Introduction

Nitric oxide (NO) plays an important role as mediator of several pathophysiological responses such as vasodilatation, platelet aggregation, immune and inflammatory diseases.1,2 Cytokines or endotoxin can induce the expression of inducible nitric oxide synthase in many murine and human cells. The subsequent production of NO can induce an array of effects ranging from cell death protection to cell death induction.3,4,5 Interleukin-1β induces apoptotic cell death in pancreatic β cells by low level of NO production.6 In contrast, a low level of NO can protect ovarian follicles from hormone-induced apoptosis.7 A high level of endogenous or exogenous NO has been shown to promote apoptotic and necrotic cell death, whereas trace levels of NO can do the reverse.8,9,10 Activated macrophages produce large amounts of NO, which is considered an essential mediator in host defence because of its cytotoxic effects on tumour cells and microorganisms.11,12 Under such conditions NO-producing macrophages themselves seem to be vulnerable toward NO toxicity.13 Recent evidence linked endogenously generated or exogenously supplied NO to characteristic apoptotic features, i.e. chromatin condensation and DNA fragmentation. NO-induced cell death has been previously described in macrophages, β-cells, thymocytes, chondrocytes and other types of cells.9 Different mechanisms of action for NO-induced apoptosis have been proposed, such as p53 accumulation,14,15,16 poly ADP (ribose) polymerase activation17,18 and Bcl-2 downregulation.19 Recent reports have provided new evidence that the transcription factor NF-κB is, at least in part, responsible for the apoptotic effect of tumour necrosis factor-alpha (TNF-α).20,21,22,23 Besides TNF-α, certain chemoterapeutic agents also induce apoptosis more effectively when NF-κB activation is inhibited.22,24 NF-κB is a member of the Rel family proteins and is typically a heterodimer of p50 and p65 subunit. In quiescent cells, NF-κB resides in the cytosol in latent form bound to inhibitory proteins, IκB-α. Stimulation of different types of cells with lipopolysaccharide, cytokines or oxidants triggers a series of signalling events that ultimately converge to the activation of one or more redox-sensitive kinase which specifically phosphorylate IκB, resulting in IκB polyubiquinatation and subsequent degradation. Once activated, the liberated NF-κB translocates into the nucleus and stimulates transcription by binding to cognate κB sites in the promoter regions of various target genes.25,26 Recent reports have demonstrated that activation of NF-κB is required for preventing apoptosis induced by over expression of ongogenic Ras mutant27,28,29 and E1A.30 On the other hand, other authors have reported that in murine B-cell line WEHI-231 the inhibition of NF-κB, which is constitutively active in these cells, leads to apoptosis. Therefore, NF-κB might have both preventive and causative role in the induction of apoptosis. NF-κB has been shown to be constitutively expressed in several monocyte cell lines at different states of differentiation.31 However, the functional role of such constitutive NF-κB has not been completely elucidated. In the present study we investigated the role of constitutive NF-κB in the control of NO-induced apoptosis in J774 macrophages.

Results

Effect of NO-releasing agents and inhibitors of NF-κB activation on cell viability

Incubation of J774 cells with SNP (1, 2, 4 and 6 mM) for 24 h caused a concentration-dependent reduction of cell viability (by 6.36±0.61, 19.87±4.22, 34.7±2.9 and 65.58±1.45%, respectively, n=6). In order to exclude that the effects of SNP on cell viability were due to cyanoid generation spent SNP, a negative control, was added to the cells. Spent SNP (1, 2, 4 and 8 mM) did not affect cell viability (94.3±1.9, 93.5±0.92, 92.86±1.33 and 90.31±1.73%, respectively, n=6). GSNO (2 mM), a structurally unrelated NO-donor, also significantly decreased cell viability (59.0±7.2%, n=6) while GSH (2 mM), which was unable to release NO, did not show significant effect (Figure 1A). TLCK (0.01, 0.1 and 1 mM) and PDTC (0.001, 0.01 and 0.1 mM) two well known inhibitors of NF-κB activation, reduced cell viability significantly and in a concentration-dependent fashion (by 17.2±9.2; 51.6±6.3; 67.7±5.2% and 6.3±3.0, 39.0±7.2, 69.4±4.5%, respectively, n=6) (Figure 1B). The treatment with SNP (1, 2, 4 and 8 mM) plus TLCK (1 mM) or PDTC (0.1 mM) caused a marked and significant decrease of cell viability compared with each compound alone (by 64.0±0.93, 67.1±0.94, 70.26±1.15, 73.61±1.23%; and 62.24±1.1, 66.68±1.0, 70.43±1.06, 73.54±1.28%, respectively, n=6) (Figure 1C,D). SNP (1, 2, 4 and 8 mM) and GSNO (2 mM) caused nitrite accumulation in the medium at 24 h (21.18±0.56, 28.52±0.59, 44.32±1.19, 60.18±1.26 nmol/106 cells; and 48.15±0.64 nmol/106 cells, respectively, n=6).

