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NF-kappaB mediates the survival of human bronchial epithelial cells exposed to cigarette smoke extract.

Liu X, Togo S, Al-Mugotir M, Kim H, Fang Q, Kobayashi T, Wang X, Mao L, Bitterman P, Rennard S - Respir. Res. (2008)

Bottom Line: Inhibition of NF-kappaB by the pharmacologic inhibitor curcumin (20 microM) or suppression of p65 by siRNA resulted in a significant increase in cell death in response to cigarette smoke exposure.The current study demonstrates that CSE activates NF-kappaB and up-regulates Bcl-XL through NF-kB activation in HBECs, and that CSE induces cell death in cells lacking p65.These results suggest that activation of NF-kappaB regulates cell survival following DNA damage by cigarette smoke in human bronchial epithelial cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Pulmonary, Critical Care and Sleep Medicine, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska, USA. srennard@unmc.edu.

ABSTRACT

Background: We have previously reported that low concentrations of cigarette smoke extract induce DNA damage without leading to apoptosis or necrosis in human bronchial epithelial cells (HBECs), and that IL-6/STAT3 signaling contributes to the cell survival. Since NF-kappaB is also involved in regulating apoptosis and cell survival, the current study was designed to investigate the role of NF-kappaB in mediating cell survival in response to cigarette smoke exposure in HBECs.

Methods: Both the pharmacologic inhibitor of NF-kappaB, curcumin, and RNA interference targeting p65 were used to block NF-kappaB signaling in HBECs. Apoptosis and cell survival were then assessed by various methods including COMET assay, LIVE/DEAD Cytotoxicity/Viability assay and colony formation assay.

Results: Cigarette smoke extract (CSE) caused DNA damage and cell cycle arrest in S phase without leading to apoptosis in HBECs as evidenced by TUNEL assay, COMET assay and DNA content assay. CSE stimulated NF-kappaB -DNA binding activity and up-regulated Bcl-XL protein in HBECs. Inhibition of NF-kappaB by the pharmacologic inhibitor curcumin (20 microM) or suppression of p65 by siRNA resulted in a significant increase in cell death in response to cigarette smoke exposure. Furthermore, cells lacking p65 were incapable of forming cellular colonies when these cells were exposed to CSE, while they behaved normally in the regular culture medium.

Conclusion: The current study demonstrates that CSE activates NF-kappaB and up-regulates Bcl-XL through NF-kB activation in HBECs, and that CSE induces cell death in cells lacking p65. These results suggest that activation of NF-kappaB regulates cell survival following DNA damage by cigarette smoke in human bronchial epithelial cells.

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Role of p65 in mediating HBEC survival in response to cigarette smoke exposure. Following transfection with p65-siRNA, HBECs were exposed to 10% CSE for 24 hours. Cells were then harvested and used for LIVE/DEAD cytotoxicity/viability assay (Panel A), COMET assay (Panel B) and colony formation assay (Panel C) as described in the methods. Data in panel A and B are one representative from 3 separate experiments, and panel C is an average of 3 separate experiments each performed in triplicate dishes. Panel B: arrow heads indicate cells with DNA damage, and arrows indicated cells undergoing apoptosis.
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Figure 5: Role of p65 in mediating HBEC survival in response to cigarette smoke exposure. Following transfection with p65-siRNA, HBECs were exposed to 10% CSE for 24 hours. Cells were then harvested and used for LIVE/DEAD cytotoxicity/viability assay (Panel A), COMET assay (Panel B) and colony formation assay (Panel C) as described in the methods. Data in panel A and B are one representative from 3 separate experiments, and panel C is an average of 3 separate experiments each performed in triplicate dishes. Panel B: arrow heads indicate cells with DNA damage, and arrows indicated cells undergoing apoptosis.

Mentions: Following transfection with p65-siRNA, HBEC cells were exposed to cigarette smoke extract for 24 hours. Cell death or survival was then evaluated by three different methods including LIVE/DEAD staining, COMET assay and clonogenic assay. Only the combination of p65-siRNA suppression and smoke exposure resulted in major cytotoxicity. In the presence of 10%CSE, the cells lacking p65 started to detach at 3–4 hrs after exposure to cigarette smoke and nearly all of the cells were detached from the culture dish after 24 hours exposure. In contrast, only a few cells in the control-siRNA treated group detached in response to cigarette smoke exposure. LIVE/DEAD staining demonstrated that very few cells were dead (red nuclei) in the non-targeting siRNA transfected cells (Figure 5A, 3.5 ± 0.3%), in the cells of p65-siRNA transfected without cigarette smoke exposure (Figure 5A, 7.0 ± 1.1%) or in non-targeting siRNA transfected cells exposed to 10% cigarette smoke (Figure 5A, 2.4 ± 1.0%). In contrast, nearly half of the cells were dead in the cells transfected with p65-siRNA and exposed to cigarette smoke extract (Figure 5A, 32.5 ± 1.8%, p < 0.01 compared to control siRNA transfected cells without exposure to cigarette smoke extract), which was similar to the cells exposed to camptothecin (data not shown). Consistent with LIVE/DEAD cytotoxicity assay, p65-depleted cells underwent severe DNA damage and apoptotic death when they were exposed to cigarette smoke as evidenced by COMET assay (Figure 5B). The apoptotic index was significantly higher in the cells lacking p65 and exposed to cigarette smoke (42.5 ± 6.5% vs 6.3 ± 1.2% of control, p < 0.01).


