title: Effects of cigarette smoke condensate on proliferation and wound closure of bronchial epithelial cells in vitro: role of glutathione.
abstract: BACKGROUND
  Increased airway epithelial proliferation is frequently observed in smokers. To elucidate the molecular mechanisms leading to these epithelial changes, we studied the effect of cigarette smoke condensate (CSC) on cell proliferation, wound closure and mitogen activated protein kinase (MAPK) activation. We also studied whether modulation of intracellular glutathione/thiol levels could attenuate CSC-induced cell proliferation.
  
  
  METHODS
  Cells of the bronchial epithelial cell line NCI-H292 and subcultures of primary bronchial epithelial cells were used for the present study. The effect of CSC on epithelial proliferation was assessed using 5-bromo-2-deoxyuridine (BrdU) incorporation. Modulation of epithelial wound repair was studied by analysis of closure of 3 mm circular scrape wounds during 72 hours of culture. Wound closure was calculated from digital images obtained at 24 h intervals. Activation of mitogen-activated protein kinases was assessed by Western blotting using phospho-specific antibodies.
  
  
  RESULTS
  At low concentrations CSC increased proliferation of NCI-H292 cells, whereas high concentrations were inhibitory as a result of cytotoxicity. Low concentrations of CSC also increased epithelial wound closure of both NCI-H292 and PBEC, whereas at high concentrations closure was inhibited. At low, mitogenic concentrations, CSC caused persistent activation of ERK1/2, a MAPK involved in cell proliferation. Inhibition of cell proliferation by high concentrations of CSC was associated with activation of the pro-apoptotic MAP kinases p38 and JNK. Modulation of intracellular glutathione (GSH)/thiol levels using N-acetyl-L-cysteine, GSH or buthionine sulphoximine (BSO), demonstrated that both the stimulatory and the inhibitory effects of CSC were regulated in part by intracellular GSH levels.
  
  
  CONCLUSIONS
  These results indicate that CSC may increase cell proliferation and wound closure dependent on the local concentration of cigarette smoke and the anti-oxidant status. These findings are consistent with increased epithelial proliferation in smokers, and may provide further insight in the development of lung cancer.
authors.affiliation: Department of Pulmonology, Leiden University Medical Center, PO Box 9600, 2300RC, Leiden, The Netherlands. luppi.fabrizio@unimo.it
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Luppi
authors.forename: Jamil
authors.initials: J
authors.lastname: Aarbiou
authors.forename: Sandra
authors.initials: S
authors.lastname: van Wetering
authors.forename: Irfan
authors.initials: I
authors.lastname: Rahman
authors.forename: Willem I
authors.initials: WI
authors.lastname: de Boer
authors.forename: Klaus F
authors.initials: KF
authors.lastname: Rabe
authors.forename: Pieter S
authors.initials: PS
authors.lastname: Hiemstra
mesh_terms: D001980:Bronchi / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology*; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism; D013638:Tars / Q000494:pharmacology*; D014945:Wound Healing / Q000187:drug effects*
chemical_list: D017382:Reactive Oxygen Species; D013638:Tars; C024746:tobacco tar; D005978:Glutathione