The effect of triethylene glycol dimethacrylate on p53-dependent G2 arrest in human gingival fibroblasts

Biomaterials 31 (2010) 8530e8538

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The effect of triethylene glycol dimethacrylate on p53-dependent G2 arrest in human gingival fibroblasts Eleni Mavrogonatou a, Theodore Eliades b, George Eliades c, Dimitris Kletsas a, * a

Laboratory of Cell Proliferation and Ageing, Institute of Biology, National Centre for Scientific Research “Demokritos”, 153 10 Athens, Greece Department of Orthodontics, School of Dentistry, Aristotle University of Thessaloniki, Thessaloniki, Greece c Department of Biomaterials, School of Dentistry, National and Kapodistrian University of Athens, Athens, Greece b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 June 2010 Accepted 21 July 2010 Available online 13 August 2010

Dental resin composites have been reported to exert adverse effects on cells of the oral cavity. In this study, we demonstrate that a non-cytotoxic concentration of the resin co-monomer triethylene glycol dimethacrylate (TEGDMA) results in the reduction of the proliferation rate of human gingival fibroblasts (HGFs), by delaying them at the G2 phase of the cell cycle, and in the sustained production of reactive oxygen species. These phenomena are accompanied by an early transient de-phosphorylation of ERK1/2 and JNKs and a late activation of the p53-p21WAF1-pRb molecular pathway. By using siRNA-mediated knocking down of the human p53 gene, we present evidence that the onco-suppressive protein p53 controls the TEGDMA-activated G2 checkpoint in HGFs and prevents their entry into mitosis, possibly in order to protect them from the detrimental genotoxic effects of the compound. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: TEGDMA Proliferation Cell cycle MAPKs DCFH-DA p53 siRNA

1. Introduction Triethylene glycol dimethacrylate (TEGDMA) constitutes a diluent co-monomer of dental composite resins, often used along with dentine bonding agents as a restorative material or adhesive. TEGDMA is nowadays identified as the main leachable compound of resin-based materials in the oral cavity due to incomplete polymerization [1,2]. Hence, a number of studies have been conducted for the determination of the possible biological effects of this monomer on oral tissues (pulp and gingival cells), as well as on cells of the innate immune system that regulate inflammatory responses in the mouth [3]. It has already been well established that TEGDMA impairs cellular redox homeostasis and is genotoxic, disturbing cell cycle distribution [3]. In fact, it has been shown to cause a depletion of the intracellular reducing agent glutathione (GSH) in human gingival and pulp fibroblasts [4e6]. Additionally, the TEGDMAinduced oxidative stress originating from the reactive oxygen species (ROS) produced by the mitochondria has been reported to be cytotoxic [7]. DNA damage caused by TEGDMA has been demonstrated in human salivary gland cells and lymphocytes using the alkaline

* Corresponding author. Tel.: þ30 210 6503565; fax: þ30 210 6511767. E-mail address: [email protected] (D. Kletsas). 0142-9612/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2010.07.074

single cell gel electrophoresis (comet assay) [8,9]. The genotoxicity of TEGDMA may or may not be linked to oxidative stress. DNA could be damaged via the generation of ROS or TEGDMA could act directly on the nucleophilic centers of the double-stranded DNA, possibly resulting in mutations [3]. Either way, it has been reported that cells respond to the genotoxic effects of TEGDMA by activating central cell cycle checkpoints, as cell cycle delay is necessary for successful DNA repair [10,11]. Cells with irreversible damage are finally driven to apoptosis [12]. The onco-suppressive protein p53 has been linked to both processes and has been reported to be the fundamental molecule that is activated by other genotoxic factors in several cell types, motivating the key responses necessary for the cells to counteract this type of stress [13]. Nevertheless, the role of this protein and of its downstream targets after exposure to TEGDMA has only scarcely been examined up to now. In the present study, we investigate the impact of a non-lethal concentration of TEGDMA on the proliferation and cell cycle regulation of human gingival fibroblasts (HGFs) and we focus on the involvement of p53 in the afore mentioned phenomena. Given that all previous studies concerning the anti-proliferative effects of TEGDMA have been mostly performed in irrelevant to the oral cavity cell types [11,14,15] or using cytotoxic concentrations of the monomer [14,16], aim of this work was to explore the mechanism of action of this commonly applied dental material on regulatory functions of a foremost oral cell type.