Figure 1
figure 1

Effect of NO-releasing agents and inhibitors of NF-κB activation on cell viability. (A) J774 macrophages were incubated with SNP (1, 2, 4 and 8 mM), GSNO (2 mM) and GSH (2 mM) for 24 h. (B) J774 macrophages were incubated for 24 h with TLCK (0.01, 0.1 and 1 mM) or PDTC (0.001, 0.01 and 0.1 mM). (C,D) J774 macrophages were incubated for 24 h with SNP (1, 2, 4 and 8 mM) in the absence (hatched bars) or presence (open bars) of TLCK (1 mM) or PDTC (0.1 mM). Thereafter cell viability was determined by MTT assay as described in Materials and Methods. Data are expressed as mean±S.E.M. of six separate experiments. *P<0.05, ***P<0.001 vs control (untreated cells); °P<0.05, °°°P<0.001 vs SNP alone

Full size image

Effect of NO-releasing agents and inhibitors of NF-κB activation on cell morphology

We investigated the effect of SNP (8 mM), GSNO (2 mM), TLCK (1 mM), PDTC (0.1 mM) and GSH (2 mM) on the morphologic changes of J774 macrophages at 8 h. The untreated cells or the GSH-treated cells showed normal morphologic aspects (Figure 2A,F). The cells treated with SNP, GSNO, TLCK or PDTC and visualised by phase-contrast microscopy were indicative of apoptosis and ApopTag-positive as illustrated in the upper panel (Figure 2B,C,D and E, respectively).

Figure 2
figure 2

Effect of NO-releasing agents or inhibitors of NF-κB activation on cell morphology. J774 macrophages were incubated for 8 h without (A) or with (B) SNP (8 mM), (C) GSNO (2 mM), (D) TLCK (1 mM), (E) PDTC (0.1 mM) and (F) GSH (2 mM), visualised by phase-contrast microscopy (magnification, 120×), analyzed for ApopTag positivity and photographed. (A) and (F) exhibited normal morphologic aspects. (B–E) were indicative of apoptosis and ApopTag positive in the upper panel

Full size image

Effect of NO-releasing agents and inhibitors of NF-κB activation on DNA fragmentation

Internucleosomal DNA degradation determined qualitatively by agarose gel electrophoresis and quantitatively by the diphenylamine reaction (Figure 3A and B, respectively), was analyzed as a parameter of apoptosis. Incubation of cells with SNP (8 mM) and GSNO (2 mM) as well as TLCK (1 mM) or PDTC (0.1 mM) for 8 h led to the appearance of oligonucleosomal fragmentation with the characteristic ladder pattern associated with apoptosis and elicited a significant increase of DNA fragmentation (by 33.1±2.31, 32.0±1.53, 36.5±1.47 and 36.7±1.37%, respectively, n=3) as compared to control untreated cells (3.5±1.35%). GSH (2 mM) did not show significant effect (4.2±1.78%).

Figure 3
figure 3

Effect of NO-releasing agents and inhibitors of NF-κB activation on DNA fragmentation. J774 macrophages were incubated with SNP (8 mM), GSNO (2 mM), GSH (2 mM), TLCK (1 mM) or PDTC (0.1 mM) for 8 h, thereafter DNA fragmentation was analyzed by agarose gel electrophoresis (A) and diphenylamine assay (B). Data illustrated in (A) are from a single experiment and are representative of three separate experiments, while data in (B) are expressed as mean±S.E.M. of three separate experiments. ***P<0.001 vs control (untreated cells)

Full size image

Effect of NO-releasing agents and inhibitors of NF-κB activation on constitutive NF-κB/DNA binding activity

