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NF-B Protection against Apoptosis Induced by HEMA

¹ Department of Oral and Maxillofacial Sciences,
² Department of Cellular and Molecular Biology and Pathology,
³ Department of Neuroscience, Unit of Physiology, and
⁴ Department of Clinical and Experimental Medicine, University of Naples "Federico II", via S. Pansini 5, 80131-Naples, Italy; and
⁵ Department of Operative Dentistry and Periodontology, University of Regensburg, 93042-Regensburg, Germany;

View larger version (30K): [in this window] [in a new window]   Figure 1. HEMA-induced apoptosis and caspases activation. (A) Dose-dependent induction of apoptosis. Cells were treated with various concentrations of HEMA for 24 hrs, and apoptotic and necrotic cells were stained with annexin V-FITC or PI, respectively. Cells were then detected and quantified by flow cytometry as a percentage of the entire population (see MATERIALS & METHODS). Results represent the means ± SEM of 4 independent experiments in duplicate (n = 4). * Significantly different from the untreated control group (one-way ANOVA followed by Bonferroni post hoc test, p < 0.05). (B) Dose-dependent caspase-3 activation. Cytosolic extracts from cells treated with 0–10 mM HEMA for 24 hrs were analyzed by Western blotting with anti-caspase-3 antibodies. (C) Time-dependence of caspase-3 activation. The cells were treated with 10 mM HEMA for the indicated periods of time, and cytosolic extracts were analyzed by Western blotting with anti-caspase-3 antibodies. (D) Caspase-8 and -9 activation. Cytosolic extracts from cells treated with 10 mM HEMA for 24 hrs were analyzed by Western blotting with anti-caspase-8 and -9 antibodies. The amounts of cell lysate were normalized with the use of polyclonal anti-actin or tubulin antibodies. Each Western blot is representative of at least 2 independent experiments.

View larger version (14K): [in this window] [in a new window]   Figure 2. Generation of ROS by HEMA in human fibroblasts. Suspended cells were incubated with 10 µM DCFH-DA for 15 min at 37°C. (A) The indicated concentrations of HEMA were added to the cells and incubated for 30 min at 37°C. (B) From 0 to 10 mM HEMA was added to the cells and incubated for 15, 30, and 60 min at 37°C. DCF fluorescence was measured by means of a flow cytometer with an FL-1 filter. Results represent the means ± SEM (n = 5). *Values are significantly different from untreated controls (one-way ANOVA followed by the Bonferroni post hoc test, p < 0.05).

View larger version (51K): [in this window] [in a new window]   Figure 3. HEMA induces IB degradation and DNA binding of NF-B. (A) The cells were treated with 10 mM HEMA for the indicated periods of time and (B) with 0–10 mM HEMA for 180 min. Cytosolic extracts were analyzed by Western blotting with anti-IB antibodies. The lower panel shows a Western blot anti-tubulin as control for protein loading. Experiments were performed 3 times, and a representative result is shown. (C) EMSA from cells treated with 10 mM HEMA for the indicated period of time with or without PDTC. Total cell extracts were prepared and analyzed by EMSA with a 32P-labeled oligonucleotide probe containing a NF-B-binding site. The middle portion of the autoradiograph shows the same cell extracts incubated with a 50-fold molar excess of unlabeled (cold) NF-B oligonucleotide.

View larger version (34K): [in this window] [in a new window]   Figure 4. Effects of PDTC on ROS production, apoptosis, and IB degradation induced by HEMA. Cells were pre-treated with 10 µM PDTC for 30 min. (A) The cells were loaded with DCFH-DA and further treated with 10 mM HEMA and PDTC for 30 min. DCF fluorescence was analyzed by flow cytometry. Results represent the means ± SEM (n = 4). (B) Apoptotic and necrotic cells were detected after 10 mM HEMA and PDTC treatments for 24 hrs. Results represent means ± SEM (n = 3). *Values are significantly different from untreated controls (one-way ANOVA, followed by the Bonferroni post hoc test, p < 0.05). (C) IB degradation was evaluated after 10 mM HEMA and PDTC treatment for 180 min. Western blot is representative of 2 independent experiments. (D) HEMA-induced apoptosis in MEF wild-type vs. p65–/– cells. MEF and p65–/– cells were treated with 8 mM HEMA for 24 hrs. Apoptotic (lower right quadrant) and necrotic (left and right upper quadrants) cells were then detected by flow cytometry. The dot plot is representative of 3 independent experiments.

Journal of Dental Research, Vol. 83, No. 11, 837-842 (2004)
DOI: 10.1177/154405910408301103


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