E. Mavrogonatou et al. / Biomaterials 31 (2010) 8530e8538 2. Materials and methods 2.1. Chemicals and reagents Collagenase, TEGDMA, dimethyl sulfoxide (DMSO), [3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazo-lium bromide] (MTT), 20 ,70 -dichlorfluorescein-diacetate (DCFH-DA), L-ascorbic acid, Propidium Iodide (PI), RNase A, protease and phosphatase inhibitors’ mixture, anti-a-tubulin, goat anti-mouse and goat anti-rabbit horseradish peroxidase-conjugated antibodies were obtained from Sigma [St. Louis, MO, USA]. Dulbecco’s Minimal Essential Medium (DMEM), phenol-red-free DMEM, penicillin and streptomycin were purchased from Biochrom AG [Berlin, Germany], while fetal bovine serum (FBS) was from Gibco BRL [Invitrogen, Paisley, UK]. OpitiMEM I and lipofectamine 2000 were also supplied by Invitrogen. [Methyl-3H]-thymidine and the ECL detection reagent were obtained from Amersham Biosciences [Buckinghamshire, UK]. Antibodies for phospho-p38 MAPK (Thr180/Tyr182), p38 MAPK, phospho-JNKs (Thr183/Tyr185) and JNKs were purchased from Cell Signaling Technology [Hertfordshire, UK]. Anti-p53 was from Santa Cruz Biotechnology [Santa Cruz, CA, USA], anti-p21WAF1, anti-phosphoERK1/2 (Thr202/Tyr204) and panERK were from BD Transduction Laboratories [Bedford, MA, USA] and anti-pRb was from BD Pharmingen [San Diego, CA, USA]. Anti-phospho-histone H3 antibody was obtained from Upstate Biotechnology [Lake Placid, NY, USA]. The sequence used for human p53 gene silencing (50 -CUACUUCCUGAAAACAACGTT-30 ) has been previously described [17] and was synthesized by Eurofins MWG Operon [Ebersberg, Germany], while the predesigned scramble (50 -UAAUGUAUUGGAACGCAUATT-30 ) was also supplied by Eurofins MWG Operon. 2.2. Isolation of human gingival fibroblasts and cell culture conditions HGFs were isolated from healthy donors and primary cultures were developed as previously described [18]. In brief, tissue specimens were chopped into small pieces and digested overnight with 1 mg/ml collagenase at 37  C. Cells were recovered by centrifugation and cultured in DMEM supplemented with penicillin (100 U/ml), streptomycin (100 mg/ml) and 10% (v/v) FBS. HGFs were routinely maintained in a humidified atmosphere of 5% CO2 at 37  C and subcultured when confluent using a trypsin/citrate solution (0.25/0.30% w/v) in a split ratio of 1:2. Experiments for this study were performed with HGFs between passages 5 and 12.

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For the determination of HGFs’ mitotic index after treatment with TEGDMA, the phosphorylation status of the known mitotic marker histone H3 on Ser10 was assessed. The assay was performed as described before [20]. Cells were fixed with 70% (v/v) ethanol at 20  C, washed three times with 0.1% (v/v) Triton X-100 in PBS and blocked for 1 h in 3% (w/v) BSA/0.1% (v/v) Triton X-100. Subsequently, they were incubated with anti-phospho-histone H3 antibody for 1 h at room temperature, washed with 0.1% (v/v) Triton X-100 in PBS, incubated for 1 h with FITC-conjugated anti-rabbit IgG and finally stained with PI. All samples were analyzed with a FACSCalibur flow cytometer equipped with an argon-ion laser (15 mW, 488 nm) using Cell Quest software [Becton Dickinson, San Jose, CA, USA]. DCF fluorescence for the measurement of ROS was recorded at logarithmic FL1 and is represented by the area under the curve on the histogram plot. In cell cycle experiments, PI fluorescence gathered at linear FL3 was used to measure DNA content, while in two-color flow cytometry, mitotic cells were represented by green fluorescence recorded at logarithmic FL1 and differences on the red fluorescence intensity collected at FL3 corresponded to the three different phases of the cell cycle. A dual FL3-Area/FL3-Width graph was used for the exclusion of aneuploid cells and nuclei doublets from further analysis. Experimental results were processed with Modfit [Verity Software House, Topsham, ME, USA] and WinMDI [Joseph Trotter, Salk Institute for Biological Studies, La Jolla, CA, USA] software. 2.6. Western blot analysis HGFs were incubated with 0.5 mM TEGDMA for several time points (0e24 h for the study of p53, p21WAF1 and pRb and 0e6 h for the kinetics analysis of the MAPK superfamily members’ phosphorylation status). Total cellular extracts were collected by scraping in Laemmli sample buffer containing protease and phosphatase inhibitors, as reported before [20]. Lysates were boiled at 100  C for 5 min, sonicated for 15 s, centrifuged at 10,000 rpm for 10 min, aliquoted and stored at 20  C until use. Samples were separated on SDS-polyacrylamide gels, transferred onto PVDF membranes and subjected to western blot analysis for phospho-p38 MAPK, phospho-ERKs, phospho-JNKs, p53, p21WAF1 and pRb. The non-phosphorylated forms of p38 MAPK, ERKs and JNKs and anti-a-tubulin were used in order to verify equal loading. Immune complexes were detected using an ECL detection reagent. 2.7. Inhibition of human p53 expression by small interfering RNA (siRNA)