The effects of different concentrations of NO-generating compounds or inhibitors of NF-κB activation on NF-κB/DNA binding activity in J774 macrophages were tested by EMSA. A constitutively basal level of NF-κB/DNA binding activity was detected in nuclear proteins from untreated macrophages. NF-κB/DNA complex consisted of two bands which were inhibited by SNP, GSNO, PDTC and TLCK. In fact, the incubation of cells with SNP (1, 2, 4 and 8 mM) for 2 h caused a concentration-dependent inhibition of NF-κB/DNA binding activity (by 9.2±7.4, 20.7±7.6, 71.5±5.4 and 89.1±3.9% respectively, n=3). GSNO (2 mM) significantly decreased NF-κB/DNA binding activity (81.6±3.8%, n=3), while GSH (2 mM) did not show effect (Figure 4). Treatment with PDTC (0.001, 0.01 and 0.1 mM) or TLCK (0.01, 0.1 and 1 mM) also caused a concentration-dependent inhibition of NF-κB/DNA binding activity (by 7.0±2.9, 62.7±2.6, 76.4±1.9%; and 9.7±2.9, 72.7±2.6, 84.6±2.5% respectively, n=3) (Figure 5). The specificity of NF-κB/DNA binding complex was evident by the complete displacement of NF-κB/DNA binding in the presence of a 50-fold molar excess of unlabelled NF-κB probe (W.T., 50×) in the competition reaction. In contrast, a 50-fold molar excess of unlabelled mutated NF-κB probe (Mut., 50×) or Sp-1 oligonucleotide (Sp-1, 50×) had no effect on this DNA-binding activity. The composition of the NF-κB complex was determined by using specific antibody against p50 (p50), p65 (p65) and c-Rel (c-Rel) subunits of NF-κB protein family. Addition of either anti-p50 or anti-p65 but not anti-c-Rel to the binding reaction resulted in a marked reduction of the intensity of NF-κB specific bands, suggesting that NF-κB complex contained predominately p50 and p65 subunits (Figure 6).

Figure 4
figure 4

Effect of NO-releasing agents on constitutive NF-κB/DNA binding. Representative EMSA of NF-κB (A) as well as densitometric analysis (B) shows the effect of SNP (1, 2, 4 and 8 mM), GSNO (2 mM) or GSH (2 mM) on constitutive NF-κB/DNA binding activity in J774 macrophages. Data illustrated in (A) are from a single experiment and are representative of three separate experiments, while data in (B) are expressed as mean±S.E.M. of three separate experiments. *P<0.05; **P<0.001; ***P<0.0001 vs control (untreated cells)

Full size image
Figure 5
figure 5

Effect of inhibitors of NF-κB activation on constitutive NF-κB/DNA binding. Representative EMSA of NF-κB (A) as well as densitometric analysis (B) shows the effect of PDTC (0.001, 0.01 and 0.1 mM) or TLCK (0.01, 0.1 and 1 mM) on constitutive NF-κB/DNA binding activity in J774 macrophages. Data illustrated in (A) are from a single experiment and are representative of three separate experiments, while data in (B) are expressed as mean±S.E.M.. of three separate experiments. ***P<0.0001 vs control (untreated cells)

Full size image
Figure 6
figure 6

Identification of constitutive NF-κB/DNA binding proteins. Nuclear extracts from J774 macrophages were prepared as described in Materials and Methods and incubated with 32P-labelled NF-κB probe. In competition reaction nuclear extracts were incubated with radiolabelled probe in absence or presence of identical but unlabelled oligonucleotides (W.T., 50×), mutated non-functional κB probe (Mut., 50×) or unlabelled oligonucleotide containing the consensus sequence for Sp-1 (Sp-1, 50×). In supershift experiments nuclear extracts were incubated with antibodies against p50 (p50), p65 (p65) or c-Rel (c-Rel) 30 min before incubation with radiolabelled NF-κB probe. Data illustrated are from a single experiment and are representative of three separate experiments

Full size image

Effect of NO-releasing agents and inhibitors of NF-κB activation on degradation of IκBα

The effects of NO-releasing agents and inhibitors of NF-κB activation on the presence of IκBα was examined by immunoblotting analysis. Untreated cells expressed a basal level of IκBα in the cytosolic fraction. When the cells were incubated with SNP (8 mM), GSNO (2 mM), PDTC (0.1 mM) or TLCK (1 mM) IκBα levels increased in cytosolic fraction (by 64.1±3.1, 55.1±3.9, 42.9±3.7 and 37.2±2.9%, respectively, n=3) as compared to control untreated cells (Figure 7).

