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Resin Monomer 2-Hydroxyethyl Methacrylate (HEMA) is a Potent Inducer of Apoptotic Cell Death in Human and Mouse Cells
A. Paranjpe,
L.C.F. Bordador,
M.-y. Wang,
W.R. Hume and
A. Jewett*
1 Division of Oral Biology and Oral Medicine, UCLA School of Dentistry, The Jane and Jerry Weintraub Center for Reconstructive Biotechnology, The Jonsson Comprehensive Cancer Center, and Dental Research Institute, 10833 Le Conte Ave., Los Angeles, CA 90095, USA;
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Figure 1. Induction of apoptotic cell death by HEMA. (A) HEMA at different molar concentrations was added to untreated and IL-2 (500 u/mL)-treated PBMCs (5 x 106 cells/mL) for 12–18 hrs at 37°C. Propidium iodide (PI) at a final concerntration of 30 µg/mL was added to each sample, and the proportion of the PI stained cells was determined by flow cytometric analysis. The average and the standard deviation are shown for 10 separate HEMA-treated samples from healthy control donors. The two-tail unpaired p value for the difference between HEMA-treated and untreated samples at 0.082 M and 0.0164 M concentrations is less than 0.0001. (B) PBMCs were treated with different concentrations of HEMA, TEGDMA, and DNCB for 12–18 hrs. The levels of DNA strand breaks, indicating apoptotic cell death, were determined by the TUNEL assay. The cursor was set based on the staining obtained by control non-treated peripheral blood mononuclear cells. The events to the right of the cursor represent positive staining for apoptosis for each of the chemical treatments. One of three representative experiments is shown in this Fig. (C) PBMCs were treated with different concentrations of HEMA and CDDP for 12–18 hrs. The levels of apoptotic cell death were determined by dual staining with PI and annexin V. The numbers in each quadrant represent the percentages of cells positive for that quadrant. Ten thousand events were analyzed for each sample.
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Figure 2. Induction of apoptotic cell death in mouse macrophages by HEMA. (A) Mouse macrophage cell line RAW 264.7 cells were left untreated or treated with different concentrations of HEMA (as indicated in the Fig.) in an overnight assay, and the levels of DNA fragmentation were determined by PI staining after ethanol fixation of the cells. (A) represents the proportion of the macrophage population at G0/G1 cell cycle, (B) represents the S phase, (C) represents the G2/M phase, and (D) represents the sub-G0/G1 peak or fragmented proportion of the cells indicating apoptotic cell death. (B) Mouse macrophage cell line RAW 264.7 cells were left untreated or treated with two different concentrations of HEMA. The levels of apoptotic cell death were determined after an overnight incubation in RAW 264.7 cells dually staining with PI and annexin V. The numbers in each quadrant represent the percentages of cells positive for that quadrant. Ten thousand events were analyzed for each sample.
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Figure 3. Decreased levels of apoptotic cell death in PBMCs obtained from HEMA-sensitized patients as compared with healthy age- and sex-matched controls. (A) PBMCs (5 x 106 cells/mL) from HEMA-sensitized patients and age- and sex-matched healthy controls were treated in the presence and absence of different concentrations of HEMA for 12–18 hrs at 37°C. The samples were analyzed for the levels of apoptotic cell death by dual PI/annexin V staining. The numbers in each quadrant represent the percentages of cells positive for that quadrant. Ten thousand events were analyzed for each sample. (B) PBMCs obtained from patient and control donors were treated with HEMA as described in Fig. 3A . The cell samples were analyzed according to two parameter of forward-angle light scatter (FLS), representing the size, and side scatter (ss), representing the granularity of the cells. Arrow is pointing to the gain in the population of cells, with decreased FLS and increased ss representing the apoptotic population in healthy control donors.
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Journal of Dental Research, Vol. 84, No. 2,
172-177 (2005)
DOI: 10.1177/154405910508400212
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