2.3. MTT cytotoxicity assay The cytotoxicity caused by TEGDMA to HGFs was estimated using the MTT assay, as reported before [19]. Briefly, cells were plated in 96-well, flat-bottomed microplates in DMEM containing 10% (v/v) FBS for 24 h. TEGDMA was then added at different concentrations between 0 and 7.5 mM using 2-fold serial dilutions and cells were incubated for another 24 and 48 h, before the replacement of the medium with phenol-red-free DMEM containing 1 mg/ml MTT. Cells were further incubated with the MTT solution for 4 h and the formazan produced by normal mitochondrial activity was solubilized in isopropanol. Optical density was measured at a wavelength of 550 nm and a reference wavelength of 690 nm. TEGDMA was solubilized in DMSO, the concentration of which in all cultures never exceeded 0.5% (v/v). 2.4. DNA synthesis assay HGFs were plated in a 48-well microplate (w20,000 cells/well) in DMEM with 10% (v/v) FBS and grown for 24 h until 70e80% confluence. TEGDMA was added at a final concentration of 0.5 mM along with [methyl-3H]-thymidine at a concentration of 0.2 mCi/ml. After a 48-h incubation, the cells were fixed with 10% (w/v) ice-cold trichloroacetic acid (TCA), DNA was solubilized using a lysis buffer containing 0.3 N NaOH and 1% (w/v) SDS and tritiated thymidine incorporation into DNA was measured by scintillation counting, as previously described [20].

siRNA transfection was performed according to the protocol previously described [20]. In short, HGFs were plated in DMEM containing 10% (v/v) FBS until they reached 60% confluence. Then, cells were transfected with 50 nM of the human p53 siRNA sequence in serum-free OpitiMEM I medium using lipofectamine 2000 at a final concentration of 0.3% (v/v). Five hours later, the transfection medium was replaced by culture medium supplemented with 10% (v/v) FBS and cells were incubated for another 48 h. In order to ensure the sequence’s specificity for p53 gene silencing, a predesigned non-specific control duplex that does not influence the expression of any known human gene (scramble) was also used in the same final concentration. Cells not expressing p53 were treated with 0.5 mM TEGDMA for different time periods and samples were collected for western blot or FACS analysis. 2.8. Statistical analysis Data presented were the mean of at least three independent experiments (standard deviation), while every single MTT and DNA synthesis experiment was performed in quadruplicates. Differences were considered significant when p < 0.05 (Student’s t test).

3. Results

2.5. Flow cytometric analysis

3.1. Effect of TEGDMA on human gingival fibroblasts’ viability

Intracellular ROS detection of living HGFs was performed using the fluorochrome DCFH-DA. HGFs were plated in 35 mm culture dishes at a confluence of 70e80% in DMEM supplemented with 10% (v/v) FBS for 24 h before the addition of TEGDMA for another 6 and 24 h. Cells were then harvested by trypsinization, washed once with phosphate buffered saline (PBS) and stained with 20 mM of DCFH-DA for 30 min at 37  C. DCFH-DA is cleaved by non-specific esterases to form DCFH, which is further oxidized by ROS to form the fluorescent compound DCF [21]. For cell cycle analysis experiments, HGFs were plated in 35 mm culture dishes at a confluence of 70e80% in DMEM containing 10% (v/v) FBS for 24 h. After incubation with 0.5 mM of TEGDMA for various time periods (6, 9, 12 and 24 h), cells were trypsinized, washed once with PBS and fixed with ice-cold ethanol 50% (v/v). The staining procedure with PI was performed as reported previously [20]. In details, cells were stained with 50 mg/ml PI in the presence of 5 mM MgCl2 and 10 mg/ml RNase A for 30 min at room temperature.

