Abstract
Objectives
The dental resin monomer triethylene glycol dimethacrylate (TEGDMA) caused a cell cycle arrest in response to DNA damage. However, the underlying mechanisms are unclear. Therefore, the influence of TEGDMA on the cell cycle was analyzed in comparison with the chemotherapeutic agents adriamycin and mitomycin C (MMC), which arrest the cell cycle through different mechanisms.
Methods
RAW264.7 mouse macrophages were exposed to TEGDMA, adriamycin, or MMC, and flow cytometry (FACS) was used for cell cycle analyses. In addition, the number of surviving cells was determined by a crystal violet assay, and viability in treated cultures was determined by FACS after staining of cells with trypan blue. Morphological changes in cells were interpreted using forward and side scatter (FSC/SSC) cell physical criteria.
Results
The exposure of cells to 1 mM TEGDMA resulted in a delay of the cell cycle in G1 phase since 85.3% of the cells were found in G1 compared with 47.4% in untreated controls. Adriamycin also increased the number of cells (72.1%) in G1 compared to controls. Caffeine, an inhibitor of the checkpoint kinases ATM (ataxia telangiectasia-mutated) and ATR (ATM and Rad3-related), had no effect on the TEGDMA and adriamycin-induced cell cycle arrest. In contrast, MMC delayed the cell cycle in G2 since cell numbers increased to 22.1% compared to 10.7% in controls. The effect of MMC on G2 was even increased by low caffeine concentrations (100–400 μM), but 1000 μM caffeine inhibited MMC activity.
Significance
Our results suggest that the mechanism of a TEGDMA-induced arrest of the cell cycle is different from the effect of the direct-acting interstrand crosslinking agent MMC. Since TEGDMA produced oxidative stress, it probably acts indirectly on the cell cycle through reactive oxygen species, unless TEGDMA–DNA adducts are shown experimentally.
1
Introduction
The process of DNA replication is tightly regulated during the eukaryotic cell cycle, which is divided into G1, S, and G2/M phases. Between each step there are checkpoints which are utilized to assure that a cell is intact before entering the next phase. In the case of DNA damage, regulatory checkpoint proteins are activated to stop the normal propagation of the cell cycle, a condition known as cell cycle arrest. DNA damage and cell cycle arrest can be induced by different stimuli like oxidative stress, UV and ionizing irradiation, or genotoxic chemicals .
Chemicals that interfere with the regulation of the cell cycle are commonly used in cancer therapy. Adriamycin (doxorubicin) is a substance which predominantly causes an arrest at the G1 to S phase transition of the cell cycle . Adriamycin has multiple effects, including the causation of DNA damage by intercalating into DNA, the formation of free radicals, and inhibition of topoisomerase II. DNA double strand breaks (DSBs) generated by adriamycin are followed by the activation of the tumor suppressor protein p53 and a subsequent arrest of the cell cycle . Other effects of adriamycin are mediated by a p38-dependent mechanism as a consequence of the production of reactive oxygen species (ROS) . In contrast to compounds like adriamycin, bifunctional molecules such as cisplatin or mitomycin C (MMC) cause DNA interstrand crosslinks (ICLs), which are among the most toxic of all DNA lesions. ICLs prevent DNA strand separation, block essential cellular processes such as DNA replication, transcription and recombination, and induce cell cycle arrest. The alkylating and direct-acting ICL agent MMC is widely used as a potent anticancer therapeutic. MMC caused DNA crosslinks followed by DSBs which finally resulted in an arrest of the cell cycle in G2 phase .
