml polydI • dC, and 35 fmol 33P-labeled oligonucleotide were run for 30 min (room temperature). The mixtures were subjected to native polyacrylamide gel electrophoresis (4%); DNA protein complexes were quantified by phosphorimaging of the gels. The following NF-B-specific oligonucleotide (Promega, Mannheim) was used in these investigations: 5`-AGTTGAGGGGACTTTCCCAGG-3`. All protein concentrations in extracts were determined by a modified Lowry assay (BioRad).
GSH determination.
GSH was determined as described (Akerboom and Sies, 1981) by quantifying the formation of thionitrobenzoic acid from free GSH and glutathione disulfide in the presence of 5,5-dithiobis-2-nitrobenzoic acid and glutathione disulfide reductase. Protein concentrations were assessed by a modified Lowry assay (BioRad).
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RESULTS |
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Smoke-Bubbled PBS Induces a Specific Kinetics of NF-
B DNA Binding That Is Not Dependent on IB-In order to elucidate the NF-
B response in smoke-bubbled PBS-treated cells, protein extracts of growth-arrested 3T3 cells exposed to 0.025 puffs/ml smoke-bubbled PBS for up to 6 h were tested for specific NF-B DNA-binding activity. As shown in Figure 1a, these experiments revealed a striking, biphasic type of kinetics: a considerable decrease of about 70% during the first 2 h of exposure was followed by a more than 2-fold (in comparison to control cells) increase in specific NF-B DNA binding after 4 to 6 h of exposure, while the amount of NF-B protein remained constant as measured by Western analysis using an antibody specific for the p65 subunit of NF-B. The CS-related increase in NF-B-DNA binding, accounting for 10% of that seen in cells treated with 20 ng/ml TNF-, was found to be statistically significant (6 h: p < 0.05, U-test) as calculated from several experiments; specific, as the retarded complex was displaced by an excess of NF-B unlabeled oligonucleotides (data not shown); and corroborated by an increased nuclear location of NF-B in CS-exposed cells as analyzed by an antibody recognizing the p65 subunit of NF-B (Fig. 1b). A candidate NF-B response gene is the proto-oncogene c-myc, which carries an NF-B-binding cis element in its promoter region (Duyao et al., 1990; LaRosa et al., 1994). We monitored c-myc expression in smoke-bubbled PBS-treated cells and compared the resulting expression pattern with the kinetics of NF-B activation in these cells. As shown in Figure 1c, c-myc mRNA levels were not elevated during the first 2 h of exposure, but then increased significantly, thus potentially matching the delayed increase in NF-B DNA binding in these cells (Fig. 1a).
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In order to find a clue for the biphasic kinetics of NF-
B activity, we monitored smoke-bubbled PBS-treated cells for changes in quantitative appearance as well as in phosphorylation status of IB-, the main regulator of NF-B. However, as demonstrated by the results shown in Figure 2, no significant quantitative alterations in the amount of IB- were detectable in these cells over a period of 6 h of exposure. In addition, using an antibody that specifically recognizes IB- phosphorylated on Ser-32, we saw no indication of the formation of this phosphor-protein in smoke-bubbled PBS-treated cells, whereas with TNF- we observed a rapidly induced phosphorylation of IB- followed by its degradation in 3T3 cells (Fig. 2). Moreover, regulation of NF-B via specific tyrosine phosphorylation of IB-, which lacks subsequent proteasome-dependent degradation of IB (Imbert et al., 1996), can also be excluded in smoke-bubbled PBS-treated cells, as no shift to forms of higher molecular weight was detectable over the whole exposure time (Fig. 2).