The effect of TEGDMA on the viability of HGFs was assessed using the MTT cytotoxicity assay (Fig. 1). We demonstrated that concentrations of TEGDMA above 0.5 mM were highly cytotoxic for HGFs even when cells were only treated for 24 h. This cytotoxicity was shown to be dose-dependent (cell viability was 77.9  8.7% at a concentration of w1 mM, 32.0  9.2% at a concentration of w2 mM and 9.5  5.0% at 3.75 mM). A similar picture was observed for the incubation time of 48 h, although with higher percentages of cell death (cell viability was 48.2  12.1% at a concentration of w1 mM, 10.4  9.9% at a concentration of w2 mM and 2.5  4.6% at 3.75 mM). The maximum TEGDMA concentration tested (7.5 mM) was 100% lethal for the cells at both 24 and 48 h of treatment.

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Fig. 1. Effect of TEGDMA on the viability of human gingival fibroblasts. Cells were grown for 24 h before the addition of various TEGDMA concentrations and were further incubated for 24 or 48 h. Cytotoxicity was evaluated using the MTT assay and numbers of viable cells are presented as percentages of the respective untreated controls. Values are the means  standard deviation of three independent experiments performed in quadruplicates.

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We further investigated the effect of TEGDMA on the proliferation of HGFs. In order to exclude the parameter of cell death from our results, we selected the highest concentration of the monomer that, according to the MTT assay, did not influence cell viability. For that reason, from this point on, all our experiments were conducted using the concentration of 0.5 mM. HGFs’ proliferative potential was estimated by measuring de novo DNA synthesis. We showed that 3H-thymidine incorporation decreased to 26  1.5% after treatment with TEGDMA for 48 h in comparison to the untreated control (Fig. 2A). To examine whether the anti-proliferative result of the monomer is due to ROS production, we detected intracellular ROS by measuring DCF fluorescence intensity. ROS production was significantly increased 6 h after treatment with TEGDMA compared to control cells and this increase persisted until 24 h (Fig. 2B). Nevertheless, pre-treatment of the cells with 200 mM of ascorbate was not able to eliminate the negative effect of TEGDMA on novel DNA synthesis (data not shown). Cell cycle analysis revealed that TEGDMA causes an accumulation of the cells at the G2/M phase (Fig. 3). This G2/M delay appeared as early as 6 h after treatment (from 11.6  5.2% to 18.4  2.1%, p ¼ 0.02) and sustained even after exposure of the cells to TEGDMA for 24 h (from 9.1  4.7% to 19.9  5.6%, p ¼ 0.02). Together, the observed G2/M arrest was accompanied by a decrease of the G0/G1 population. Subsequently, the expression or activation of known cell cycle regulators was investigated using western blot analysis (Fig. 4). We showed that p53 was up-regulated in response to TEGDMA 3 h after treatment. A decrease of this up-regulation was observed at posterior time points, though a slight induction was still obvious after 9 h of incubation. In accordance to p53 over-expression, higher levels of the cyclin-dependent kinase inhibitor (CKI)

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Fig. 2. Proliferative potential and ROS production of TEGDMA-treated human gingival fibroblasts. A. Cells were grown until sub-confluence before the addition of 0.5 mM of TEGDMA along with [methyl-3H]-thymidine at a final concentration of 0.2 mCi/ml. 3 H-thymidine incorporation was measured by scintillation counting after a 48-h incubation and was normalized to the value of untreated cells. Results are expressed as mean values  standard deviation and the asterisk represents a statistically significant difference in comparison to the control (t test, p < 0.05). B. HGFs were treated with 0.5 mM of TEGDMA for 6 and 24 h and stained with DCFH-DA. Basal and TEGDMAinduced ROS amounts were analyzed using a flow cytometer. The observed shift of the curves to the right due to increased fluorescence indicates an increase in the intracellular levels of ROS. Histogram plots of DCF fluorescence are representative of three independent experiments.

p21WAF1 were also monitored. The kinetics of p21WAF1 up-regulation followed that of p53 (starting at 6 h, with a peak at 9 h), indicating a p53-mediated regulation of the CKI under these conditions. p53 and p21WAF1 over-expression was accompanied by the hypo-phosphorylation of the retinoblastoma protein pRb that coincides with the appearance of the G2/M block demonstrated in Fig. 3.

