Exposure of Human Lung Cells to Tobacco Smoke Condensate Inhibits the Nucleotide Excision Repair Pathway

Exposure to tobacco smoke is the number one risk factor for lung cancer. Although the DNA damaging properties of tobacco smoke have been well documented, relatively few studies have examined its effect on DNA repair pathways. This is especially true for the nucleotide excision repair (NER) pathway which recognizes and removes many structurally diverse DNA lesions, including those introduced by chemical carcinogens present in tobacco smoke. The aim of the present study was to investigate the effect of tobacco smoke on NER in human lung cells. We studied the effect of cigarette smoke condensate (CSC), a surrogate for tobacco smoke, on the NER pathway in two different human lung cell lines; IMR-90 lung fibroblasts and BEAS-2B bronchial epithelial cells. To measure NER, we employed a slot-blot assay to quantify the introduction and removal of UV light-induced 6–4 photoproducts and cyclobutane pyrimidine dimers. We find a dose-dependent inhibition of 6–4 photoproduct repair in both cell lines treated with CSC. Additionally, the impact of CSC on the abundance of various NER proteins and their respective RNAs was investigated. The abundance of XPC protein, which is required for functional NER, is significantly reduced by treatment with CSC while the abundance of XPA protein, also required for NER, is unaffected. Both XPC and XPA RNA levels are modestly reduced by CSC treatment. Finally, treatment of cells with MG-132 abrogates the reduction in the abundance of XPC protein produced by treatment with CSC, suggesting that CSC enhances proteasome-dependent turnover of the protein that is mediated by ubiquitination. Together, these findings indicate that tobacco smoke can inhibit the same DNA repair pathway that is also essential for the removal of some of the carcinogenic DNA damage introduced by smoke itself, increasing the DNA damage burden of cells exposed to tobacco smoke

Exposure of Human Lung Cells to Tobacco Smoke Condensate Inhibits the Nucleotide Excision Repair Pathway

<div><p>Exposure to tobacco smoke is the number one risk factor for lung cancer. Although the DNA damaging properties of tobacco smoke have been well documented, relatively few studies have examined its effect on DNA repair pathways. This is especially true for the nucleotide excision repair (NER) pathway which recognizes and removes many structurally diverse DNA lesions, including those introduced by chemical carcinogens present in tobacco smoke. The aim of the present study was to investigate the effect of tobacco smoke on NER in human lung cells. We studied the effect of cigarette smoke condensate (CSC), a surrogate for tobacco smoke, on the NER pathway in two different human lung cell lines; IMR-90 lung fibroblasts and BEAS-2B bronchial epithelial cells. To measure NER, we employed a slot-blot assay to quantify the introduction and removal of UV light-induced 6–4 photoproducts and cyclobutane pyrimidine dimers. We find a dose-dependent inhibition of 6–4 photoproduct repair in both cell lines treated with CSC. Additionally, the impact of CSC on the abundance of various NER proteins and their respective RNAs was investigated. The abundance of XPC protein, which is required for functional NER, is significantly reduced by treatment with CSC while the abundance of XPA protein, also required for NER, is unaffected. Both XPC and XPA RNA levels are modestly reduced by CSC treatment. Finally, treatment of cells with MG-132 abrogates the reduction in the abundance of XPC protein produced by treatment with CSC, suggesting that CSC enhances proteasome-dependent turnover of the protein that is mediated by ubiquitination. Together, these findings indicate that tobacco smoke can inhibit the same DNA repair pathway that is also essential for the removal of some of the carcinogenic DNA damage introduced by smoke itself, increasing the DNA damage burden of cells exposed to tobacco smoke.</p></div

CSC inhibits NER in IMR-90 human lung fibroblasts.

<p>(A) Cell viability after CSC treatment. Confluent IMR-90 cells were treated with CSC (or mock treated with DMSO) with the concentrations shown for 24 h and the percentage of viable cells was measured using Trypan blue dye exclusion. The data represent the mean ± SE (Standard Error) from four independent experiments (except for 60 μg/ml CSC which included two independent experiments). (B) Removal of 6–4 PPs. Cells were treated with the concentrations of CSC shown or with DMSO for 24 h and irradiated with 20 J/m² UVC to introduce photolesions. After irradiation, cells were either lysed immediately or after incubation in medium containing CSC or DMSO for the times (h) shown to permit repair. The immunoblot assay to detect 6–4 PPs was performed and samples were loaded in duplicate for each repair time point. (C) Removal of 6–4 PPs. A graphical representation of results obtained from multiple immunoblots measuring the removal of 6–4 PPs is shown. Each data point represents the mean ± SE of three repeats from two independent experiments. (D) Removal of CPDs. IMR-90 cells were treated with 200 μg/mL CSC or with DMSO for 24 h and irradiated with 2 J/m² UVC to introduce photolesions. After irradiation, cells were either lysed immediately or after incubation in medium containing CSC or DMSO for the times shown to permit repair. An immunoblot assay to detect CPD lesions was performed and samples were loaded in triplicate for each time point. (E) Removal of CPDs. A graphical representation of results obtained from multiple immunoblots measuring the removal of CPDs is shown. Each data point represents the mean ± SE of three repeats from two independent experiments.</p

