A549 and Beas-2b lung epithelial cells were purchased from American Tissue Cell Culture ATCC, Manassas, VA, USA. A549 cells were cultured in Ham's F-12 medium (Gibco, Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum and 0.1% gentamicine (Gibco, Invitrogen, Carlsbad, USA) in a cell culture environment (humidified atmosphere of 5% CO2/balanced air at 37°C). Beas-2b cells were cultured according to the ATCC prescription using the serum-free medium BEGM (Cambrex, East Rutherford, NJ, USA). Before the experiments all cells were exposed 'overnight' to serum- or growth factor free (BEBM, Cambrex, East Rutherford, NJ, USA) media.
Chemicals and Reagents
All reagents were purchased from Sigma (Sigma-Aldrich Corp. St. Louis, MO, USA) unless otherwise mentioned. Hemin (ferriprotoporphyrin-IX chloride) was dissolved in DMSO to a stock solution of 10mM, which was kept in -20°C until use. For experiments, the stock solution of hemin was dissolved to a working concentration of 10mM. Lipopolysaccharide (LPS) was freshly prepared and used at a concentration of 100ng/mL in the experiment described.
Western blot analysis
The following antibodies were used for immunoblotting: anti-heme oxygenase-1 (1:2000) (Stressgen, Victoria, BC, Canada), anti-cytochrome-c, anti-caspase-3, anti-caspase-9, anti-Bax, anti-apoptosis inducing factor etc….etc….. (Santa Cruz, Santa Cruz, CA, USA), anti-6A7/Bax (BD) (all 1:1000). Briefly, cells were washed twice in ice-cold PBS and scraped of in cell lysis buffer (1% Nomidet P40, 150mM NaCl, 20mM Tris pH 8.0, 1mM Na3VO4, 1mM phenylmethylsulfonyl fluoride and 10mg/ml aprotinin with complete protease inhibitor mixture (Roche, Indianapolis, IN, USA)). Cellular protein extracts were electropheresed under denaturing conditions (10-12.5% polyacrylamide gels) and transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA). After that, membranes were incubated overnight with blocking buffer (5% nonfat dry milk in TTBS (10% Tween in Tris buffered saline) and washed with TTBS. The blots were incubated with anti HO-1 for one hour, and with all the other primary antibodies 'overnight', and washed again with TBS containing 0.05% Tween 20. Blots were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000). Bound IgGs were visualized using an enhanced chemiluminescence detection kit (Amersham, Uppsala, Sweden).
Mitochondrial Isolation
A549 and Beas-2b cells were harvested in 0.05% digitonin in an extraction buffer containing 10 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM EGTA, and 1.5 mM MgCl2. The cell extracts were spun at 700g for 10 min. The supernatants were transferred to new tubes and centrifuged again at 30,000g at 4 °C for 30 min. The resulting supernatants were removed, and the pellets were either retained for western blotting, or for assessment of HO-enzyme activity.
Carbon monoxide exposure
For cell culture experiments, CO at a concentration of 1% (10,000 parts per million (ppm)) in compressed air was mixed with compressed balanced air containing 5% CO2 in a stainless steel mixing cylinder before being delivered at 250 ppm into the exposure chamber, with a flow into the chamber of 2 L/min. The chamber was dark, humidified and maintained at 37°C. A CO analyzer (Interscan, Chatsworth, CA) was used to measure CO levels in the chamber and there were no fluctuations in the CO concentrations after the chamber had equilibrated.
Preparation of cigarette smoke extract (CSE)
Kentucky 1R3F research-reference filtered cigarettes (The Tobacco Research Institute, University of Kentucky, Lexington, KY) where smoked using a peristaltic pump (VWR Int. West Chester, PA, USA). Just before the experiments, the filters were cut form the cigarettes. The smoke was directly bubbled through cell growth medium. Each cigarette was smoked in 6 minutes with a 17 mm but remaining. Four cigarettes were bubbled through 20 ml of medium, and this solution was regarded as 100% strength CSE. The 100% CSE was adjusted to a pH of 7.45 and was used within 15 minutes after preparation.