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CS-Induced NF-
B DNA Binding Is Paralleled by NF-B/Trx Complex Formation and Expression of TrxRThe DNA-binding activity of NF-
B is critically dependent on the presence of the reduced thiol function of Cys-62 of p50, which renders NF-B subject to redox regulation (Flohé et al., 1997; Matthews et al., 1992; Okamoto et al., 1992). Reduction of Cys-62 via a dithiol-disulfide exchange reaction is ensured by its physiological reducing catalyst, Trx, a small, ubiquitous protein with two redox-active half-cystine residues in an exposed active center (Holmgren, 1985). In order to test whether the kinetics of NF-B DNA-binding in smoke-bubbled PBS-treated cells might be influenced by the availability of reduced Trx, we determined next the formation of Trx/NF-B complexes, which is shown to precede enhanced NF-B DNA binding under oxidative stress conditions (Hirota et al., 1999). Trx/NF-B complexes were immunoprecipitated with an anti-Trx polyclonal antibody and then analyzed by immunoblotting with a polyclonal antibody recognizing the p65 subunit of NF-B (Fig. 3). Co-immunoprecipitation of Trx and NF-B was detectable in untreated cells, decreased considerably in extracts prepared from cells treated for 1 or 2 h with 0.03 puffs/ml smoke-bubbled PBS, and then increased again in extracts derived from cells exposed for 4 to 6 h to levels slightly above those seen in control extracts. As shown by Western blotting, cellular amounts of both total Trx (oxidized plus reduced) and NF-B remained nearly constant over the whole exposure period (Fig. 3), indicating that Trx/NF-B complex formation is not due to quantitative alterations in the amount of these proteins. Hence, the results from these experiments would account for the biphasic kinetics of specific NF-B DNA-binding in CS-treated cells, as depicted in Figure 1a
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Oxidized Trx must become reduced to again function as a reducing agent. This is accomplished by the presence of thioredoxin reductase (TrxR) and the cellular reductant NADPH. Three TrxR isozymes have been described so far (Sun et al., 1999
), and the gene encoding TrxR-1 has been shown to be inducible under oxidative stress conditions. Monitoring the expression of this candidate stress response gene in smoke-bubbled PBS-treated cells by quantitative reverse transcriptase-PCR, we observed a significant increase in TrxR mRNA after 4 to 6 h of exposure (Fig. 4), which perfectly matches the kinetics of re-formation of NF-B/Trx complexes in CS-treated cells (Fig. 3).
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In order to evaluate cells exposed for up to 8 h to smoke-bubbled PBS for the availability of reducing equivalents provided by NADPH, we chose to determine the relative concentration of reduced glutathione (GSH) in these cells, as the reduction of oxidized GSH via GSH reductase is likewise dependent on the presence of NADPH and the level of reduced GSH also represents a general indicator for the redox condition of cellular sulfhydryl groups. As shown in Figure 5
, levels of reduced GSH drop immediately in CS-exposed cells, but then recover to near initial levels within the next 6 h. If we assume a similar kinetics for the availability of reduced Trx, these results would explain the biphasic kinetics of Trx/NF-B complex formation and, consequently, the kinetics of NF-B DNA binding in smoke-bubbled PBS-treated cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CS is a general inducer of oxidative stress and has been implicated in many pulmonary diseases including inflammatory disorders and cardiovascular disease. On a molecular basis, all these lesions have been associated, at least in part, with the anomalous induction of signaling pathways in cells of targeted tissues, resulting in the activation of stress-responsive trans-activating transcription factors, of which the proinflammatory transcription factor NF-
B is of major relevance. For example, earlier experiments demonstrated the increased release of the chemokine IL-8 from bronchial epithelial cells in culture exposed to aqueous extracts of CS, as well as augmented concentrations of IL-8 in the bronchoalveolar lavage from smokers (Mio et al., 1997). More recent studies identified CS-dependent superoxide formation and NF-B activation as being responsible for these effects (Nishikawa et al., 1999). With regard to CS-related cardiovascular disease, evidence is accumulating that NF-B–driven increased expression of cyclooxygenase-2 participates in the genesis of atherosclerotic plaques (Yan et al., 2000).
According to the results presented here, the activity of NF-B in CS-exposed cells is subject mainly to a redox-controlled mechanism dependent on the availability of reduced Trx rather than governed by its main regulator IB-. This conclusion is based on the strict correspondence between the kinetics of NF-B DNA binding (Fig. 1a), Trx/NF-B complex formation (Fig. 3), TrxR expression (Fig. 4), and GSH depletion and repletion (Fig. 5), along with the lack of IB- phosphorylation and degradation (Fig. 2). Hence, we propose the following scenario in which NF-B deactivation and reactivation in CS-exposed cells is a consequence of perturbations of the cellular redox conditions (Fig. 6): immediate loss of reduced Trx (analogous to GSH), together with an oxidized status of Cys-62 of NF-B, results in decreased NF-B DNA-binding rates, whereas the reappearance of reduced Trx (due to the induction of TrxR and the availability of NADPH) at incubation times > 2 h activates an IB-–independent response of NF-B by rebinding to DNA. However, due to the lack of IB- inactivation, it remains to be elucidated by which mechanism the 2-fold increase, as compared to control cells, in NF-B DNA binding at longer incubation times is achieved in CS-treated cells.