Figure 7
figure 7

Effect of NO-releasing agents and inhibitors of NF-κB activation on IκBα levels. Representative Western blot of IκB-α (A) as well as the densitometric analysis (B) shows the effect of SNP (8 mM), GSNO (2 mM), GSH (2 mM), PDTC (0.1 mM) or TLCK (1 mM) on IκBα levels. Data illustrated in (A) are from a single experiment and are representative of three separate experiments, while data in (B) are expressed as mean±S.E.M. of three separate experiments. ***P<0.0001 vs control (untreated cells)

Full size image

Discussion

A role for NO on apoptosis pathway has been extensively studied in the recent years. However, the molecular mechanisms by which NO modulates apoptosis are not yet elucidated.9,10,32 The results of our study demonstrate that SNP and GSNO, two different NO donors, induce apoptosis in murine/macrophage cell line J774 as evaluated by biochemical markers and morphologic parameters, i.e. DNA ladder formation, ApopTag positivity and loss of cell viability. The effect is likely to be mediated by the release of NO, and not another metabolite, since the two compounds are structurally unrelated. In addition, apoptotic alterations typified by DNA fragmentation and cell morphology were absent in the cells treated with GSH (the parent compound of GSNO) which was unable to release NO. Inhibition by GSNO was more effective than that exhibited by SNP. In fact, the NO-induced DNA fragmentation, ApopTag positivity and loss of cell viability were elicited at same extent by 2 mM GSNO and 8 mM SNP. Our results are in agreement with several other reports that previously demonstrated that exogenous NO induces apoptosis in a macrophage cell line.14,15,17 In our experiments a fairly large amount of SNP and GSNO was required to induce apoptosis in J774 cells compared to concentration used by other authors in different macrophage cell lines, such as RAW 264.716,18,19 or murine peritoneal macrophages.17,18 However, it has been previously shown that NO-induced apoptosis in J774 macrophages requires high concentration of NO donors, suggesting a lower susceptibility of this cell line to NO.33 The variable responses could also be due to differences in the cellular anti-oxidant machinery.4 For example, upregulation of specialised resistance genes, such as manganese superoxide dismutase, glucose 6 phosphate dehydrogenase and glutathione, may counteract the destruction by high output of NO.34,35,36 We demonstrate that the antioxidant PDTC and the serine protease inhibitor TLCK, two well-known inhibitors of NF-κB activation,37,38,39,40,41 also induced a significant DNA fragmentation, ApopTag positivity and loss of cell viability. Moreover, it is interesting to note that SNP (even at the concentrations of 1 mM) plus TLCK or PDTC treatment caused a more marked loss of cell viability suggesting that NF-κB is important in protecting cells from cell death. A growing body of evidence have shown that NF-κB/Rel is involved in programmed cell death.24,31 First, many potent NF-κB-activating stumuli induce apoptosis, such as TNF-α,42 H2O2,43,44 LPS and serum withdrawal. Second, apoptosis can be prevented by conditions that at the same time suppress NF-κB activation. This finding has been shown either for the antioxidants, such as N-acetyl-L-cysteine and PDTC,45 or for the serine protease inhibitor TLCK.43,46,47 Finally, several genes induced upon apoptosis are transcriptionally induced by stimuli activating NF-κB and their promoter regions contain potential NF-κB-binding motifs, including the Fas/APO-1 ligand,48,49 c-myc,50 p5351 and interleukin-1 β converting enzyme.52 Several studies have demonstrated that NF-κB protects the cells from TNF-α induced cell death, suggesting an anti-apoptotic role of this transcription factor.20,21,22,23 Our results, achieved by EMSA, show that J774 cells express constitutively active NF-κB. Such constitutive NF-κB/DNA binding activity has been previously described for different cell types and has been correlated with progression of breast cancer, melanoma and juvenile myelomonocytic leukaemia.53,54,55,56 How NF-κB is constitutively activated in some tumour cells and which role it plays in induction of resistance to apoptosis is, to date, still not clear. Here, we show that treatment of J774 cells with both SNP and GSNO inhibits NF-κB/DNA binding activity. PDTC and TLCK also suppressed constitutive NF-κB activation only at concentrations that resulted apoptotic. All together these findings suggest a link between constitutive NF-κB/DNA binding activity and protection against NO-induced apoptosis. Previous reports have shown that exogenous NO donors inhibit NF-κB/DNA binding.57,58,59,60,61 Exogenous NO has been shown to stabilise NF-κB by inhibiting the dissociation of the IκBα inhibitor and simultaneously increasing IκBα mRNA levels in human vascular endothelial cells.57,61 Other studies have shown that ex vivo biochemical modification of NF-κB p50 inhibits DNA binding as determined by the gel shift assay. Particularly, S-nytrosylation of the redox sensitive NF-κB p50 C62 residue was associated with the inhibition of p50 binding to its consensus DNA target sequence.58,59 Our results, in agreement with those of Peng et al.57 and Spiecker et al.,61 suggest that SNP and GSNO prevent constitutive NF-κB/DNA binding activity by increasing IκBα protein levels. Furthermore, PDTC and TLCK, which have been shown to prevent NF-κB activation by blocking proteolytic degradation of IκBα, also significantly increased IκBα protein levels. Our results suggest that NO may cause apoptosis in J774 macrophages by increasing IκBα level which, in turn, inhibits constitutive NF-κB/DNA binding activity. However, several other mechanisms may be also involved in the regulation of constitutive NF-κB/DNA binding activity by NO. It has been shown that exogenously generated or endogenously produced NO increases both the mRNA and protein content of the anti-apoptotic protein Bcl-2 in rat aortic endothelial cells.62 Bcl-2 has been shown to down-regulate the activity of NF-κB induced upon apoptosis.63 Therefore, it is possible to hypothesise that NO may up-regulate Bcl-2 expression which, in turn, blocks constitutive NF-κB/DNA binding activity. Moreover, other studies have demonstrated that NO-releasing compounds induced the expression of heat shock protein 70 (HSP-70) in rat hepatocytes.64 Recent findings have shown that HSP-70 inhibited cytokine-induced NF-κB activation in MLE-15 murine lung epithelium65 and brain glial cells66 suggesting a possible involvement of HSP-70 in the NO-mediated down-regulation of constitutive NF-κB/DNA binding activity. Finally, it has been proposed that reactive oxygen species (ROS) are involved in protection of cells from apoptosis by sustaining constitutive NF-κB/DNA binding activity.67 On the other hand, it is well recognised that NO can avidly react with several ROS leading to a modification of cell redox state.68 Therefore, it is also possible to hypothesise that NO, altering cell redox state, may block constitutive NF-κB/DNA binding activity and thus induce apoptosis. In conclusion, we demonstrate that SNP and GSNO inhibit constitutive NF-κB/DNA binding activity suggesting, for the first time, a novel pathway for NO-induced apoptosis. Nevertheless, delineation of the mechanism(s) of negative regulation of NF-κB/DNA binding activity by NO remains to be shown. In any case, the down-regulation of NF-κB/DNA binding activity by exogenous or endogenous NO may represent a negative regulatory mechanism in modulating the sustained NF-κB activation that occurs in many pathological events, such as HIV-infected monocytes that play a key role in HIV virus replication.69,70