Viability
Cell viability was assessed using the trypan blue exclusion method and the crystal violet uptake method. For the trypan blue exclusion method, cell monolayers were washed with phosphate buffered saline (PBS). The cells were detached from the culture dishes utilizing 0.05% trypsin/0.5 mM EDTA solution, and a similar volume of fetal bovine serum inhibited proteolytic action. Thereafter, 10ml of the cell solution was added to 10ml 0.4% (w/v) trypan blue solution and the cells were counted, using a hemocytometer under light microscopy. Cells failing to exclude the dye were considered non-viable cells. Data are expressed as percentage of viable cells.
For the crystal violet dye uptake method, briefly 0.5% (w/v) crystal violet was dissolved in 30% ethanol and 8% formaldehyde in water. Cultured cells were washed twice with PBS and enough crystal violet was added to cover the bottom of the plate, and was incubated while gently shaken for 15 min. Thereafter the plates were washed with PBS till absolutely clean and the plates were dried overnight. The next day a 1% SDS solution was added, and after 8h viability was assessed by triplicate measurements of each sample using a 96 well plate, read at 550nm in a plate reader (Spectramax 190, Molecular Devices, Sunnyvale, CA, USA). Data are expressed as percentage of the control sample media.
AnnexinV-FITC propidium iodide (PI) staining
Using the fluorescein isothiocyanate (FITC)-labelled annexinV kit from BD Pharmingen (San Diego, CA, USA), we followed the manufacturer's protocol. Briefly, cell monolayers were washed with PBS and detached from the culture dishes utilizing 0.05% trypsin/0.5 mM EDTA solution. Then the cells were washed twice with cold PBS and resuspended with binding buffer (10 mM HEPES/NaOH (pH 7.4), 140 mM NaCl, and 2.5 mM CaCl2) before transferring 1 x 105 cells to a 5-ml tube. Then 5 µl of annexinV-FITC and 5 µl (100µg/ml) of PI were added, and cells were incubated for 15 min in the dark. Finally binding buffer (400 µl) was added to each tube before the labelled cells were analysed using a FACS-Calibur (BD Biosciences, San Diego, CA, USA).
Heme oxygenase enzyme activity assay
For meausurement of HO enzyme activity in whole cell lystates, cells were washed with PBS and then scraped in a sucrose-Tris pH 7.4 buffer. The cell suspensions were then sonicated one ice (3 x 10 sec). The mitochondrial protein fraction was prepared as described above. After ultra centrifuging, the pellet was resuspended in sucrose buffer. The protein concentration of cellular and mitochondrial fractions was determined using the Coomassie blue dye-binding assay (BioRad, Hercules, CA, USA). The HO activity was measured by the spectrophotometric determination of bilirubin production, as described previously. For HO activity, final reaction concentrations were: 25 µM heme, 2 mM glucose 6-phosphate, 2 U glucose 6-phosphate dehydrogenase (Sigma, Type XV from Baker's Yeast), 1 mM ß-NADPH and 2 mg/mL partially purified bovine liver biliverdin reductase preparation (prepared according to the protocol of Tenhunen et al.[ ]). Reaction mixtures were incubated for 60 min in a 37°C water bath in the dark. The reactions were terminated by addition of 1 volume of chloroform. Bilirubin concentration in the chloroform extracts was determined on a DU640 scanning spectrophotometer (Beckman Coulter, Fullerton, CA, USA) by measuring O.D. (464-530 nm). HO activity is reported as pmol bilirubin/mg protein/h assuming an extinction coefficient of 40 mM-1 cm-1 for bilirubin in chloroform. Both cell and mitochondrial fractions were prepared and run through the same experiment each time. Three independent experiments were performed, each with triplicate bilirubin determinations.
