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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 human bronchial epithelial cell DNA damage and survival. Human bronchial epithelial cells were exposed to 10% cigarette smoke extract for 24 hours or to 0.5 μM camptothecin for 4 hours, respectively, in LHC-D/RPMI medium. Both floating and attached cells were harvested, combined and used for TUNEL assay (Panel A), DNA content profiling and cell cycle analysis (Panel B), COMET assay (Panel C) and colony formation assay (Panel D). Data presented in panels A, C and D is one representative experiment from at least 4 replicates in both BEAS-2B and HBEC cells. Panel B is an average of 8 different experiments for "control and CSE", and 4 different experiments for camptothecin (CPT) treated. * p < 0.05, ** p < 0.01 compared to control. Panel C: arrow heads indicate cells with DNA damage, and arrows indicate cells undergoing apoptosis with a typical fan-like tail and small head.
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Figure 1: Effect of cigarette smoke on human bronchial epithelial cell DNA damage and survival. Human bronchial epithelial cells were exposed to 10% cigarette smoke extract for 24 hours or to 0.5 μM camptothecin for 4 hours, respectively, in LHC-D/RPMI medium. Both floating and attached cells were harvested, combined and used for TUNEL assay (Panel A), DNA content profiling and cell cycle analysis (Panel B), COMET assay (Panel C) and colony formation assay (Panel D). Data presented in panels A, C and D is one representative experiment from at least 4 replicates in both BEAS-2B and HBEC cells. Panel B is an average of 8 different experiments for "control and CSE", and 4 different experiments for camptothecin (CPT) treated. * p < 0.05, ** p < 0.01 compared to control. Panel C: arrow heads indicate cells with DNA damage, and arrows indicate cells undergoing apoptosis with a typical fan-like tail and small head.

Mentions: Cigarette smoke extract induces DNA damage without leading to apoptosis Cigarette smoke induced DNA damage in human bronchial epithelial cells as evidenced by TUNEL positivity (Figure 1A). To further determine if apoptosis occurred in these DNA-damaged cells, confluent HBECs were treated with 10% CSE or 0.5 μM camptothecin. DNA content by FACS analysis and DNA damage/apoptosis by COMET assay were then evaluated. Compared to medium alone (control), 10% cigarette smoke extract did not increase apoptosis in human bronchial epithelial cells as evaluated by either DNA content or COMET assay (Figure 1B and 1C). In contrast, camptothecin induced apoptosis in these cells as evidenced by a sub-G1 peak (14.6 ± 1.8%, Figure 1B) or cells with a small DNA head and fan-like tail in the COMET image (apoptotic index: 43.5% ± 8.9%, Figure 1C). Cigarette smoke, however, led to cell cycle arrested in S phase (Figure 1B, 40.2 ± 3.8% vs 25.9 ± 2.9% of control, p < 0.01). Furthermore, cells with DNA damage, if allowed to recover, were able to proliferate and form colonies in subsequent culture (Figure 1D). In addition, necrosis was not induced by cigarette smoke extract (10% or lower) as evidenced by both MTT and LDH assay (data not shown).


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 human bronchial epithelial cell DNA damage and survival. Human bronchial epithelial cells were exposed to 10% cigarette smoke extract for 24 hours or to 0.5 μM camptothecin for 4 hours, respectively, in LHC-D/RPMI medium. Both floating and attached cells were harvested, combined and used for TUNEL assay (Panel A), DNA content profiling and cell cycle analysis (Panel B), COMET assay (Panel C) and colony formation assay (Panel D). Data presented in panels A, C and D is one representative experiment from at least 4 replicates in both BEAS-2B and HBEC cells. Panel B is an average of 8 different experiments for "control and CSE", and 4 different experiments for camptothecin (CPT) treated. * p < 0.05, ** p < 0.01 compared to control. Panel C: arrow heads indicate cells with DNA damage, and arrows indicate cells undergoing apoptosis with a typical fan-like tail and small head.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Effect of cigarette smoke on human bronchial epithelial cell DNA damage and survival. Human bronchial epithelial cells were exposed to 10% cigarette smoke extract for 24 hours or to 0.5 μM camptothecin for 4 hours, respectively, in LHC-D/RPMI medium. Both floating and attached cells were harvested, combined and used for TUNEL assay (Panel A), DNA content profiling and cell cycle analysis (Panel B), COMET assay (Panel C) and colony formation assay (Panel D). Data presented in panels A, C and D is one representative experiment from at least 4 replicates in both BEAS-2B and HBEC cells. Panel B is an average of 8 different experiments for "control and CSE", and 4 different experiments for camptothecin (CPT) treated. * p < 0.05, ** p < 0.01 compared to control. Panel C: arrow heads indicate cells with DNA damage, and arrows indicate cells undergoing apoptosis with a typical fan-like tail and small head.
Mentions: Cigarette smoke extract induces DNA damage without leading to apoptosis Cigarette smoke induced DNA damage in human bronchial epithelial cells as evidenced by TUNEL positivity (Figure 1A). To further determine if apoptosis occurred in these DNA-damaged cells, confluent HBECs were treated with 10% CSE or 0.5 μM camptothecin. DNA content by FACS analysis and DNA damage/apoptosis by COMET assay were then evaluated. Compared to medium alone (control), 10% cigarette smoke extract did not increase apoptosis in human bronchial epithelial cells as evaluated by either DNA content or COMET assay (Figure 1B and 1C). In contrast, camptothecin induced apoptosis in these cells as evidenced by a sub-G1 peak (14.6 ± 1.8%, Figure 1B) or cells with a small DNA head and fan-like tail in the COMET image (apoptotic index: 43.5% ± 8.9%, Figure 1C). Cigarette smoke, however, led to cell cycle arrested in S phase (Figure 1B, 40.2 ± 3.8% vs 25.9 ± 2.9% of control, p < 0.01). Furthermore, cells with DNA damage, if allowed to recover, were able to proliferate and form colonies in subsequent culture (Figure 1D). In addition, necrosis was not induced by cigarette smoke extract (10% or lower) as evidenced by both MTT and LDH assay (data not shown).

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