Thirdhand smoke exposure causes replication stress and impaired transcription in human lung cells

Abstract

Thirdhand cigarette smoke (THS) is a newly described toxin that lingers in the indoor environment long after cigarettes have been extinguished. Emerging results from both cellular and animal model studies suggest that THS is a potential human health hazard. DNA damage derived from THS exposure could have genotoxic consequences that would lead to the development of diseases. However, THS exposure-induced interference with fundamental DNA transactions such as replication and transcription, and the role of DNA repair in ameliorating such effects, remain unexplored. Here, we found that THS exposure increased the percentage of cells in S-phase, suggesting impaired S-phase progression. Key DNA damage response proteins including RPA, ATR, ATM, CHK1, and BRCA1 were activated in lung cells exposed to THS, consistent with replication stress. In addition, THS exposure caused increased 53BP1 foci, indicating DNA double-strand break induction. Consistent with these results, we observed increased micronuclei formation, a marker of genomic instability, in THS-exposed cells. Exposure to THS also caused a significant increase in phosphorylated RNA Polymerase II engaged in transcription elongation, suggesting an increase in transcription-blocking lesions. In agreement with this conclusion, ongoing RNA synthesis was very significantly reduced by THS exposure. Loss of nucleotide excision repair exacerbated the reduction in RNA synthesis, suggesting that bulky DNA adducts formed by THS are blocks to transcription. The adverse impact on both replication and transcription supports genotoxic stress as a result of THS exposure, with important implications for both cancer and other diseases.

Keywords: DNA damage response; DNA double-strand breaks; DNA repair; micronuclei frequency; stalled RNA polymerase; tobacco smoking.

Fig. 1.

Growth rate, cell cycle distribution and proliferation analysis following exposure of BEAS-2B cells to THS. (A) Growth rate was analyzed following exposure to 5% THS. Cells were counted after 24, 48, 72, 96 and 144 h of exposure and plotted as fold increase vs time. (B) Cell cycle distributions were analyzed by PI staining protocol and plotted. Quantification shows accumulation of S-phase cells following exposure to 2.5% THS. CTRL represents mock exposure. Bars represent the mean from 3 experiments. (C) Cells were exposed to 5% or 2.5% THS for 24 h followed by EdU incorporation (4 h), fixed, permeabilized, conjugated with Alexa Fluor 488 by the Click-iT reaction, and imaged. Mock (only DMEM) exposure done in parallel served as control. (D) Quantification shown as percent EdU positive cells. Data are the mean ± SD from N=3.

Fig. 2.

Loss of NER reduces the stimulation of DNA synthesis by THS exposure. (A) DNA synthesis following 24 h exposure to 5% THS was measured by EdU incorporation in BEAS-2B cells treated with a non-specific control siRNA (siCTRL) or with siRNA specific for XPA. (B) EdU fluorescence intensity of 100 cells each from siCTRL and siXPA cultures was monitored and plotted. Data represent the mean of ±SD for N=3. (C) Knock-down (KD) of XPA in BEAS-2B cells by treatment with siXPA vs siCTRL, analyzed by western. XPB was used as a loading control

Fig. 3.

Replication stress following exposure of lung cells to THS as evidenced by RPA phosphorylation. (A) BEAS-2B cells were exposed to various doses of THS as indicated and RPA phosphorylation was monitored by western analysis with phospho-RPA32 (S4/S8) antibody. GAPDH was used as a loading control for normalization of pRPA32 signal at each time point in order to calculate fold increase for each THS dose compared to mock treated control. The same samples were also analyzed with anti-RPA32 antibody to assess the amount of total RPA32. (B). Time course of RPA phosphorylation in BEAS-2B cells exposed to 5% THS for various lengths of time. Cells were collected at the indicated treatment times followed by western analysis with phospho-RPA32 (S4/S8) antibody. Fold increase in RPA phosphorylation was calculated after normalization with GAPDH. (C) hPFs were exposed to the indicated doses of THS and RPA phosphorylation was monitored as in panel A. (D) Recovery of hPFs cells from replication stress following exposure to various doses of THS. Cells were treated with THS for 12h or mock treated, then allowed to recover for 12h by changing to complete medium. Induction of pRPA32 and recovery as indicated by its loss were followed by western analysis using phospho-RPA32 (S4/S8) antibody.

Fig. 4.

THS exposure causes replication stress and activates DSB response machineries. (A) Replication stress response was monitored following exposure of hPFs to the indicated doses of THS for 12 h followed by western analysis with anti-phosphoATR Thr1989 antibody. Tubulin was used as loading control. (B) Induction of DSBs by exposure of hPFs to 2.5% THS for 24 h was analyzed by IF detection of 53BP1 protein, which accumulates in foci at DSBs. SHS (0.4 PE) treatment was used as a positive control. (C) Activation of ATM following exposure of hPFs to 10%, 5% and 2.5% THS for 12 h was analyzed by western with anti-pATM (Ser1981). The same membrane was probed for phosphorylation of histone H2AX using anti-γH2AX antibody (Ser139) as an indicator of DSB formation and for pRPA32 (as in Fig. 3) to monitor replication stress. (D) Micronuclei formation in BEAS-2B cells following exposure to 5% THS for 48 h. Data represent the mean of ±SD for N=2.

Fig. 5.

Basal transcription is impaired following exposure to THS. (A) BEAS-2B cells were exposed to THS for 24 h with the indicated doses or mock treated, and RNA polymerase II was analyzed by western using anti-RNAP II antibody (8wg16), which recognizes both hypo- and hyper-phosphorylated forms of the polymerase. 20 J/m2 UV- irradiation followed by 1h incubation was used as a positive control. TFIIH subunit XPB served as a loading control. Band intensities for RNAPIIo and IIa were quantified and the fraction of RNAP as the IIo form was calculated for each condition. (B) Time course of RNAPII phosphorylation in BEAS-2B cells treated for various times with 5% THS was analyzed by western with anti-RNAPII CTD phosphoSer2 antibody (Covance; H5), which recognizes the phosphorylated form of RNAP II that is engaged in transcription elongation. GAPDH was the loading control. (C) Ongoing RNA synthesis was measured by EU incorporation after exposing BEAS-2B cells to 5% or 2.5% THS for 24 h. As a positive control for transcription inhibition, cells were irradiated with 20 J/m2 UV and EU was added 1 h after exposure. Representative images obtained at 20x magnifications are shown. (D) Quantification by ImageJ of the EU fluorescence intensity of 100 cells from mock, THS exposed, or UV irradiated samples. Data represent the mean of ± SD for N=3

Fig. 6.

THS exposure severely impairs basal transcription in NER defective cells. (A) Dermal fibroblasts from an XPA patient (XP12BE) were exposed to 5% or 2.5% THS for 24 h followed by EU incorporation and imaging as in Fig. 5. 40x magnification images are shown. (B) Quantification using ImageJ of the average fluorescence intensity of 10 cells from mock treated or THS-exposed XP12BE samples. Data represent the mean of ± SD for N=3. (C) Absence of XPA protein detectable with anti-XPA antibody in the XP-A patient cell line XP12BE is shown. The human fibroblast strain HCA2 was a normal control. XPB protein was used as a loading control.