Adenosine Triphosphate (ATP) assay
ATP was measured by using a ATP-luciferace assay kit (Calbiochem, San Diego, CA, USA) according to the manufactures instructions. Briefly, Beas-2b cells were seeded at a concentration of 5*103 cells/well in white coated, tissue culture suitable, 96-well microplates (BD Falcon, San Jose, CA, USA) and grown under standard cell culture conditions for 48h until the experiments. A stock solution of 'ATP monitoring enzyme' with an 'enzyme reconstitution buffer' was made and stored in small samples in -80°C to prevent thaw/freezing. After the cell experiments, the cells were gently shaken in 100ml of nuclear releasing agent for 5 min. Thereafter 1ml of the pre-diluted ATP monitoring enzyme was put in each well and luciferase activity was measured within one minute using a luminometer (Lmax, molecular devices, Sunnyvale, CA, USA).. An ATP standard calibration curve was made using the kits ATP standard solution.
Immunofluorescence labeling and confocal laser microscopy
Beas-2b cells were grown on 35mm/10mm glass bottom culture dishes (MatTek Corp. Ashland, MA, USA) for immunofluorescence labeling. Living cells were, using growth media conditions at 37°C stained for mitochondria using the Mitotracker Red dye (M-7513, Molecular Probes Inc. Eugene, OR, USA), at a concentration of 1mM for 45 min. After this mitochondrial staining, cells were washed with PBS and fixed and permeabilised in 2% paraformaldehyde with 0.1% Triton-X100. Thereafter cells were washed with PBS and washbuffer (0.5% bovine serum albumin, 0.15% glycin in PBS) and blocked with 10% goatserum in washbuffer. Immunostaining was performed using a polyclonal HO-1 antibody (1:1000; Stressgen, Victoria, BC, Canada). After rinsing the cells in washbuffer, bound primary antibodies were visualized with goat anti rabbit Alexa 488 conjugated secondary antibodies (1:1000; Jackson ImmunoResearch Laboratories, Inc. PA, USA). Cells were viewed with an Olympus Fluoview BX 61 confocal microscope and images were collected using a DC-330S cooled CCD camera (DAGE-MTI Inc., Michigan, USA).
Transfection protocols
Adenovirus-HO-1 (Ad-HO-1) and adenovirus-LacZ (Ad-LacZ) were supplied by the Center for Biotechnology & Bioengineering, University of Pittsburgh, PA, USA (www.vectorcore.pitt.edu). Briefly, beas-2b cells were grown until sub-confluency and starved overnight. Ad-HO-1 and Ad-LacZ were added using 50 pfu/cell. After 6h the medium containing the virus was changed to regular growth medium and the experiments were performed the next day.
Transition Electron Microscopy
Beas-2b cells were after the experiments washed with PBS and fixed in 2.5% glutaraldehyde in PBS. The cells were photographed using a JEOL JEM 1210 transmission electron microscope (Peabody, MA, USA) at 80 or 60 kV onto electron microscope film (Kodak, ESTAR thick base, Rochester, NY) and printed onto photographic paper. Rabbit anti-HO-1 polyclonal antibody (Stressgen, Victoria, BC, Canada) suitable for IHC was used for gold-immunostaining……..
Results
HO-1 is expressed in the mitochondria
Because of the supposed effects of CSE at the mitochondrial level and the possible role in the defense against oxidative stress/CSE by HO-1 we set out to study the localization of HO-1 in the cell. At first we exposed A549 and Beas-2b cells with 10mM hemin, a potent inducer of HO-1, for 18h. We observed an inducible HO-1 protein expression in the mitochondrial fractions of both cell lines (figure&&.). To show that this induction is not only substrate dependent (hemin) we exposed beas-2b cells to both LPS (0.1mg/ml) and CSE. Both stimuli were also able to induce mitochondrial HO-1, with a striking increase in the CSE induced mitochondrial HO-1 (figure&.). The mitochondrial extraction was furthermore also performed by a different technician (M vd Toorn) using a different extraction protocol (&&&&) in a different lab (data not shown). HO-1 appeared also inducible in whole cell substrates in a dose and time dependent manner when using CSE (figure &&.). We confirmed the mitochondrial localization of HO-1 using immunogold labeling on electron microscopy (figure&&.) and immunofluorescent staining for confocal laser microscopy (figure&&..).