The effect of CSC on the abundance of XPC and XPA RNA in IMR-90 cells.

<p>Cells were treated with the indicated concentrations of CSC (or DMSO) for 24 hours. RNA was isolated and Real Time PCR was performed as described in the methods section. The expression of XPC or XPA RNA was normalized to GAPDH RNA for each treatment. The data presented are the mean ± SE of one analysis from three independent experiments.</p

CSC inhibits NER and the abundance of XPC protein in BEAS-2B cells.

<p>(A) Cells were treated with the concentrations of CSC shown for 16 h and the percentage of viable cells was measured using Trypan blue dye exclusion. The data presented are the mean ± SE of four biological experiments. (B) Cells were treated with 175 μg/mL CSC (or DMSO) for 16 h, and irradiated with 20 J/m² UVC to introduce photolesions. After irradiation, cells were either lysed immediately or after incubation in medium containing CSC or DMSO for increasing periods of time to permit repair. An immunoblot assay was performed and samples were loaded in duplicate to measure the removal of 6–4 PPs. (C) A graphical representation of multiple immunoblots for 6–4 repair is shown. Each data point represents the mean ± SE of four repeats from one experiment. (D) Cells were treated with the different concentrations of CSC shown for 24 h, lysed, and the abundance of XPC and XPA were examined by western analysis. (E) A graphical representation of multiple western blots for XPC and XPA protein is shown. The data presented are the mean ± SE of two repeats from two independent experiments for XPC and three repeats of one experiment for XPA. XPC and XPA values were normalized to β-actin.</p

The impact of CSC on XPC protein and NER efficiency depends on treatment duration.

<p>(A) IMR-90 Cells were treated with 200 μg/ml of CSC for the times indicated and XPC expression was examined by western blot analysis. (B) A graphical representation of multiple western blots examining the time course of inhibition for XPC expression after CSC treatment is shown. The data presented are the mean ± SE of three repeats from one experiment, and XPC expression was normalized to β-actin. (C) Results of an immunoblot showing the time course of the effect of CSC on repair of 6–4 PPs in IMR-90 cells. Cells were treated with 200 μg/mL CSC for 8, 16, or 24 h (or DMSO for 24 h) and irradiated with 20 J/m² UVC to introduce photolesions. After irradiation, cells were either lysed immediately or after incubation in medium containing CSC or DMSO for the times shown (3, 6 and 8 h) to permit repair. An immunoblot assay was performed and samples were loaded in duplicate to measure the removal of 6–4 PPs. (D) A graphical representation of multiple immunoblots for 6–4 repair is shown. Each data point represents the mean ± SE of three repeats from one experiment.</p

Involvement of the proteasome in the reduced expression of XPC protein in IMR-90 cells after treatment with UV or CSC.

<p>(A) Cells were treated with 10 μM MG-132 or untreated for 4 hours, and then irradiated with 20 J/m² UV-C. After irradiation, cells were either lysed immediately or after incubation for increasing periods of time in the same type of medium as was used for the pretreatment; medium containing MG-132 or not containing MG-132. XPC expression was measured using Western blot analysis. (B) A graphical representation of the experiments from (A) is shown. XPC expression was normalized to β-actin. The data presented are the mean of two repeats from one experiment. The percent XPC protein was calculated by comparing post-UV time points to the appropriate 0 h (MG-treated or not MG-treated) time point. Treatment with MG-132 for 4 h had a negligible impact on XPC expression before irradiation, so both 0 h values were set to 100%. (C) Cells were treated with a combination of CSC and/or MG-132 at the indicated concentrations for 16 h. XPC levels were measured by Western blot analysis and normalized to Actin. (D) A graphical representation of the average obtained from two different blots for the experiments in (C) is shown; treatment with 3 μM MG-132 was not included. XPC expression was normalized to β-actin.</p