Materials and Methods

Cell culture

The murine monocyte/macrophage cell line J774 (American Tissue Culture Catalogue T1B pag. 231) was cultured at 37°C in humidified 5% CO2/95% air in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% foetal bovine serum, 2 mM glutamine, 100 UI/ml penicillin and 100 μg/ml streptomycin.

MTT viability assay

The cells were plated in 96 culture wells at a density of 250 000 cells/ml and allowed to adhere for 2 h. Thereafter the medium was replaced with fresh medium and cells were incubated with SNP (1, 2, 4 and 8 mM), GSNO (2 mM), TLCK (0.01, 0.1 and 1 mM), PDTC (0.001, 0.01 and 0.1 mM) or GSH (2 mM) alone or in association. In parallel experiments spent SNP (1, 2, 4 and 8 mM) was added to the cells. After 24 h, supernatant (100 μl) was harvested for nitrite determination and cell viability was determined by using 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) conversion assay.71 Briefly, 25 μl of MTT (5 mg/ml in complete DMEM) were added to the cells and incubated for additional 3 h. After this time point the cells were lysed and the dark blue crystals solubilised with 100 μl of a solution containing 50% (v:v) N,N-dymethylformamide, 20% (w:v) SDS with an adjusted pH of 4.5. The optical density (OD) of each well was measured with a microplate spectrophotometer (Titertek Multiskan MCCC/340) equipped with a 620 nm filter. The cell viability in response to treatment with test compounds was calculated as % cell viability=(OD treated/OD control)×100.