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3.3. Phosphorylation of the stress-induced mitogen-activated protein kinases (MAPKs) Members of the MAPK superfamily have often been connected with cell proliferation and cellular responses to stress [22,23]. The phosphorylation status of MAPKs after treatment with TEGDMA was evaluated (Fig. 5). We showed that both ERKs and JNKs were rapidly (after 30 min) inactivated by de-phosphorylation in HGFs exposed to the monomer. The phosphorylation of ERKs increased again at 3 h and returned to basal levels after 6 h, in parallel to that of JNKs that was also shown to recover to some extent at 3 and 6 h. Surprisingly, no change in the phosphorylated levels of p38 MAPK in response to TEGDMA was evident. 3.4. Effect of p53 depletion by siRNA on human gingival fibroblasts’ response to TEGDMA The pathway p53-p21WAF1-pRb has been previously reported to regulate the manifestation of a G1 cell cycle arrest [24]. On the

contrary, in our cell model we showed the activation of the G2 cell cycle checkpoint by TEGDMA, in parallel to the up-regulation of p53 and p21WAF1 and the hypo-phosphorylation of pRb. We followed by investigating the role of p53 on the TEGDMA-induced G2/M arrest in HGFs. For that reason, p53 was inhibited using a specific siRNA sequence. Transfection of the cells with this p53 siRNA led to over 90% reduction in the protein’s endogenous levels, accompanied by an almost 100% down-regulation of p21WAF1 (Fig. 6A). This finding verifies that p21WAF1 lies downstream of p53 in this cell type. p53 knocking down by siRNA in HGFs also hindered the TEGDMAstimulated up-regulation of the latter protein, as well that of p21WAF1 (Fig. 6B). Accordingly, in p53-depleted cells the hyperphosphorylated form of pRb could still be detected after exposure to the compound (Fig. 6B). Flow cytometric analysis of HGFs with suppressed p53 revealed that just the inhibition of the protein leads to a higher percentage of cells accumulating at the G2/M phase of the cell cycle (from 14.0  1.2% for control cells and 15.3  1.3% for cells treated with scrambled siRNA to 19.1  0.9% for p53-siRNA treated cells, p ¼ 0.04 and p ¼ 0.03, respectively) (Fig. 7A). Exposure to TEGDMA further

Fig. 4. TEGDMA-induced activation of known cell cycle regulators. Cells were grown in the presence of serum and were exposed to 0.5 mM of TEGDMA for various time periods. Total cellular extracts were collected and subjected to western blot analysis for p53, p21WAF1 and pRb. Representative blots of four independently performed experiments are shown here. The underphosphorylated (pRb) and hyperphosphorylated (ppRb) forms of pRb are indicated. a-tubulin was used as loading control.

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Fig. 5. Regulation of MAPKs’ phosphorylated levels by TEGDMA in human gingival fibroblasts. TEGDMA was added to HGFs for the indicated time periods before protein extraction. Cell lysates were immunoblotted using antibodies raised against the phosphorylated forms of p38 MAPK, ERK1/2 and JNKs. The non-phosphorylated forms of the kinases served as loading controls. Western blot analyses were in all cases repeated three times and a representative experiment is presented here.

enhanced this delay (from 22.6  1.6% for control cells and 22.0  2.3% for cells treated with scrambled siRNA to 26.6  1.7% for p53 siRNA-treated cells, p ¼ 0.03 and p ¼ 0.04, respectively) (Fig. 7A). Since staining with PI is not able to distinguish mitotic cells from cells being at the G2 phase of the cell cycle, for this purpose we used an antibody against the phosphorylated form of histone H3 at Ser10, which is a known mitotic marker [25]. The population of mitotic cells was found to be increased after treatment with TEGDMA (from 0.24  0.15% to 0.58  0.26%, p ¼ 0.01 and 0.28  0.11% to 0.40  0.81%, p ¼ 0.03 for control cells and cells treated with scrambled siRNA, respectively) (Fig. 7B). Moreover, we demonstrated that in the absence of p53 HGFs were released to M phase, having an elevated mitotic index in comparison to control cells (0.47  0.17%, p ¼ 0.01), while this percentage was even higher when p53-depleted cells were further exposed to TEGDMA (1.37  0.39%, p ¼ 0.0002) (Fig. 7B). 4. Discussion

Fig. 6. Effect of p53 loss-of-expression on the activation of the TEGDMA-stimulated p53-p21WAF1-pRb pathway. Cells were treated with 50 nM of the specific for the human p53 gene siRNA or the scrambled sequence for 48 h. A. Western blot analysis was performed for the evaluation of p53 and p21WAF1 expression, using the appropriate antibodies. B. Pre-treated with p53-siRNA HGFs were exposed to 0.5 mM of TEGDMA. Cell lysates were collected at 3 and 9 h and subjected to immunoblot analysis for p53 or p21WAF1 and pRb, respectively. Immunoblot for a-tubulin was realized in order to verify equal loading of the samples.

