The human bronchial epithelial cell line, BEAS-2B, was obtained from the American Type Culture Collection (CRL-9609; Rockville, MD). Normal human bronchial epithelial cells (HBECs) were acquired from bronchial biopsies of smokers with normal lung function using a previously published method with modifications 9 under a protocol approved by the University of Nebraska Human Studies Committee. Both BEAS-2B and HBEC cells were cultured in 100 mm tissue culture dishes (Falcon; BD Bioscience Discovery Labware, Bedford, MA), which were coated with 0.03 mg/ml collagen (Vitrogen 100, Angiotech BioMaterials, Palo Alto, CA), in a 1:1 mixture of LHC-9/RPMI 1640 1011. Cells were passaged once weekly at a 1:3 ratio. HBECs between the 3rd and 10th passage were used for experiments.

Following primary antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA): anti-Stat3, anti-p65, anti-p50/p105, anti-Bcl-XL, anti-Bad, anti-Bax, anti-Bcl2, and anti-XIAP antibodies. Anti-β-actin antibody (A5441) was purchased from Sigma (St. Louise, MO).

Cigarette smoke extract (CSE) was prepared with a modification of the method of Carp and Janoff 12. Briefly, one 100 mm long cigarette without filter (R1 Research Grade Cigarette, University of Kentucky; these were obtained prior to their being discontinued by the manufacturer and were stored (4°C) until use.) was combusted with a Mini-Pump Variable Flow (Fisher Scientific, Pittsburgh, PA). The smoke was bubbled through 25 ml double distilled water (ddH2O) at a speed of 70 cc/minute till the unburned butt is less than 1 cm long. The resulting suspension was in yellowish color and the optical density (OD) value at 405 nm wave-length was 0.120 ± 0.020. This solution was filtered through a 0.22 μm pore filter (Lida Manufacturing Corp., Kenosha, WI) to remove bacteria and large particles. This filtered solution was considered to be 100% CSE and diluted with LHC-D/RPMI 1640 medium 1011 within 30 minutes of preparation to obtain the desired concentration used in each experiment. The LHC-D/RPMI 1640 medium was a 1:1 mixture of LHC-D and RPMI 1640, which contained no serum or growth factors.

DNA strand breaks were evaluated quantitatively with a colorimetric kit (Titer TACS, Trevigen, Gaithersburg, CA) that utilizes TUNEL stain in a 96-well format, following the manufacturer's instructions. Briefly, cells were cultured in 96-well plates till sub-confluent and treated with desired concentrations of cigarette smoke extract. Cells were then fixed with 3.7% Buffered Formaldehyde for 5 minutes followed by washing once with PBS. The cells were post-fixed with 100% methanol for 20 minutes and washed once with PBS. Cells were then labeled by TUNEL following the manufacturer's instructions. Absorbance at 450 nm was measured with a Benchmark microplate reader (Bio-Rad, Hercules, CA). For comparison, data are expressed as % of Positive Control (DNAse treated cells), according to the formula: (Sample OD value-blank)/(Positive Control OD value-blank) × 100.

To determine the presence of apoptotic cells, DNA content was measured by flow cytometry as reported previously 2. Cells were cultured in 6-well plates till confluent. After treatment with CSE or as a positive control, the DNA topoisomerase inhibitor camptothecin (CPT), medium was harvested to collect floating cells and attached cells were detached from the tissue culture dishes with trypsin/EDTA. Both floating and attached cells were then pelleted together and fixed with 70% ethanol at 4°C for 30 minutes. After staining with propidium iodide (50 μg/10⁶ cells, Sigma, St. Louis, MO), DNA content/cell cycle analysis was performed by flow cytometry. Cells with less DNA content than that of G1 cells (sub-G1 peaks or A₀ cells) were considered apoptotic.

Comet assay was performed using the CometAssay™ Kit (Trevigen, Inc. Gaithersburg, MD). Briefly, cells were cultured in 6-well plates and treated with cigarette smoke extract or camptothecin as described above. Floating and attached cells were harvested, pelleted and re-suspended with cold PBS at 10⁵ cells/ml. The resulting cell suspension (50 μL) was mixed with 500 μL of LMAgarose and 75 μL of the agarose/cells was pipetted over the sample area of the COMET Slides. The samples were lysed, electrophoresed and stained following the manufacturer's instructions. Cells were then viewed with an epifluorescence microscopy (NIKON Eclipse E800) and photographed with a digital camera (OPTRONICS) under 200× magnification. Apoptotic index = number of cells with small DNA head with fan-like tail/total cell number × 100%.