Ataxia telangiectasia-mutated (ATM), a member of the phosphoinositide 3-kinase-like family of serine/threonine protein kinases (PIKK) is a component of cell cycle checkpoints, and plays a central role in the cellular response to DSBs. The generation of DSBs leads to an ATM-mediated activation of downstream regulatory target proteins including p53 followed by an arrest of the cell cycle to allow either for DNA repair or apoptosis . ATR (ATM and Rad3-related kinase) is a related PIKK with functions at cell cycle checkpoints and partially overlaps with ATM. ATM and ATR activities are inhibited by the methyl-xanthine alkaloid caffeine. Caffeine inhibits phosphorylation of Chk2/Cds1 by ATM, and thereby abrogates an arrest of the cell cycle in G2 phase in response to DNA damage.
It has recently been shown that monomers released from dental resin materials are capable of interfering with cellular regulatory networks. Resin monomers like triethylene glycol dimethacrylate (TEGDMA) and 2-hydroxy ethyl methacrylate (HEMA) modify basic cell functions, and genotoxic effects like the induction of DNA strand breaks, gene mutation or chromosomal mutations were detected in bacteria and mammalian cells as well . As a consequence, there is experimental evidence that TEGDMA and HEMA are causative agents of a cell cycle arrest . Depending on the cell line, the concentration of the monomer, and the exposure period, an arrest of the cell cycle caused by TEGDMA was observed in both G1 and G2 phases. Human fibroblasts responded very quickly to the exposure to TEGDMA through the induction of a cell cycle arrest, and it appeared that the activation of the cell cycle checkpoint at G1 was an immediate response to cell damage. Noteworthy is that high TEGDMA concentrations increased the number of cells in G2 phase after long exposure . It appeared that the activation of a G1 checkpoint was dependent on p53 function since the cell cycle of p53-deficient V79 hamster fibroblasts was arrested in G2 phase only. Since these effects were attenuated by the ROS scavenger N-acetylcysteine (NAC), a monomer-induced cell cycle arrest was most likely the consequence of oxidative stress and oxidative DNA damage . Yet, it is still possible that the generation of mutations and a cell cycle arrest by TEGDMA is caused by its covalent binding to genomic DNA as previously suggested . In this case, the beta carbon of the two double bonds in the bifunctional TEGDMA molecule would directly react with nucleophilic centers in the DNA via Michael addition . This binding could result in the formation of intra-strand DNA crosslinks similar to the mechanism discussed for MMC.
The characterization of the mechanisms of biological effects of dental materials will allow for the improvement of basic strategies for the protection of oral tissues . A more detailed understanding of the possible adverse effects provides a better estimation of the risks that could be associated with a particular dental therapy. The aim of this study was to further clarify a possible mechanism behind the effect of the dental resin monomer TEGDMA on the regulation of the mammalian cell cycle. Therefore, the influence of the monomer on the cell cycle was first analyzed in relation to adriamycin and MMC, which arrest the cell cycle in the G1 or G2 phase through contrasting mechanisms. Second, the use of the ATM/ATR inhibitor caffeine should add additional information about a specific pathway initiated by the three different chemicals.
2
Materials and methods
2.1
Chemicals and reagents
Triethylene glycol dimethacrylate (TEGDMA; CAS No. 109-16-0), adriamycin (doxorubicin; CAS No. 25316-40-9), mitomycin C (CAS No. 50-07-7), caffeine (CAS No. 58-08-2), propidium iodide (PI), RNase (R-4875), and bovine serum albumin (BSA) were purchased from Sigma–Aldrich (Taufkirchen, Germany). RPMI 1640 medium containing l -glutamine and 2.0 g/l NaHCO 3 was obtained from PAN Biotech (Aidenbach, Germany). Trypan blue (TB), foetal bovine serum (FBS), penicillin/streptomycin, and calcium–magnesium free phosphate-buffered saline (CMF-PBS) supplemented with 5 mM EDTA (PBS-EDTA) came from Life Technologies, Gibco BRL (Eggenstein, Germany).
