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Mitogenic and functional responses by nicotine and hydrogen peroxide in AR42J cells: a comparative study.

Walker A, Udupa KB, Chowdhury P - Tob Induc Dis (2008)

Bottom Line: Nicotine-induced increase in the proliferation of AR42J cells was significantly higher in comparison to H2O2 exposed cells.CCK-stimulated cell function induced by nicotine was significantly higher in AR42J cells as compared to the response by H2O2.These results suggest that nicotine- induced mitogenic and functional response in AR42J cells are associated with ERK signaling and increase in reactive oxygen species production.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR, USA. PChowdhury@uams.edu.

ABSTRACT
The aim of the current study was to investigate the oxidative effects of nicotine by examining the mitogenic and functional responses in AR42J cells. As a control and for comparison, hydrogen peroxide (H2O2) was used as a source of known oxidative biomarker. Responses were examined by determining cell proliferation through the activation of ERK signaling, basal and CCK-stimulated cell function and measuring lipid peroxidation. AR42J cells have been exposed to either a non-cytotoxic dose of 20 muM H2O2 for 15 min or to 100 muM of nicotine for 3 min respectively. Nicotine and H2O2 at these dose and time intervals produced similar levels of malondialdyde (MDA) production and p-ERK1/2 activation. Immunofluorescence studies employing specific antibody to p-ERK1/2 confirmed the latter. Nicotine-induced increase in the proliferation of AR42J cells was significantly higher in comparison to H2O2 exposed cells. CCK-stimulated cell function induced by nicotine was significantly higher in AR42J cells as compared to the response by H2O2. These results suggest that nicotine- induced mitogenic and functional response in AR42J cells are associated with ERK signaling and increase in reactive oxygen species production. The data suggests that nicotine-induced mitogenic response in AR42J cells closely identifies the response induced by an oxidative biomarker.

No MeSH data available.


Related in: MedlinePlus

Dose and time dependent induction of ERK1/2 in AR42J cells. Cell lysates were loaded onto an SDS gel, separated by electrophoresis, blocked in 1% fat free milk and probed with antibodies to total and phosphorylated (p) ERK1/2. Horseradish peroxidase-coupled anti IgG was used as a secondary antibody. Bands were visualized with ECL-plus and quantified using a STORM Imager. The data shown as means ± SEM of n = 5 experiments. A: Induction of ERK1/2 in cells exposed to 20 μM H2O2 for 10–60 min compared to control untreated cells. B: Induction of ERK in cells exposed to 10–20 μM H2O2 for 15 min. C: Band intensity showing the fold increase in the time dependent induction of ERK1/2. D: Band intensity showing the fold increase in the dose dependent induction of ERK1/2.
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Figure 3: Dose and time dependent induction of ERK1/2 in AR42J cells. Cell lysates were loaded onto an SDS gel, separated by electrophoresis, blocked in 1% fat free milk and probed with antibodies to total and phosphorylated (p) ERK1/2. Horseradish peroxidase-coupled anti IgG was used as a secondary antibody. Bands were visualized with ECL-plus and quantified using a STORM Imager. The data shown as means ± SEM of n = 5 experiments. A: Induction of ERK1/2 in cells exposed to 20 μM H2O2 for 10–60 min compared to control untreated cells. B: Induction of ERK in cells exposed to 10–20 μM H2O2 for 15 min. C: Band intensity showing the fold increase in the time dependent induction of ERK1/2. D: Band intensity showing the fold increase in the dose dependent induction of ERK1/2.

Mentions: It has been shown that in other cells, ERK1/2 is activated by H2O2 treatment [20,21]. To determine the effects of H2O2 on ERK1/2 activation in AR42J cells, the cells were exposed to H2O2 (20 μM) for various times. Cell lysates were prepared and measured for total and p-ERK1/2 activation employing the specific antibodies to total and p-ERK1/2 and analyzed by Western blots. With 20 μM H2O2, a 3 fold increase in band intensity was observed at 15 min which was significantly higher (p < 0.02) when compared to the control cells (Figure 3A). Expressing band intensity as the fold increase above the control, a steady increase in p-ERK activation was observed with increasing concentrations of H2O2 at 15 min of incubation, attaining a 7-fold increase with 100 μM H2O2 (Fig. 3B). Total ERK1/2 in all instances showed no alteration with H2O2 incubation and indicated a uniformity of loading of the wells with samples.


