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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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Related in: MedlinePlus

Effect of cigarette smoke on NF-κB and DNA binding. Cells were exposed to 0, 5 and 10% CSE or TNF-a (10 ng/ml) for 30 minutes in LHC-D/RPMI medium. Nuclear proteins were extracted and subjected for EMSA as described in the methods. Data presented are one representative of 3 separate experiments with similar results.
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Figure 2: Effect of cigarette smoke on NF-κB and DNA binding. Cells were exposed to 0, 5 and 10% CSE or TNF-a (10 ng/ml) for 30 minutes in LHC-D/RPMI medium. Nuclear proteins were extracted and subjected for EMSA as described in the methods. Data presented are one representative of 3 separate experiments with similar results.

Mentions: Since NF-κB is involved in regulating cell death and survival in variety of cell types, activation of NF-κB in response to cigarette smoke exposure was investigated by EMSA. Cigarette smoke extract (5% and 10%) stimulated DNA binding activity of NF-κB as evidenced by the electrophoresis mobility shift assay (Figure 2).


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)

Effect of cigarette smoke on NF-κB and DNA binding. Cells were exposed to 0, 5 and 10% CSE or TNF-a (10 ng/ml) for 30 minutes in LHC-D/RPMI medium. Nuclear proteins were extracted and subjected for EMSA as described in the methods. Data presented are one representative of 3 separate experiments with similar results.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Effect of cigarette smoke on NF-κB and DNA binding. Cells were exposed to 0, 5 and 10% CSE or TNF-a (10 ng/ml) for 30 minutes in LHC-D/RPMI medium. Nuclear proteins were extracted and subjected for EMSA as described in the methods. Data presented are one representative of 3 separate experiments with similar results.
Mentions: Since NF-κB is involved in regulating cell death and survival in variety of cell types, activation of NF-κB in response to cigarette smoke exposure was investigated by EMSA. Cigarette smoke extract (5% and 10%) stimulated DNA binding activity of NF-κB as evidenced by the electrophoresis mobility shift assay (Figure 2).

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