2.2
Exposure of cell cultures
RAW264.7 mouse macrophages (ATCC TIB71) were maintained in RPMI 1640 medium containing l -glutamine and 2.0 g/l NaHCO 3 supplemented with 10% FBS, penicillin (100 units/ml), and streptomycin (100 μg/ml) at 37 °C and 5% CO 2 . Suspensions of 1 × 10 6 cells in 20 ml medium were seeded into cell culture plates (15 cm in diameter) and left untreated for 24 h at 37 °C. Subsequently, the medium was discarded and the cells were treated with fresh medium containing either 1 mM TEGDMA, 1 μM adriamycin or 0.5 μg/ml mitomycin C (MMC) dissolved in medium. For caffeine treatment cells were preincubated with 100, 400 or 1000 μM caffeine for 1 h at 37 °C. Then, all samples were incubated for another 24 h in the presence of 1 mM TEGDMA, 1 μM adriamycin or 0.5 μg/ml MMC. Untreated cell cultures were used as controls. The concentrations of TEGDMA, adriamycin, MMC, and caffeine used here had been determined in preliminary range finding experiments.
2.3
Cell cycle analysis and flow cytometry
The cells were harvested and washed twice in CMF-PBS supplemented with 2% BSA. The cells were fixed and perforated with 70% MeOH at 4 °C over night. MeOH was then removed after centrifugation, and the cells were washed twice with CMF-PBS with 2% BSA. After that the cells were incubated with 0.1 mg/ml RNase in CMF-PBS with 2% BSA for 30 min at 37 °C. Subsequently 50 μg/ml PI was added. The DNA content of the cells, which is proportional to the mean fluorescence value, was measured in a FACSCanto™ flow cytometer (Becton Dickinson, Heidelberg, Germany). Twenty thousand events for each sample were collected with FACSDiva™ software (version 5.0.2) and the data were stored as list mode files. The percentages of cells in G1, S, and G2 phases of the cell cycle were calculated after mathematical modeling using ModFit LT™ software (Verity Software House, Topsham, ME, USA).
2.4
FSC and SSC measurements and cell viability
Forward (FSC) and side light scatter (SSC) measurements can be useful tools in studying the morphological characteristics of cells by flow cytometry . Whereas FSC indicates cell size, SSC is an indicator of the granularity of a cell. Here, FSC and SSC were measured at 488 nm and signals were collected in linear mode. FSC/SSC density plots were generated using FACSDiva™ software (version 5.0.2). To detect possible changes in these parameters in cell cultures exposed to the various chemicals, the main population of untreated cells was marked by a region of interest called P1 (gating). The gate settings were not changed during the course of an experiment. To compare measurements, the percentage of the gated events that could be assigned to these regions was used.
Cell viability after exposure to TEGDMA, adriamycin, or mitomycin C alone or in combination with caffeine as well as in untreated controls was determined by flow cytometry using trypan blue (TB) as a dye. Aliquots for TB measurements were taken from the same cell cultures analyzed for the distribution of cells among the different phases of the cell cycle. The cells were harvested and washed two times in buffer (1% BSA and 0.1% NaN 3 in CMF-PBS) by centrifugation at 300 × g for 5 min. After resuspending the cells in 100 μl buffer, TB was added to a final concentration of 0.013% and incubated for 5 min at RT in the dark, followed by two washes as described above. Then, mean fluorescence intensities (MFI) were measured in a FACSCanto™ flow cytometer with an excitation wavelength of 633 nm and a detector equipped with a 780/60 nm band pass filter. Necrotic cells were distinguished from viable cells by higher fluorescence intensities, which result in two distinct peaks in a histogram. For each sample, 20,000 events were counted and analyses were performed with FACSDiva™ 5.0.2 software.