Mitogenic and functional responses by nicotine and hydrogen peroxide in AR42J cells: a comparative study.

Walker A, Udupa KB, Chowdhury P - Tob Induc Dis (2008)

Dose and time dependent induction of ERK1/2 in AR42J cells. Cell lysates were loaded onto an SDS gel, separated by electrophoresis, blocked in 1% fat free milk and probed with antibodies to total and phosphorylated (p) ERK1/2. Horseradish peroxidase-coupled anti IgG was used as a secondary antibody. Bands were visualized with ECL-plus and quantified using a STORM Imager. The data shown as means ± SEM of n = 5 experiments. A: Induction of ERK1/2 in cells exposed to 20 μM H2O2 for 10–60 min compared to control untreated cells. B: Induction of ERK in cells exposed to 10–20 μM H2O2 for 15 min. C: Band intensity showing the fold increase in the time dependent induction of ERK1/2. D: Band intensity showing the fold increase in the dose dependent induction of ERK1/2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2556029&req=5

Figure 3: Dose and time dependent induction of ERK1/2 in AR42J cells. Cell lysates were loaded onto an SDS gel, separated by electrophoresis, blocked in 1% fat free milk and probed with antibodies to total and phosphorylated (p) ERK1/2. Horseradish peroxidase-coupled anti IgG was used as a secondary antibody. Bands were visualized with ECL-plus and quantified using a STORM Imager. The data shown as means ± SEM of n = 5 experiments. A: Induction of ERK1/2 in cells exposed to 20 μM H2O2 for 10–60 min compared to control untreated cells. B: Induction of ERK in cells exposed to 10–20 μM H2O2 for 15 min. C: Band intensity showing the fold increase in the time dependent induction of ERK1/2. D: Band intensity showing the fold increase in the dose dependent induction of ERK1/2.
Mentions: It has been shown that in other cells, ERK1/2 is activated by H2O2 treatment [20,21]. To determine the effects of H2O2 on ERK1/2 activation in AR42J cells, the cells were exposed to H2O2 (20 μM) for various times. Cell lysates were prepared and measured for total and p-ERK1/2 activation employing the specific antibodies to total and p-ERK1/2 and analyzed by Western blots. With 20 μM H2O2, a 3 fold increase in band intensity was observed at 15 min which was significantly higher (p < 0.02) when compared to the control cells (Figure 3A). Expressing band intensity as the fold increase above the control, a steady increase in p-ERK activation was observed with increasing concentrations of H2O2 at 15 min of incubation, attaining a 7-fold increase with 100 μM H2O2 (Fig. 3B). Total ERK1/2 in all instances showed no alteration with H2O2 incubation and indicated a uniformity of loading of the wells with samples.

Bottom Line: Nicotine-induced increase in the proliferation of AR42J cells was significantly higher in comparison to H2O2 exposed cells.CCK-stimulated cell function induced by nicotine was significantly higher in AR42J cells as compared to the response by H2O2.These results suggest that nicotine- induced mitogenic and functional response in AR42J cells are associated with ERK signaling and increase in reactive oxygen species production.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR, USA. PChowdhury@uams.edu.

ABSTRACT
The aim of the current study was to investigate the oxidative effects of nicotine by examining the mitogenic and functional responses in AR42J cells. As a control and for comparison, hydrogen peroxide (H2O2) was used as a source of known oxidative biomarker. Responses were examined by determining cell proliferation through the activation of ERK signaling, basal and CCK-stimulated cell function and measuring lipid peroxidation. AR42J cells have been exposed to either a non-cytotoxic dose of 20 muM H2O2 for 15 min or to 100 muM of nicotine for 3 min respectively. Nicotine and H2O2 at these dose and time intervals produced similar levels of malondialdyde (MDA) production and p-ERK1/2 activation. Immunofluorescence studies employing specific antibody to p-ERK1/2 confirmed the latter. Nicotine-induced increase in the proliferation of AR42J cells was significantly higher in comparison to H2O2 exposed cells. CCK-stimulated cell function induced by nicotine was significantly higher in AR42J cells as compared to the response by H2O2. These results suggest that nicotine- induced mitogenic and functional response in AR42J cells are associated with ERK signaling and increase in reactive oxygen species production. The data suggests that nicotine-induced mitogenic response in AR42J cells closely identifies the response induced by an oxidative biomarker.

No MeSH data available.


Related in: MedlinePlus