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.

Figure 1

Acute versus Chronic CSE treatment of OKF6 induces apoptotic resistance. a) CSE treatment of OKF6 results in chronic mitochondrial membrane potential depolarization. b) Chronic CSE treatment of OKF6 results in decreased cell death over time (Normalized to cells in normal incubator). c) Chronic CSE treated OKF6 are increasingly resistant to valinomycin induced depolarization. d) CSE treated OKF6 are increasingly resistant to mitochondrial membrane potential depolarization induced apoptosis. Values were normalized to normal incubator cells.

Figure 2

Chronic CSE treatment induces mild resistance to apoptosis induced by staurosporine a nonselective protein kinase inhibitor. Staurosporine induced apoptosis occurs via pathways that are different from valinomycin.

Figure 3

Chonic CSE treatment induces chronic activation of apoptotic mediators. a) Mitochondrial cytochrome c at baseline and in response to valinomycin treatment shows that at baseline, chronically treated cells retain less cytochrome c. b) Caspase 3 activation at baseline and in response to valinomycin treatment. CSE treated cells show virtually no response to valinomycin treatment with regard to caspase-3 activation.

Figure 4

Chronic CSE treatment induces amino acid changing mitochondrial DNA mutations. Chronic CSE treatment induces a greater number of all types of mutations, but as shown, its shows the greatest difference in the amino acid changing mutations.

Figure 5

CSE treatment of cells acute versus chronic. The number of total mutations and mutations in the coding region of the genome induced by .1% CSE over time increased.