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Inhibition of Phosphatidylinositol 3-Kinase Amplifies TEGDMA-induced Apoptosis in Primary Human Pulp Cells

¹ Department of Oral and Maxillo-Facial Sciences and
³ Department of Cellular and Molecular Biology and Pathology, University of Naples "Federico II", via S. Pansini 5, 80131-Naples, Italy; and
² Department of Operative Dentistry and Periodontology, University of Regensburg, D-93042 Regensburg, Germany;

View larger version (44K): [in this window] [in a new window]   Figure 1. The effect of TEGDMA on the induction of apoptosis and necrosis in human primary pulp cells. Cell cultures were incubated with annexin V-FITC (apoptotic cells) and PI (necrotic cells) and analyzed by flow cytometry. The representative dual-parameter fluorescence histograms were derived from cell cultures exposed to various TEGDMA concentrations for 24 hrs. The numbers indicate the percentages of the viable (annexin V-; PI-) cell population (lower left quadrant), the apoptotic (annexin V+) cell population (lower right quadrant), and the necrotic cell populations in the upper right (annexin V+; PI+) and the upper left quadrants (annexin V-; PI +) in one typical experiment.

View larger version (15K): [in this window] [in a new window]   Figure 2. Dose-dependent induction of apoptosis and necrosis in human pulp cells. Cell cultures were treated with various concentrations of TEGDMA in duplicate for 24 hrs. The percentages of apoptotic () and necrotic () cell populations were analyzed by flow cytometry. Values (means ± SD) from 6 independent experiments are presented (n = 6); * indicates significant (p 0.05), and ** indicates highly significant (p 0.01) differences from untreated control cultures (Mann-Whitney U-test).

View larger version (14K): [in this window] [in a new window]   Figure 3. Time-dependent induction of apoptosis by TEGDMA. Primary human pulp cell cultures were continuously exposed to 1 mmol/L TEGDMA for 6, 12, and 24 hrs. The percentages of the apoptotic cell population in untreated controls () and TEGDMA-treated cultures (•) were analyzed by flow cytometry. Values (means ± SD) from 3 independent experiments in duplicate are presented (n = 3); * indicates a significant (p 0.05) difference from untreated control cultures (Mann-Whitney U-test).

View larger version (30K): [in this window] [in a new window]   Figure 4. The effects of the inhibition of MAPK/ERK and PI3K pathways in human pulp cells. (a) Total extracts derived from human primary pulp cells treated with 1 mmol/L TEGDMA or 3 mmol/L TEGDMA for 15 min were separated on 10% SDS-PAGE, and immunoblotted with anti-P-Akt (ser 473) or anti-P-ERK 1/2. The amounts of total protein present in the extracts were determined by immunoblotting with anti-Akt, anti-tubulin, and anti-ERK 2. Experiments were performed 3 times, and a representative result is shown. (b) The ratio between P-Akt and total Akt in pulp cells after treatment with TEGDMA determined by densitometry. Asterisk indicates significant differences from untreated cell cultures (n = 3). (c) Induction of apoptosis and necrosis in human pulp cells. Cells were pre-treated with 50 µmol/L LY294002 and 40 µmol/L PD98056 for 30 min, and then further exposed to 1 mmol/L TEGDMA for 24 hrs. Apoptotic (annexin V+) and necrotic (PI+) cell populations were analyzed by flow cytometry as described. Viability of a cell population is expressed as the difference between the total cell population and the populations which stained with annexin V-FITC and PI. Values (means ± SD) from at least 4 independent experiments in duplicate are presented (n = 4); * indicates significant (p 0.05), and ** indicates highly significant (p 0.01) differences from untreated control cultures (Mann-Whitney U-test). (d) Inhibition of Akt phosphorylation in human pulp cells. The cells were treated with 40 mmol/L LY294002 for 30 min, and then exposed to 1 mmol/L TEGDMA for 15 min. Cell lysates were separated on SDS-PAGE and blotted with anti-P-Akt or total Akt antibodies.

Journal of Dental Research, Vol. 83, No. 9, 703-707 (2004)
DOI: 10.1177/154405910408300909


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