Control (10000x) Hemin 10mM (10000x) Hemin 10mM (25000x)
HO-1 Mitotracker Red Merge
Mitochondrial HO-1 is largely inducible and highly active
To study the functional activity of the mitochondrial HO-enzyme, we used an HO enzyme activity reaction. After exposure of Beas-2b cells for 18h with 10mM hemin or 15% CSE we isolated mitochondrial protein and whole cell lysates. As expected both hemin and CSE induced the HO-activity in whole cell lysates from baseline (175±28 pmol bilirubin/mg protein/h) four to five fold (hemin: 849±114; CSE: 675±35 pmol bilirubin/mg protein/h) (figure 19). Surprisingly HO activity in the mitochondrial fractions increased 13 fold (for hemin) to 19 fold (for CSE) compared to the baseline mitochondrial HO activity (308±58 pmol bilirubin/mg protein/h).
Cigarette smoke extract induces both apoptosis and necrosis in lung epithelial cells.
Until now the mechanisms of CS induced epithelial cell death remain unclear. Hoshino et al. reported that low concentrations of CS-extract (CSE) induced apoptosis, and high concentrations of CSE in A549 cells induced necrosis, both in a dose and time dependent manner [12]. They speculate that the effects seen might be due to an interaction between aldehydes and oxidants present in the CSE, or formed in the CSE exposed EC. This same observation has also been made in cultured human fibroblasts: lower CSE%: apoptosis, higher %: necrosis [13]. Aoshiba et al. reported also apoptosis induces by CSE in alveolar macrophages, both in vitro and in vivo. In their study apoptosis was associated with increased oxidative stress, Bax accumilation, mitochondrial dysfunction, cytochrome-c release, but was independent of p53, Fas and caspase activation [14]. Furthermore, CS has been shown to induce apoptosis in rat bronchial/bronchiolar epithelial cells model [15].
In non-pulmonary cells, induction of apoptosis by CSE at low concentrations and necrosis at higher concentrations has been described in a human premonocytic line (U-937) [16]. Furthermore, exposure to CSE induced apoptosis in rat gastric mucosa cells through a ROS mediated (and p53-indipendent pathway) [17], and in a human gastric adenocarcinoma cell line, apoptosis could be induced by exposure to CSE via inhibition of BCL-2 and the activation of a mitochondria related pathway and caspase-3 dependency [18]. Also has CSE been shown to induce caspase-3 activity and apoptosis in a human umbilical venous endothelial cell line [19].
In contrast to all previous studies, Wickenden et al. reported recently that CSE exposure to A549 cells causes only necrosis, and even prevents apoptosis [20]. They suggested that this CSE induced apoptosis prevention takes possibly place at an early step in the caspase cascade thereby not activating caspase-3 and 9, and it is furthermore hypothesize that CSE treatment may alter the levels of pro- and antiapoptogenic factors downstream of the mitochondria to inhibit active apoptosome formation.
To investigate the CSE induced cell death process in lung epithelial cells, Beas-2b cells we exposed to 50% CSE for 0-5h, and apoptosis and necrosis were assessed by using the annexinV-FITC/propidium iodide (PI) assay (figure ….). By using this assay we showed that already after 1h a peak in the annexinV-FITC signal could be noticed, decreasing in the hours thereafter. The percentage of PI (necrotic cells) however, increased after the first hour, to keep on increasing until the last hour measured (5h). To investigate if there is a dose dependency in the occurrence apoptosis and necrosis we exposed the beas-2b cells to different concentrations of CSE for 3h (figure ….). Below a concentration of 30% CSE, almost only AnnexinV-FITC positive cells (apoptosis) could be shown, whereas from a 30% strength CSE and on necrosis started. We furthermore confirmed the occurrence of CSE induced apoptosis in beas-2b cells by showing occurrence of a time dependent splicing of active caspase-3 when using 30% CSE (figure….)
To be done II:
Final experiments on mitochondrial apoptotic factors (Wang/Choi):
Cells Induced with +control (PMA or menadion or H2O2 or….) and with CSE (30%)
Intervention with AdHO-1
Assessement of: -Cytochrome c
-Bax
