title: Repeated exposure to whole cigarette smoke promotes primary human gingival epithelial cell growth and modulates keratin expression.
abstract: OBJECTIVE
  The gingiva is the first oral tissue directly exposed to cigarette smoke (CS). Exposure to CS compromises the structure and function of gingival tissue. Damaging or altering the gingival epithelium leads to a compromised protective barrier of the periodontium, resulting in several diseases. The aim of this study was to assess the effect of repeated exposure to CS on gingival epithelial cell growth and on expression of apoptotic protein and keratin.
  
  
  METHODS
  Primary human gingival epithelial cells were seeded on a collagen scaffold for 5 d to allow growth and stratification. The cells were then exposed for 5 min to whole CS for 3, 6 and 9 d. At the end of each exposure period, cell proliferation [using (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (MTT) and 5-bromo-2'-deoxyuridine (BrdU) assays], gene expression [by quantitative reverse transcription polymerase chain reaction (qRT-PCR)] and protein production (by western blot analysis) were investigated.
  
  
  RESULTS
  Higher metabolic activity was found in the CS-exposed cells than in the nonexposed cells, specifically after 3 and 6 d of exposure to CS. At 9 d there was no significant difference between CS-exposed and nonexposed cells. Metabolic activity was supported by the BrdU cell-proliferation analyses, which showed increased cell growth at 3 d compared with the control. However, at 6 and 9 d, cell proliferation in the CS-exposed culture was comparable to that in the nonexposed culture. Interestingly, the Bax/Bcl-2 protein ratios decreased with increased CS exposure, suggesting cell resistance. Moreover, protein analyses showed that CS decreased expression of keratin(K) 5 at 3, 6 and 9 d, and increased expression of K14 at 6 and 9 d. Finally, mRNA analyses showed significant decreases of K1, K6, K10 and K16 in CS-exposed cultures, correlating, at times, with a decrease of protein production.
  
  
  CONCLUSIONS
  CS was shown to increase epithelial cell proliferation, which may involve cell resistance to apoptosis. This is supported by the modulation of expression of different keratin genes and proteins. Altogether, these data may explain the hyperplasia reported in gingival tissue, as well as periodontal disease, in smokers.
authors.affiliation: Groupe de Recherche en Écologie Buccale, Faculté de Médecine Dentaire, Université Laval, Québec, QC, Canada.
authors.forename: I A
authors.initials: IA
authors.lastname: Alharbi
authors.affiliation: Groupe de Recherche en Écologie Buccale, Faculté de Médecine Dentaire, Université Laval, Québec, QC, Canada.
authors.forename: M
authors.initials: M
authors.lastname: Rouabhia
mesh_terms: D000293:Adolescent; D000328:Adult; D017209:Apoptosis / Q000187:drug effects; D001973:Bromodeoxyuridine; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism*; D004848:Epithelium / Q000187:drug effects; D005786:Gene Expression Regulation; D005881:Gingiva / Q000166:cytology* / Q000254:growth & development*; D006801:Humans; D007633:Keratins / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D012906:Smoke / Q000009:adverse effects*; D013778:Tetrazolium Salts; D013844:Thiazoles; D013997:Time Factors; D014026:Nicotiana / Q000009:adverse effects*; D055815:Young Adult; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism
chemical_list: C494826:BAX protein, human; C000595707:BCL2 protein, human; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D012906:Smoke; D013778:Tetrazolium Salts; D013844:Thiazoles; D051028:bcl-2-Associated X Protein; D007633:Keratins; C022616:thiazolyl blue; D001973:Bromodeoxyuridine
keywords: cigarette smoke; epithelial cell; keratin; periodontium; proliferation