Nitrite determination

NO was measured as nitrite (NO2−, nmol/106 cells) accumulated in the incubation medium after 24 h. A spectrophotometric assay based on the Griess reaction was used. Briefly, Griess reagent (1% sulphanilamide, 0.1% naphthylethylenediamine in phosphoric acid) was added to an equal volume of cell culture supernatant and the absorbance at 550 nm was measured after 10 min. The nitrite concentration was determined by reference to a standard curve of sodium nitrite.

Morphological investigation

J774 macrophages (1×105 cells/ml) were plated in 35×10 mm diameter culture dishes and incubated with SNP (8 mM), PDTC (0.1 mM) or TLCK (1 mM) for 8 h. Thereafter, the cells were washed three times with PBS, fixed with 3% formaldehyde in PBS, and then visualised by phase-contrast microscopy. The apoptotic cells were detected by ApopTag In Situ Kit.72

Gel electrophoresis of fragmented DNA

Cells (2×106) were seeded on 10 cm diameter Petri dishes and allowed to adhere for 2 h. Thereafter the medium was replaced with fresh medium and the cells were incubated with SNP (8 mM), GSNO (2 mM), GSH (2 mM), TLCK (1 mM) or PDTC (0.1 mM) for 8 h. For analysis of genomic DNA, cells were gently scraped off and collected together with non-attached cells in the supernatant. Cells were resuspended in 250 μl of Tris-Borate EDTA (TBE) buffer containing 0.25% NP-40 and 100 μg/ml RNAse A. After incubation at 37°C for 30 min, extracts were treated with 1 mg/ml proteinase K for additional 30 min at 37°C. Then equal amounts (10 μl) of the extracts were loaded on a 1% agarose gel containing (1 μg/ml) ethidium bromide and run for 1 h in 1×TBE at 90 V. The gel was photographed with Polaroid type 55 film using an UV transilluminator and an MP-3 Polaroid camera set-up.

Quantification of DNA fragmentation

DNA fragmentation was performed essentially as previously described by Messmer and Brune15 with some modification. Briefly, cells (4×106) were seeded on 10 cm diameter culture dishes and incubated with SNP (8 mM), GSNO (2 mM), GSH (2 mM), TLCK (1 mM) or PDTC (0.1 mM) for 8 h. Thereafter, the cells were scraped off the culture plates, centrifuged at 180×g for 10 min, resuspended in 250 μl of TE-buffer (10 mM Tris, 1 mM EDTA, pH 8.00), and incubated with an additional volume of lysis buffer (5 mM Tris, 1 mM EDTA, pH 8.00, 0.5% Triton X-100) for 30 min at 4°C. After lysis, the intact chromatin (pellet) was separated from DNA fragments (supernatant) by centrifugation for 15 min at 13 000 r.p.m. Pellet was resuspended in 500 μl of TE-buffer and samples were precipitated by adding 500 μl of 25% trichloroacetic acid at 4°C. Samples were pelleted at 3000×g for 10 min and the supernatant was removed. After addition of 300 μl of 5% trichloroacetic acid, samples were boiled for 15 min. DNA contents were quantified by using the diphenylamine (DPA) reagent (0.3 g DPA, 0.2 ml of sulphuric acid, 0.1 ml of 1.6% acetaldehyde, 10 ml of acetic acid glacial). The percentage of fragmented DNA was calculated as the ratio of the DNA content in the supernatant to the amount in the pellet.