4.1. TEGDMA cytotoxicity It has been previously reported that TEGDMA may diffuse through dentin and cause damages to the cells of the oral cavity [26]. Even though the cytotoxic effect of TEGDMA on HGFs has been previously investigated, the lowest lethal concentration of the compound for this cell type could not be deduced from the literature. According to Janke et al., TEGDMA was not cytotoxic until 2.5 mM, as determined by the DNA-intercalating dye Hoechst 33342Ô [27]. On the other hand, it has been published that concentrations higher than 0.5 mM led to a decrease in the percentage of viable HGFs exposed to TEGDMA for 24 h and that 50% of toxicity was obtained with 1e3.5 mM of TEGDMA for different lines of gingival fibroblasts [5,28,29]. In the present investigation, TEGDMA concentrations up to 0.5 mM did not reduce cell viability after 24 and 48 h exposure periods, in agreement with Volk et al. [29]. All other concentrations tested were cytotoxic for HGFs. The clinical importance of this finding for the common practice is high and should be taken under consideration, given that the concentration of TEGDMA in the pulp after diffusion across the dentin can reach the value of 4 mM [3].

4.2. Effect of TEGDMA on HGFs’ proliferation In order to explore the role of TEGDMA on the proliferative potential of HGFs, we selected the highest concentration of the compound that did not cause cell death. Using novel DNA synthesis as a pre-requisite of cell proliferation, we observed that a TEGDMA concentration of 0.5 mM was able to reduce HGFs’ proliferation rate at a percentage that exceeded 70%, when cells were treated with the substance for 48 h. This result is in accordance with previous reports describing a significant decrease of cellular proliferation at TEGDMA concentrations  0.25 mM and a complete inhibition at higher concentrations, both in HGFs and THP-1 monocytes [27,30,31]. The TEGDMA-induced oxidative stress has been well documented [4,6,7,14,29] and it has been reported that the compound disturbs the redox state of HGFs, influencing intracellular GSH concentration and cell viability [4,7]. These phenomena have already been shown to be connected with ROS production, since use of the radical-scavenging antioxidant ascorbic acid at a final

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Fig. 7. Effect of p53 inhibition on the TEGDMA-induced G2/M cell cycle arrest. HGFs were transfected with 50 nM scramble or p53 siRNA for 48 h and then exposed to 0.5 mM of TEGDMA. A. Cells were fixed after a 24-h incubation, stained with PI and analyzed by flow cytometry. Results are presented as mean values  standard deviation of three independent experiments. B. TEGDMA-treated cells not expressing p53 were fixed, simultaneously stained with PI and anti-phospho-histone H3 antibody and analyzed by two-color flow cytometry. Percentages of phospho-histone H3-positive cells gathered in gate R2 were calculated using WinMDI software. Density plots are representative of five independent experiments and values used for the graph are the means  standard deviation. Asterisks represent differences with statistical significance in comparison to the respective untreated control (t test, p < 0.05).

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concentration of 200 mM abrogated TEGDMA-mediated GSH depletion and cytotoxicity in both human gingival and pulp fibroblasts [5]. In the present study, we attempted to examine the effect of increased intracellular ROS on the proliferation of HGFs. Hitherto, ROS production due to TEGDMA has been mainly studied within a time-frame up to 4 h [5,16,32]. We demonstrated that TEGDMAdependent increase of intracellular ROS is not a transient phenomenon, since DCF fluorescence of HGFs exposed to the monomer was higher than that of control cells even after 24 h of treatment. Nevertheless, pre-treated with ascorbate cells could not regain their ability for novel DNA synthesis, suggesting the existence of a ROS-independent biochemical pathway that leads to the reduction of the proliferation rate. Accordingly, the proliferative potential of the cells seems to be regulated by other factors as well, than just by the maintenance of the intracellular redox state’s equilibrium. Since the TEGDMA concentration used in our experiments was not lethal, reduction in HGFs’ proliferation could only be explained by a cell cycle delay. The effect of TEGDMA on cell cycle regulation has been investigated before in various cell types with contentious results. It has been reported that exposure of mouse macrophages to 1 mM of TEGDMA resulted in a delay of the cell cycle at the G1 phase [33]. In a recent study regarding the differential gene expression of normal human skin fibroblasts in response to TEGDMA, it was shown that concentrations of 1 and 3 mM led to a G1 delay at 2, 24 and 48 h of treatment, accompanied by a slight reduction of the G2 population [15], while in another work a G1 and a G2 arrest were detected in the same cell type [11]. The majority of TEGDMA-treated p53-deficient V79 Chinese hamster lung fibroblasts exhibited a cell cycle delay in G2 phase [11] that was annulled when cells were co-treated with N-acetyl-cysteine [10]. THP-1 cells exposed to TEGDMA were delayed at the G1 and G2 phases of the cell cycle, in parallel to a progressive reduction of the S phase population [14]. Finally, human primary pulp cells have been reported to be delayed at the G2 phase after exposure to TEGDMA [11]. Measurement of the cellular DNA content of HGFs in the presence or absence of TEGDMA by flow cytometry revealed that the compound led to an increase of the G2/M population, accompanied by a decrease of cells being in G1 phase, and did not affect the percentage of S phase cells. Activation of cell cycle checkpoints is a cellular response to DNA damage in order to allow cells to appreciate if this injury is reversible and can be repaired or if they must trigger the process leading to apoptosis [34]. Many molecules have been implicated in the regulation of cell cycle progression after exposure to genotoxic stress. The onco-suppressive protein p53 has been accounted as the “guardian of the genome” [35], participates in DNA damage responses and cell cycle perturbations [36] and its dysfunction has been linked to genomic instability and cancer [37]. The CKI p21WAF1 is a major target of activated p53 [38], known to induce cell cycle arrest [39], while pRb is located at the end of this cascade, typically eventuating in G1 checkpoint activation [40]. The development of DNA double strand breaks by TEGDMA has yet been established in primary HGFs [41]. Noticeably, the induction of the above mentioned proteins, known to be stimulated by DNA damage, has not been studied in this cell model before. There is only one recent work concerning the potential use of p53, p63 and p16 immunohistochemical expression as a biomarker of genotoxicity, with no concluding results [42]. In our study, we showed that p53 and p21WAF1 were up-regulated after exposure of HGFs to TEGDMA. Accordingly, pRb was also found to be hypo-phosphorylated. It is worth mentioning that, even though these molecules are unbreakably bonded to the manifestation of a G1 cell cycle delay, their implication in G2 arrest has often been reported [43e48].