NF-kappaB mediates the survival of human bronchial epithelial cells exposed to cigarette smoke extract.

Liu X, Togo S, Al-Mugotir M, Kim H, Fang Q, Kobayashi T, Wang X, Mao L, Bitterman P, Rennard S - Respir. Res. (2008)

Role of p65 in mediating HBEC survival in response to cigarette smoke exposure. Following transfection with p65-siRNA, HBECs were exposed to 10% CSE for 24 hours. Cells were then harvested and used for LIVE/DEAD cytotoxicity/viability assay (Panel A), COMET assay (Panel B) and colony formation assay (Panel C) as described in the methods. Data in panel A and B are one representative from 3 separate experiments, and panel C is an average of 3 separate experiments each performed in triplicate dishes. Panel B: arrow heads indicate cells with DNA damage, and arrows indicated cells undergoing apoptosis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2567966&req=5

Figure 5: Role of p65 in mediating HBEC survival in response to cigarette smoke exposure. Following transfection with p65-siRNA, HBECs were exposed to 10% CSE for 24 hours. Cells were then harvested and used for LIVE/DEAD cytotoxicity/viability assay (Panel A), COMET assay (Panel B) and colony formation assay (Panel C) as described in the methods. Data in panel A and B are one representative from 3 separate experiments, and panel C is an average of 3 separate experiments each performed in triplicate dishes. Panel B: arrow heads indicate cells with DNA damage, and arrows indicated cells undergoing apoptosis.
Mentions: Following transfection with p65-siRNA, HBEC cells were exposed to cigarette smoke extract for 24 hours. Cell death or survival was then evaluated by three different methods including LIVE/DEAD staining, COMET assay and clonogenic assay. Only the combination of p65-siRNA suppression and smoke exposure resulted in major cytotoxicity. In the presence of 10%CSE, the cells lacking p65 started to detach at 3–4 hrs after exposure to cigarette smoke and nearly all of the cells were detached from the culture dish after 24 hours exposure. In contrast, only a few cells in the control-siRNA treated group detached in response to cigarette smoke exposure. LIVE/DEAD staining demonstrated that very few cells were dead (red nuclei) in the non-targeting siRNA transfected cells (Figure 5A, 3.5 ± 0.3%), in the cells of p65-siRNA transfected without cigarette smoke exposure (Figure 5A, 7.0 ± 1.1%) or in non-targeting siRNA transfected cells exposed to 10% cigarette smoke (Figure 5A, 2.4 ± 1.0%). In contrast, nearly half of the cells were dead in the cells transfected with p65-siRNA and exposed to cigarette smoke extract (Figure 5A, 32.5 ± 1.8%, p < 0.01 compared to control siRNA transfected cells without exposure to cigarette smoke extract), which was similar to the cells exposed to camptothecin (data not shown). Consistent with LIVE/DEAD cytotoxicity assay, p65-depleted cells underwent severe DNA damage and apoptotic death when they were exposed to cigarette smoke as evidenced by COMET assay (Figure 5B). The apoptotic index was significantly higher in the cells lacking p65 and exposed to cigarette smoke (42.5 ± 6.5% vs 6.3 ± 1.2% of control, p < 0.01).

Bottom Line: Inhibition of NF-kappaB by the pharmacologic inhibitor curcumin (20 microM) or suppression of p65 by siRNA resulted in a significant increase in cell death in response to cigarette smoke exposure.The current study demonstrates that CSE activates NF-kappaB and up-regulates Bcl-XL through NF-kB activation in HBECs, and that CSE induces cell death in cells lacking p65.These results suggest that activation of NF-kappaB regulates cell survival following DNA damage by cigarette smoke in human bronchial epithelial cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Pulmonary, Critical Care and Sleep Medicine, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska, USA. srennard@unmc.edu.

ABSTRACT

Background: We have previously reported that low concentrations of cigarette smoke extract induce DNA damage without leading to apoptosis or necrosis in human bronchial epithelial cells (HBECs), and that IL-6/STAT3 signaling contributes to the cell survival. Since NF-kappaB is also involved in regulating apoptosis and cell survival, the current study was designed to investigate the role of NF-kappaB in mediating cell survival in response to cigarette smoke exposure in HBECs.

Methods: Both the pharmacologic inhibitor of NF-kappaB, curcumin, and RNA interference targeting p65 were used to block NF-kappaB signaling in HBECs. Apoptosis and cell survival were then assessed by various methods including COMET assay, LIVE/DEAD Cytotoxicity/Viability assay and colony formation assay.

Results: Cigarette smoke extract (CSE) caused DNA damage and cell cycle arrest in S phase without leading to apoptosis in HBECs as evidenced by TUNEL assay, COMET assay and DNA content assay. CSE stimulated NF-kappaB -DNA binding activity and up-regulated Bcl-XL protein in HBECs. Inhibition of NF-kappaB by the pharmacologic inhibitor curcumin (20 microM) or suppression of p65 by siRNA resulted in a significant increase in cell death in response to cigarette smoke exposure. Furthermore, cells lacking p65 were incapable of forming cellular colonies when these cells were exposed to CSE, while they behaved normally in the regular culture medium.

Conclusion: The current study demonstrates that CSE activates NF-kappaB and up-regulates Bcl-XL through NF-kB activation in HBECs, and that CSE induces cell death in cells lacking p65. These results suggest that activation of NF-kappaB regulates cell survival following DNA damage by cigarette smoke in human bronchial epithelial cells.

Show MeSH
Related in: MedlinePlus