Preparation of cytosolic and nuclear extracts

Extracts of macrophages incubated for 8 h with SNP (1, 2, 4 and 8 mM), GSNO (2 mM) GSH (2 mM), PDTC (0.001, 0.01 and 0.1 mM) or TLCK (0.01, 0.1 and 1 mM) were prepared as described.73 Briefly, harvested cells (2×107) were washed two times with ice-cold PBS and centrifuged at 180×g for 10 min at 4°C. The cell pellet was resuspended in 100 μl of ice-cold hypotonic lyses buffer (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM phenylmethylsulphonylfluoride, 1.5 μg/ml soybean trypsin inhibitor, 7 μg/ml pepstatin A, 5 μg/ml leupeptin, 0.1 mM benzamidine, 0.5 mM DTT) and incubated on ice for 15 min. The cells were lysed by rapid passage through a syringe needle five or six times and the cytoplasmic fraction was then obtained by centrifugation at 13 000×g for 1 min. The nuclear pellet was resuspended in 60 μl of high salt extraction buffer (20 mM HEPES pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% v/v glycerol, 0.5 mM phenylmethylsulphonylfluoride, 1.5 μg/ml soybean trypsin inhibitor, 7 μg/ml pepstatin A, 5 μg/ml leupeptin, 0.1 mM benzamidine, 0.5 mM DTT) and incubated with shaking at 4°C for 30 min. The nuclear extract was then centrifuged for 15 min at 13 000×g and supernatant was aliquoted and stored at −80°C. Protein concentration was determined by the Bio-Rad protein assay kit.

Electrophoretic mobility shift assay (EMSA)

Double stranded oligonucleotides containing the NF-κB recognition sequence (5′-AGT TGA GGG GAC TTT CCC AGG-3′) were end-labelled with 32P-γ-ATP. Nuclear extracts containing 7.5 μg proteins were incubated for 30 min with radiolabelled oligonucleotides (2.5–5.0×104 c.p.m.) in 20 μl reaction buffer containing 2 μg poly dI-dC, 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 μg/ml bovine serum albumin, 10% (v/v) glycerol. Nuclear protein-oligonucleotide complexes were resolved by electrophoresis on a 6% non-denaturing polyacrylamide gel in 1×TBE buffer at 150 V for 2 h at 4°C. The gel was dried and autoradiographed with intensifying screen at −80°C for 20 h. Subsequently, the relative bands in nuclear fractions were quantified by densitometric scanning of the X-ray films with a GS 700 Imaging Densitometer (Bio-Rad) and a computer programme (Molecular Analyst, IBM).

Western blot analysis

Immunoblotting analysis of IκBα was performed on J774 cells incubated with SNP (8 mM), GSNO (2 mM), GSH (2 mM), PDTC (0.1 mM) or TLCK (1 mM) for 2 h. Cytosolic fraction proteins were mixed with gel loading buffer (50 mM Tris/10%, SDS/10%, glycerol/10%, 2-mercaptoethanol/2 mg of bromophenol//ml) in a ratio of 1 : 1, boiled for 3 min and centrifuged at 10 000×g for 10 min. Protein concentration was determined and equivalent amounts (75 μg) of each sample were electrophoresed in 12% discontinuous polyacrylamide minigel. The proteins were transferred onto nitro-cellulose membranes, according to the manufacturer's instructions (Bio-Rad). The membranes were saturated by incubation at 4°C overnight with 10% non fat dry milk in PBS and then incubated with (1 : 1000) anti-IκBα antibody for 1 h at room temperature. The membranes were washed three times with 1% Triton 100-X in PBS and then incubated with anti-rabbit immunoglobulins coupled to peroxidase (1 : 1000). The immunocomplexes were visualised by the ECL chemiluminescence method (Amersham). Subsequently, the relative expression of IκBα protein in cytosolic fraction was quantified by densitometric scanning of the X-ray films with a GS 700 Imaging Densitometer (Bio-Rad) and a computer programme (Molecular Analyst, IBM).

Statistics

Results were expressed as the mean±S.E.M. of n experiments. Statistical analysis was determined by Student's unpaired t-test with P<0.05 considered significant.

Reagents

Phosphate buffer saline was from Celbio (Milan, Italy). DL-Dithiothreitol, phenylmethylsulfonylfluoride, soybean trypsin inhibitor, pepstatin A, leupeptin and benzamidine were from Calbiochem (Milan, Italy). 32P-γATP was from Amersham (Milan, Italy). Poly dI-dC was from Boehringer-Mannheim (Milan, Italy). Anti-IκBα, anti-p50, anti-p65 and anti c-Rel antibodies were from Santa Cruz (Milan, Italy). Non fat dry milk was from Bio-Rad (Milan, Italy). Tib Molbiol, Boehringer-Mannheim (Genova, Italy), performed oligonucleotide synthesis to our specifications. ApopTag kit was from Oncor (Maryland, USA). All the other reagents were from Sigma (Milan, Italy).