Cells were lysed with lysing buffer (50 mM Tris buffer, pH 7.4, containing 10 mM EDTA, 2 mM EGTA, 2 mM benzamidine, 2.5 mM dithiothreitol, 2 μg/ml soybean trypsin inhibitor, 100 μM tosyl-L-lysine chloromethyl ketone, 200 μM leupeptin, and 50 μM phenylmethylsulphonyl fluoride). The cell lysate was centrifuged at 12,000 g for 10 minutes at 4°C and the precipitates were discarded. Protein concentrations in supernatants were determined by a protein dye-binding assay (Bio-Rad, Hercules, CA). Proteins were then subjected to immunoblot analysis. After heating for 3 minutes at 95°C, 10 μg of total protein was mixed with 2× sample buffer (0.5 M Tris-HCL, pH 6.8, 10% SDS, 0.1% bromphenol blue, 20% glycerol, 2% b-mercaptoethanol) and loaded into each well before performing electrophoresis with the Mini-protein 3 Cell System^® (Bio-Rad, Hercules, CA). The proteins were transferred to PVDF membranes (Bio-Rad, Hercules, CA) in transfer buffer (20 mM Tris, pH 8.0, 150 mM glycine, 20% methanol) at 20 V for 40 minutes with the semi-dry electrophoretic transfer system (Bio-Rad, Hercules, CA). The membrane was blocked with 5% dry-milk in PBS-Tween at room temperature for 1 hour and then exposed to primary antibodies at 4°C overnight. Target proteins were subsequently detected using horseradish peroxidase conjugated IgG with an enhanced chemiluminescence detection system (ECL, Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, England).

Electrophoretic mobility shift assay (EMSA) was performed with a kit (Panomics, Inc, Redwood city, CA) following the manufacturer's instructions. Briefly, nuclear extract (5 μg) was incubated with 10 ng biotin-labeled NF-κB (p65) probe. For the "cold probe" assay, 20 ng of unlabeled (cold) NF-kB probe was mixed with sample 5 minuets before adding 10 ng biotin-labeled NF-kB probe (cold vs labeled probe ratio was 2:1). Protein-DNA complexes were then resolved by non-denaturing polyacrylamide gel electrophoresis (PAGE). After transfer to Pall Biodyne B^® membrane (Pall Corporation, East Hills, NY), proteins were immobilized in UV cross-linker. After blocking, avidin-HRP was applied and detected by enhanced chemiluminescence (ECL, Amersham).

Targeting p65 siRNA and non-targeting control siRNA (Dharmcon, Inc, Lafayette, CO) were introduced into the cells using a method previously described with modification 13. Briefly, normal human bronchial epithelial cells were plated into V30-coated 60 mm dishes at a density of 10⁶ /dish so that the cells were 60–70% confluent after 1–2 days. At that time, after washing with PBS, cells were treated with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) containing p65-siRNA, STAT3-siRNA or non-targeting control siRNA (final concentration 100 nM in Opti-MEM) for 6 hours followed by re-feeding with LHC-9/RPMI medium. On the next day, cells were trypsinzed and counted. One half million cells were used to examine the silencing effect. The remainder of the cells were plated into 6-well plates and exposed to cigarette smoke on the next day.

Cell viability was evaluated by ethidium homodimer-1 dye exclusion using the LIVE/DEAD Kit, following the manufacturer's instructions (Invitrogen, Carlsbad, CA). Briefly, cells were detached by trypsinization and incubated in LHC-D/RPMI medium containing Calcein AM (Green) and ethidium homodimer-1 (EthD-1, Red) at 37°C for 15 minutes. Cells were then spun onto slides by cytospin followed by observation under fluorescence microscopy within 24 hrs. Nuclei stained by EthD-1, which appeared red, were counted as dead cells. Cell number was counted in 5 randomly chosen fields and expressed as percent of dead cells (number of red nuclear stained cells/total cell number). In general, at least 300 cells were counted.

Assay of clongenic growth was performed with a modification of the previously reported methods 14. Briefly, cells were cultured in 6-well plates and treated with CSE or camptothecin for 24 hours. Both floating and attached cells were harvested. After counting the cell number, cells were plated in non-coated 60 mm tissue culture dishes at 10³ cells/ml, 5 ml/dish in LHC-9/RPMI. Cells were maintained in culture for 7–10 days with medium changes every 2–3 days. Cells were then fixed with PROTOCOL (Fisher Diagnostics. Middletown, VA) and photographed. All colonies present in each well, defined as a cluster of 20 or more cells, were counted visually.