4.3. MAPKs’ phosphorylation in response to TEGDMA MAPKs control vital cellular functions, including proliferation and responses to a variety of stressful stimuli [22,23]. In addition, it has been demonstrated in the past that ROS may interact with MAPKs [49]. The MAPK superfamily is a large family of protein kinases that includes the extracellular signal-regulated kinases ERK1/2, the c-jun N-terminal kinases or stress-activated protein kinases JNKs/SAPKs and p38 MAPK. ERKs are mainly associated with the regulation of cell proliferation [23], while JNKs and p38 MAPK are known to be activated in cells exposed to stress [50,51]. As mentioned above, TEGDMA was demonstrated to reduce the proliferation rate of HGFs and to increase intracellular ROS. Therefore we investigated the direct influence of the compound on the activation of all the MAPK family members. The activated state of MAPKs is regulated through post-translational modifications and more specifically through phosphorylation that could be achieved very rapidly [52]. This is the reason why the MAPK-mediated cascades belong to the arsenal of the acute cellular responses against environmental stresses. TEGDMA resulted in a fast and transient de-phosphorylation and thus de-activation of ERKs in HGFs. The reduction in the phosphorylated levels of the kinases is consistent with the observed decreased proliferation rate of the cells in our study and has also been reported for mouse macrophages [53], but is not in accordance with the induction of ERKs described in a salivary gland cell line and in human pulp-derived cells subsequent to TEGDMA exposure [16,53]. This controversy could result from tissue-specific traits (i.e. salivary gland and pulp vs. gingival cells) or just be explained by the difference in the TEGDMA concentrations used in these studies (i.e. 2 and 3 mM vs. 0.5 mM). Actually, ERKs induction described by Samuelsen et al. [16] may be related only to the survival role of the kinases [54], as this work concerns the apoptotic response of salivary gland cells to TEGDMA. Spagnuolo et al. have reported no activation of ERKs in primary human pulp cells after treatment with TEGDMA, as well [12]. In addition to ERKs de-activation, a decrease of JNKs phosphorylated levels was evident in our cell system after exposure to TEGDMA. JNKs have been shown to be activated in response to stressful conditions [22], their de-phosphorylation though has also been described in kidney cells exposed to hypertonic stress [55]. TEGDMA has been shown to activate JNKs in mouse macrophages and human pulp-derived cells at a higher concentration (3 mM) and only after treatment for 24 h [53]. Finally, we showed that in TEGDMA-treated HGFs the levels of phosphop38 MAPK remained stable during the entire incubation period (from 30 min to 6 h). Our data are in agreement with previous reports in salivary gland cells, THP-1 monocytes and human pulpderived cells in which no alteration in the basal phosphorylated levels of p38 MAPK was detected within this time-frame, even after exposure to higher TEGDMA concentrations [14,16,53]. 4.4. Implication of p53 in the G2 arrest To evaluate the possible regulatory role of p53 on the TEGDMAinduced G2 checkpoint activation in HGFs, a specific sequence for the silencing of the human p53 gene was used. siRNA-mediated knocking down of the latter protein inhibited both the up-regulation of p53 and p21WAF1, as well as the hypo-phosphorylation of pRb in cells treated with TEGDMA. These results support the existence of a p53-p21WAF1-pRb axis in HGFs, which is motivated under these experimental conditions and may launch a cascade of downstream signaling events. Abrogation of this molecular pathway by p53 depletion resulted in an increased accumulation of HGFs in G2/M phase, which was even higher after treatment with TEGDMA. An augmented G2/M population after p53 inhibition has

