Multiplexed quantitative high content screening reveals that cigarette smoke condensate induces changes in cell structure and function through alterations in cell signaling pathways in human bronchial cells

• Published in: Toxicology (Amsterdam) Vol. 261; no. 3; pp. 89 - 102
• Main Authors: Carter, Charleata A, Hamm, Jonathan T
• Format: Journal Article
• Language: English
• Published: Kidlington Elsevier Ireland Ltd 10.07.2009; Elsevier
• Subjects: Actins - metabolism; Apoptosis; Apoptosis - drug effects; Biological and medical sciences; Bronchi - drug effects; Bronchi - metabolism; Bronchi - pathology; Caspase 3; Caspase 3 - metabolism; Cell Adhesion - drug effects; Cell Line, Transformed; Cell Movement - drug effects; Cell Size - drug effects; Cell Survival - drug effects; Dose-Response Relationship, Drug; Emergency; Enzyme Activation; F-actin; Humans; Inhibitory Concentration 50; Medical sciences; Membrane Potential, Mitochondrial - drug effects; Mitochondria - drug effects; Mitochondria - metabolism; NF-kappa B - metabolism; NF-κB; Nicotiana; Protein kinase C; Protein Kinase C-alpha - metabolism; Signal Transduction - drug effects; Smoke - adverse effects; Smoking - adverse effects; Stress Fibers - drug effects; Stress Fibers - metabolism; Tobacco, tobacco smoking; Toxicology; Vinculin; Vinculin - metabolism; 12-O-tetradecanoylphorbol 13-acetate; cigarette smoke condensate; TPA; DMSO; dimethylsulfoxide; activator protein-1; 4′-6-Diamidino-2-phenylindole; DAPI; nuclear factor-kappa B; PKC; AP-1; NF-κB; CSC; FITC; high content screening; F-actin; Vinculin; Caspase 3; HCS; fluorescein isothiocyanate; protein kinase C; COPD; chronic obstructive pulmonary disease; Apoptosis; Protein kinase C; Structure function; Cell function; Toxicity; Cysteine endopeptidases; Tobacco smoking; Respiratory system; Respiratory tract; Cigarette; Signal transduction; Signaling pathway; Condensates; Transcription factor NFκB; Fumes; Quantitative analysis; Human; Cell structure; Enzyme; Transferases; Bronchus; Medical screening; Peptidases; Screening; Tobacco; Cell death; Hydrolases
• ISSN: 0300-483X; 1879-3185
• DOI: 10.1016/j.tox.2009.04.039

Abstract Human bronchial cells are one of the first cell types exposed to environmental toxins. Toxins often activate nuclear factor-κB (NF-κB) and protein kinase C (PKC). We evaluated the hypothesis that cigarette smoke condensate (CSC), the particulate fraction of cigarette smoke, activates PKC-α and NF-κB, and concomitantly disrupts the F-actin cytoskeleton, induces apoptosis and alters cell function in BEAS-2B human bronchial epithelial cells. Compared to controls, exposure of BEAS-2B cells to doses of 30 μg/ml CSC significantly activated PKC-α, while CSC doses above 20 μg/ml CSC significantly activated NF-κB. As NF-κB was activated, cell number decreased. CSC treatment of BEAS-2B cells induced a decrease in cell size and an increase in cell surface extensions including filopodia and lamellipodia. CSC treatment of BEAS-2B cells induced F-actin rearrangement such that stress fibers were no longer prominent at the cell periphery and throughout the cells, but relocalized to perinuclear regions. Concurrently, CSC induced an increase in the focal adhesion protein vinculin at the cell periphery. CSC doses above 30 μg/ml induced a significant increase in apoptosis in BEAS-2B cells evidenced by an increase in activated caspase 3, an increase in mitochondrial mass and a decrease in mitochondrial membrane potential. As caspase 3 increased, cell number decreased. CSC doses above 30 μg/ml also induced significant concurrent changes in cell function including decreased cell spreading and motility. CSC initiates a signaling cascade in human bronchial epithelial cells involving PKC-α, NF-κB and caspase 3, and consequently decreases cell spreading and motility. These CSC-induced alterations in cell structure likely prevent cells from performing their normal function thereby contributing to smoke-induced diseases.