All data are expressed as mean ± standard error of the mean. Statistical comparisons of multi-group data were analyzed by analysis of variance (ANOVA) followed by student's t test for values that appeared different with the Tukey's (one-way) or Bonferroni's (two-way) post-test correction for multiple comparisons using PRISM4 software. P < 0.05 was considered significant.

Since NF-κB is involved in regulating cell death and survival in variety of cell types, activation of NF-κB in response to cigarette smoke exposure was investigated by EMSA. Cigarette smoke extract (5% and 10%) stimulated DNA binding activity of NF-κB as evidenced by the electrophoresis mobility shift assay (Figure 2).

To investigate the role of NF-κB in modulating cell survival, both the pharmacological inhibitor of NF-κB, curcumin, and RNA interference targeting p65 were used in the current study. Neither 10% CSE nor curcumin (up to 20 μM) alone affected cell viability as examined by LIVE/DEAD Cytotoxicity and Viability assay (Figure 3). When cigarette smoke extract and curcumin were added together, however, the number of dead cells was significantly increased (41.4 ± 1.2% of CSE plus curcumin vs 11.9 ± 1.8% of control, p < 0.01, Figure 3B).

The role of NF-κB in mediating cell survival was further investigated by small RNA inference of p65. Following transfection with p65-siRNA, the expression of p65 was suppressed nearly 100%; in contrast, there was little effect on p50 or β-actin, but an apparent increase in expression of STAT3, which was specifically suppressed by STAT3 siRNA (Figure 4).

Following transfection with p65-siRNA, HBEC cells were exposed to cigarette smoke extract for 24 hours. Cell death or survival was then evaluated by three different methods including LIVE/DEAD staining, COMET assay and clonogenic assay. Only the combination of p65-siRNA suppression and smoke exposure resulted in major cytotoxicity. In the presence of 10%CSE, the cells lacking p65 started to detach at 3–4 hrs after exposure to cigarette smoke and nearly all of the cells were detached from the culture dish after 24 hours exposure. In contrast, only a few cells in the control-siRNA treated group detached in response to cigarette smoke exposure. LIVE/DEAD staining demonstrated that very few cells were dead (red nuclei) in the non-targeting siRNA transfected cells (Figure 5A, 3.5 ± 0.3%), in the cells of p65-siRNA transfected without cigarette smoke exposure (Figure 5A, 7.0 ± 1.1%) or in non-targeting siRNA transfected cells exposed to 10% cigarette smoke (Figure 5A, 2.4 ± 1.0%). In contrast, nearly half of the cells were dead in the cells transfected with p65-siRNA and exposed to cigarette smoke extract (Figure 5A, 32.5 ± 1.8%, p < 0.01 compared to control siRNA transfected cells without exposure to cigarette smoke extract), which was similar to the cells exposed to camptothecin (data not shown). Consistent with LIVE/DEAD cytotoxicity assay, p65-depleted cells underwent severe DNA damage and apoptotic death when they were exposed to cigarette smoke as evidenced by COMET assay (Figure 5B). The apoptotic index was significantly higher in the cells lacking p65 and exposed to cigarette smoke (42.5 ± 6.5% vs 6.3 ± 1.2% of control, p < 0.01).

To further evaluate cell survival and ability to proliferate, clonogenic assay was performed. As shown in Figure 5C, neither cigarette smoke exposure of the non-targeting siRNA transfected cells (92.6 ± 14.9 colonies/dish) nor the p65-siRNA transfection alone (80.6 ± 8.1 colonies/dish) altered the ability of the cells to form colonies. However, cigarette smoke exposure of the p65-suppressed cells resulted in a significant decrease in colony formation (3.4 ± 1.4 colonies/dish, p < 0.01 compared to non-targeting siRNA transfection without cigarette smoke exposure 85.6 ± 9.6 colonies/dish). Similarly, colony formation was significantly decreased in camptothecin-treated cells (3.6 ± 1.1 colonies/dish, p < 0.01 compared to non-targeting siRNA transfection without cigarette smoke exposure).

To explore the mechanism of NF-κB prevent HBEC from death in response to cigarette smoke exposure, effect of cigarette smoke on anti-apoptotic protein synthesis was investigated. As shown in Figure 6A, cigarette smoke significantly stimulated anti-apoptotic protein Bcl-XL with strongest effect at 5%CSE. CSE also slightly stimulated Bcl2, another anti-apoptotic protein, but did not affect levels of pro-apoptotic proteins, Bax and Bad. Furthermore, up-regulation of Bcl-XL by CSE in HBECs was significantly reduced in the cells lacking of p65 (Figure 6B).