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also been described in normal human fibroblasts exposed to DNAdamaging agents and has been attributed to a decrease of the fraction of cells that stop to G1 phase [56]. However, a G2/M arrest could also be explained by an increase in the M phase cells and not just by the abrogation of the G1/S block of the cell cycle. When the mitotic index of HGFs was calculated, we observed that inhibition of p53 led to a more than 2-fold increase of mitotic cells in the absence of TEGDMA and approximately 8-fold increase in the presence of the compound. This finding is in agreement with an earlier report depicting the requirement of p53 and p21WAF1 for the maintenance of a G2 arrest after DNA damage caused by ionizing radiation [43], which has been connected to centrosome amplification [57]. It appears that p53 protects TEGDMA-treated HGFs from entering mitosis, in order to evade putative mutations. 5. Conclusions In the current study we investigated the effect of the commonly used resin monomer TEGDMA on the proliferation and cell cycle regulation of gingival fibroblasts that constitute a representative cell type of the oral cavity. We demonstrated that even a non-lethal concentration of the compound (that can be easily attained after diffusion across the dentin) has anti-proliferative properties by delaying the cells at the G2 phase of the cell cycle. We present evidence that this TEGDMA-induced G2 arrest in HGFs is p53dependent and we reveal that the functional p53 can play a protective role against the detrimental genotoxicity of the monomer by preventing entry of the cells into mitosis. Our results could have applications on the clinical practice, since obtaining insight into the cytostatic mode of action of dental materials is required for the development of effective therapeutic approaches that take under consideration the protection of oral tissues. Appendix Figures with essential color discrimination. Fig. 7 in this article has parts that are difficult to interpret in black and white. The full color images can be found in the online version, at doi:10.1016/j. biomaterials.2010.07.074. References [1] Spahl W, Budzikiewicz H, Geurtsen W. Determination of leachable components from four commercial dental composites by gas and liquid chromatography/mass spectrometry. J Dent 1998;26:137e45. [2] Geurtsen W. Substances released from dental resin composites and glass ionomer cements. Eur J Oral Sci 1998;106:687e95. [3] Schweikl H, Spagnuolo G, Schmalz G. Genetic and cellular toxicology of dental resin monomers. J Dent Res 2006;85:870e7. [4] Engelmann J, Leyhausen G, Leibfritz D, Geurtsen W. Effect of TEGDMA on the intracellular glutathione concentration of human gingival fibroblasts. J Biomed Mater Res 2002;63:746e51. [5] Stanislawski L, Lefeuvre M, Bourd K, Soheili-Majd E, Goldberg M, Perianin A. TEGDMA-induced toxicity in human fibroblasts is associated with early and drastic glutathione depletion with subsequent production of oxygen reactive species. J Biomed Mater Res A 2003;66:476e82. [6] Volk J, Engelmann J, Leyhausen G, Geurtsen W. Effects of three resin monomers on the cellular glutathione concentration of cultured human gingival fibroblasts. Dent Mater 2006;22:499e505. [7] Lefeuvre M, Amjaad W, Goldberg M, Stanislawski L. TEGDMA induces mitochondrial damage and oxidative stress in human gingival fibroblasts. Biomaterials 2005;26:5130e7. [8] Kleinsasser NH, Schmid K, Sassen AW, Harreus UA, Staudenmaier R, Folwaczny M, et al. Cytotoxic and genotoxic effects of resin monomers in human salivary gland tissue and lymphocytes as assessed by the single cell microgel electrophoresis (Comet) assay. Biomaterials 2006;27:1762e70. [9] Kleinsasser NH, Wallner BC, Harreus UA, Kleinjung T, Folwaczny M, Hickel R, et al. Genotoxicity and cytotoxicity of dental materials in human lymphocytes as assessed by the single cell microgel electrophoresis (comet) assay. J Dent 2004;32:229e34.

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