title: Chronic cigarette smoke extract treatment selects for apoptotic dysfunction and mitochondrial mutations in minimally transformed oral keratinocytes.
abstract: Cigarette smoke demonstrates a carcinogenic effect through chronic exposure, not acute exposures. However, current cell line models study only the acute effects of cigarette smoke. Using a cell line model, we compared the effects of acute versus chronic cigarette smoke extract (CSE) on mitochondria in minimally transformed oral keratinocytes (OKF6). OKF6 cells were treated with varying concentrations of CSE for 6 months. Cells were analyzed monthly by flow cytometry for mitochondrial membrane potential (MMP), cytochrome c release, caspase 3 activation and viability after CSE exposure. At each time point, the same assays were performed after 24 hr of valinomycin (MMP-depolarizing agent) treatment. The mitochondrial DNA of chronically CSE-treated cells was sequenced. After 6 months of CSE treatment, the cells were increasingly resistant to CSE-mediated and valinomycin-induced cell death. In addition, chronic CSE treatment caused chronic depolarization of MMP, cytochrome c release and caspase activation. Cells grown in the presence of only CSE vapor also exhibited the same resistance and chronic baseline apoptotic activation. Mitochondrial DNA sequencing found that chronic CSE-treated cells had more amino acid-changing mitochondrial mutations than acutely treated cells. CSE treatment of normal cells select for apoptotic dysfunction as well as mitochondrial mutations. These findings suggest that chronic tobacco exposure induces carcinogenesis via selection of apoptosis resistance and mitochondrial mutation in addition to previously known genotoxic effects that were found by acute treatments. Chronic models of tobacco exposure on upper aerodigestive epithelia may be more insightful than models of acute exposure in studying head and neck carcinogenesis.
authors.affiliation: Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins Medical Institutions, Baltimore, MD 21287-0910, USA.
authors.forename: Steven S
authors.initials: SS
authors.lastname: Chang
authors.forename: Wei Wen
authors.initials: WW
authors.lastname: Jiang
authors.forename: Ian
authors.initials: I
authors.lastname: Smith
authors.forename: Chad
authors.initials: C
authors.lastname: Glazer
authors.forename: Wen-Yue
authors.initials: WY
authors.lastname: Sun
authors.forename: Suhail
authors.initials: S
authors.lastname: Mithani
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Califano
mesh_terms: D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism; D002461:Cell Line, Transformed; D045304:Cytochromes c / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000235:genetics*; D004789:Enzyme Activation; D005434:Flow Cytometry; D006801:Humans; D015603:Keratinocytes / Q000166:cytology* / Q000201:enzymology; D008564:Membrane Potentials; D009154:Mutation; D012906:Smoke; D014634:Valinomycin / Q000494:pharmacology
chemical_list: D004272:DNA, Mitochondrial; D012906:Smoke; D014634:Valinomycin; D045304:Cytochromes c; D053148:Caspase 3