2.5
Cytotoxicity testing
Cell numbers in treated and untreated cultures were determined as a measure of cytotoxicity using a crystal violet assay as described earlier . The RAW mouse macrophages (7.5 × 10 3 /well) were cultivated in 96 well plates for 24 h at 37 °C. Then, the cell cultures were preincubated with caffeine (100, 400 or 1000 μM) for 1 h or directly treated with 200 μl cell culture medium containing either 1 mM TEGDMA, 1 μM adriamycin or 0.5 μg/ml MMC. Exposure of the cell cultures was stopped after 24 h, the cells were fixed with 1% glutaraldehyde for 30 min, and stained with crystal violet (0.02% in water) for 15 min. The amount of crystal violet bound to the cells was dissolved with 70% ethanol and optical densities were measured at 600 nm in a multiwell spectrophotometer (Infinite 200, TECAN, Crailsheim, Germany).
2.6
Data analyses
For the analysis of cytotoxicity, four replicate cell cultures were exposed to each concentration of the tested chemicals in repeated independent experiments. Cell numbers were calculated from individual optical density readings and normalized to untreated controls (=100%) as specified in the legend of Fig. 5 . The percentage of cells stained with trypan blue (TB) for the analysis of cell viability using FACS was determined in at least four independent experiments. The ratios of viable and necrotic cells in treated cultures were normalized to those obtained in untreated control cultures (=100%). Likewise, the distribution of cells among the different phases of the cell cycle (G1, S, and G2) was calculated from individual histograms obtained in at least four independent experiments. Median values plus 25% and 75% quartiles were calculated from individual measurements. Differences between median values were statistically analyzed using the Mann-Whitney U test (SPSS 15.0, SPSS, Chicago, IL, USA) for pairwise comparisons among groups at the 0.05 level of significance.
2
Materials and methods
2.1
Chemicals and reagents
Triethylene glycol dimethacrylate (TEGDMA; CAS No. 109-16-0), adriamycin (doxorubicin; CAS No. 25316-40-9), mitomycin C (CAS No. 50-07-7), caffeine (CAS No. 58-08-2), propidium iodide (PI), RNase (R-4875), and bovine serum albumin (BSA) were purchased from Sigma–Aldrich (Taufkirchen, Germany). RPMI 1640 medium containing l -glutamine and 2.0 g/l NaHCO 3 was obtained from PAN Biotech (Aidenbach, Germany). Trypan blue (TB), foetal bovine serum (FBS), penicillin/streptomycin, and calcium–magnesium free phosphate-buffered saline (CMF-PBS) supplemented with 5 mM EDTA (PBS-EDTA) came from Life Technologies, Gibco BRL (Eggenstein, Germany).
2.2
Exposure of cell cultures
RAW264.7 mouse macrophages (ATCC TIB71) were maintained in RPMI 1640 medium containing l -glutamine and 2.0 g/l NaHCO 3 supplemented with 10% FBS, penicillin (100 units/ml), and streptomycin (100 μg/ml) at 37 °C and 5% CO 2 . Suspensions of 1 × 10 6 cells in 20 ml medium were seeded into cell culture plates (15 cm in diameter) and left untreated for 24 h at 37 °C. Subsequently, the medium was discarded and the cells were treated with fresh medium containing either 1 mM TEGDMA, 1 μM adriamycin or 0.5 μg/ml mitomycin C (MMC) dissolved in medium. For caffeine treatment cells were preincubated with 100, 400 or 1000 μM caffeine for 1 h at 37 °C. Then, all samples were incubated for another 24 h in the presence of 1 mM TEGDMA, 1 μM adriamycin or 0.5 μg/ml MMC. Untreated cell cultures were used as controls. The concentrations of TEGDMA, adriamycin, MMC, and caffeine used here had been determined in preliminary range finding experiments.
2.3
Cell cycle analysis and flow cytometry
The cells were harvested and washed twice in CMF-PBS supplemented with 2% BSA. The cells were fixed and perforated with 70% MeOH at 4 °C over night. MeOH was then removed after centrifugation, and the cells were washed twice with CMF-PBS with 2% BSA. After that the cells were incubated with 0.1 mg/ml RNase in CMF-PBS with 2% BSA for 30 min at 37 °C. Subsequently 50 μg/ml PI was added. The DNA content of the cells, which is proportional to the mean fluorescence value, was measured in a FACSCanto™ flow cytometer (Becton Dickinson, Heidelberg, Germany). Twenty thousand events for each sample were collected with FACSDiva™ software (version 5.0.2) and the data were stored as list mode files. The percentages of cells in G1, S, and G2 phases of the cell cycle were calculated after mathematical modeling using ModFit LT™ software (Verity Software House, Topsham, ME, USA).