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It is well established that aqueous extracts of CS contain large amounts of H2O2 (Pryor and Stone, 1993
). H2O2 itself is the source of hydroxyl radicals produced via the Fenton Reaction, and studies on catalase or inhibitors of the Fenton Reaction clearly demonstrate that CS-dependent DNA strand breaks are of this origin (Cosgrove et al., 1985; Müller and Gebel, 1994; Nakayama et al., 1985). Reactive oxygen species of the type produced by the Fenton Reaction are discussed even as physiological mediators of NF-B activation via IB kinase complex activation, resulting in IB- phosphorylation and degradation (for review see, Epinat and Gilmore, 1999, and references cited therein); therefore, it seems surprising that this pathway is obviously not triggered in smoke-bubbled PBS-treated cells (Fig. 2). However, with regard to the signaling properties of H2O2, the effects induced may be interfered with by the strong sulfhydryl activity also observed in aqueous extracts of CS. Previous studies in our laboratory have shown that activation of the pathway resulting in the expression of c-fos is not inhibitable by either catalase or inhibitors of the Fenton Reaction, but is mainly dependent on the strong sulfhydryl oxidant peroxynitrite in association with CS-dependent aldehydes (Müller and Gebel, 1998). Thus it cannot be ruled out that oxidation of critical sulfhydryl residues by peroxynitrite and/or CS-dependent aldehydes may inhibit or compete for key elements in H2O2-dependent signal transduction such as kinases or phosphatases. However, there are also reports (Brennan and O'Neill, 1995; Israel et al., 1992) describing the inability of H2O2 to induce NF-B activation in some cell lines.
Inhibition of NF-B DNA-binding in cells exposed to aqueous extracts of CS has already been described by Vayssier et al. (1998) in human monocytes. In accordance with results reported here, they describe a decrease in NF-B DNA binding in U937 cells exposed to smoke-bubbled PBS for 2 h, while not presenting data for longer incubation times. According to their results, the CS-dependent inhibition of NF-B DNA binding is linked to the expression of heat shock protein 70 (Hsp70) via the activation of heat shock transcription factor(s) (Vayssier et al., 1998). However, there is no evidence for such a mechanism in our system; at the CS doses used, we do not see any significant expression of Hsp70, either on a transcriptional or on a translational level (data not shown). Although this difference may be related to cell type specificities and/or smoke-bubbled PBS concentrations applied [considerably higher doses of CS, up to more than 10-fold, were used by Vayssier et al. (1998)], others have reported a lack of increased Hsp70 expression in vivo following exposure to mainstream or sidestream CS (Wong et al. 1997; Wong et al., 1995).
A stronger clue to understanding the kinetics of NF-B DNA binding in smoke-bubbled PBS-treated cells may be found in the recent publication by Horton et al. (1999), which shows that the CS-related aldehyde acrolein causes a transient decrease in NF-B DNA binding in A549 cells that is independent of IB-. As seen for smoke-bubbled PBS (Fig. 5), deactivation of NF-B by acrolein is paralleled by GSH depletion (Horton et al. 1999). Recently, we have demonstrated that CS-related aldehydes formaldehyde, acetaldehyde, and acrolein are mainly responsible for GSH depletion in smoke-bubbled PBS-treated cells (Müller and Gebel, 1998).
In summary, we have characterized the NF-B response in cells exposed to smoke-bubbled PBS. We obtained a distinct pattern of deactivation and reactivation, which appears to be induced mainly by the perturbation of the cellular homeostasis of key thiol functional compounds. As NF-B functions as a key element in crucial pathways controlling survival (Beg and Baltimore, 1996), tumor promotion (Young et al., 1999), and apoptosis (Kasibhatla et al., 1998), the CS-dependent effects on the activity of NF-B may have profound consequences for exposed cells.
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ACKNOWLEDGMENTS |
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We thank L. Conroy for expert editorial support and V. Böhm, T. Mankart, and B. Buttlies (INBIFO GmbH) for skillful technical assistance. Supported by Philip Morris, U.S.A.
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NOTES |
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1 To whom correspondence should be addressed. Fax: +49-2203-303362. E-mail: th.w.mueller{at}t-online.de.
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