2.4
FSC and SSC measurements and cell viability
Forward (FSC) and side light scatter (SSC) measurements can be useful tools in studying the morphological characteristics of cells by flow cytometry . Whereas FSC indicates cell size, SSC is an indicator of the granularity of a cell. Here, FSC and SSC were measured at 488 nm and signals were collected in linear mode. FSC/SSC density plots were generated using FACSDiva™ software (version 5.0.2). To detect possible changes in these parameters in cell cultures exposed to the various chemicals, the main population of untreated cells was marked by a region of interest called P1 (gating). The gate settings were not changed during the course of an experiment. To compare measurements, the percentage of the gated events that could be assigned to these regions was used.
Cell viability after exposure to TEGDMA, adriamycin, or mitomycin C alone or in combination with caffeine as well as in untreated controls was determined by flow cytometry using trypan blue (TB) as a dye. Aliquots for TB measurements were taken from the same cell cultures analyzed for the distribution of cells among the different phases of the cell cycle. The cells were harvested and washed two times in buffer (1% BSA and 0.1% NaN 3 in CMF-PBS) by centrifugation at 300 × g for 5 min. After resuspending the cells in 100 μl buffer, TB was added to a final concentration of 0.013% and incubated for 5 min at RT in the dark, followed by two washes as described above. Then, mean fluorescence intensities (MFI) were measured in a FACSCanto™ flow cytometer with an excitation wavelength of 633 nm and a detector equipped with a 780/60 nm band pass filter. Necrotic cells were distinguished from viable cells by higher fluorescence intensities, which result in two distinct peaks in a histogram. For each sample, 20,000 events were counted and analyses were performed with FACSDiva™ 5.0.2 software.
2.5
Cytotoxicity testing
Cell numbers in treated and untreated cultures were determined as a measure of cytotoxicity using a crystal violet assay as described earlier . The RAW mouse macrophages (7.5 × 10 3 /well) were cultivated in 96 well plates for 24 h at 37 °C. Then, the cell cultures were preincubated with caffeine (100, 400 or 1000 μM) for 1 h or directly treated with 200 μl cell culture medium containing either 1 mM TEGDMA, 1 μM adriamycin or 0.5 μg/ml MMC. Exposure of the cell cultures was stopped after 24 h, the cells were fixed with 1% glutaraldehyde for 30 min, and stained with crystal violet (0.02% in water) for 15 min. The amount of crystal violet bound to the cells was dissolved with 70% ethanol and optical densities were measured at 600 nm in a multiwell spectrophotometer (Infinite 200, TECAN, Crailsheim, Germany).
2.6
Data analyses
For the analysis of cytotoxicity, four replicate cell cultures were exposed to each concentration of the tested chemicals in repeated independent experiments. Cell numbers were calculated from individual optical density readings and normalized to untreated controls (=100%) as specified in the legend of Fig. 5 . The percentage of cells stained with trypan blue (TB) for the analysis of cell viability using FACS was determined in at least four independent experiments. The ratios of viable and necrotic cells in treated cultures were normalized to those obtained in untreated control cultures (=100%). Likewise, the distribution of cells among the different phases of the cell cycle (G1, S, and G2) was calculated from individual histograms obtained in at least four independent experiments. Median values plus 25% and 75% quartiles were calculated from individual measurements. Differences between median values were statistically analyzed using the Mann-Whitney U test (SPSS 15.0, SPSS, Chicago, IL, USA) for pairwise comparisons among groups at the 0.05 level of significance.
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