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Global Gene Expression Analyses of Mouse Fibroblast L929 Cells

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Global Gene Expression Analyses of Mouse Fibroblast L929 Cells Dental Materials Journal 25（2）：205－213, 2006 Global Gene Expression Analyses of Mouse Fibroblast L929 Cells Exposed to IC50 MMA by DNA Microarray and Confirmation of Four Detoxification Genes’ Expression by Real-time PCR Atsuko ISHIKAWA1, Satoshi JINNO2, Tomoo SUZUKI2, Tatsuhide HAYASHI3, Tatsushi KAWAI3, Tatsuya MIZUNO1, Takashi MORI1 and Masami HATTORI1 1The Second Department of Prosthodontics, School of Dentistry, Aichi-gakuin University, 2-11 Suemori-dori, Chikusaku, Nagoyashi, Aichi-ken 464-8651, Japan 2Department of Periodontology, School of Dentistry, Aichi-gakuin University, 2-11 Suemori-dori, Chikusaku, Nagoyashi, Aichi-ken 464-8651, Japan 3 Department of Dental Material Science, School of Dentistry, Aichi-gakuin University, 1-100 Kusumoto-cho, Chikusaku, Nagoyashi, Aichi-ken 464-0045, Japan Corresponding author, Atsuko Ishikawa E-mail:[email protected] Received November 9, 2005/Accepted January 17, 2006 ―――――――――――――――――――――――――――――――――――――――――――――――――――――― Methyl methacrylate（MMA）is the main component of methyl methacrylic resin, which is widely used in dentistry. Previ- ous studies have investigated whether MMA has any adverse effects on growth and gene expression in mouse fibroblast L929 cells. The present study was designed to further understand the effects of MMA by focusing on cDNA microarray data after L929 cells were exposed to MMA. MMA was found to inhibit cell growth and induce detoxification response genes in L929 cells. One of the most highly up-regulated genes was glutathione S-transferase, alpha 1（Ya）（Gsta1）, which has recently been shown to participate in Nrf2 regulation and is considered to be related to detoxification response. Molecular biological data obtained in the present study may therefore provide useful insights into the effects of MMA on living tissue. Key words : MMA, cDNA microarray, Gene expression ―――――――――――――――――――――――――――――――――――――――――――――――――――――― INTRODUCTION MATERIALS AND METHODS Methyl methacrylic resin is an important material Experimental procedure for dental and medical purposes; it is relatively easy The experimental procedure employed in this study is to handle, and has flexible formability and excellent summarized in Fig. 1. reparability. On the other hand, residual monomer after polymerization is sometimes more than 20％ Cell viability test and the cytotoxicity thereof may cause inflammatory Mouse fibroblast cell line L929（ATCC＃ CCL-1）was symptoms or allergies1,2）. Due to these cytotoxicity maintained in Minimum Essential Medium-Earle concerns, the adverse effects of methyl methacrylate （Invitrogen, Japan）supplemented with 10％ fetal bo- （MMA）present in dental materials have been thor- vine serum（Equitech Bio Inc.）at 37℃ and 5％ CO2. oughly tested in vitro3－15）. L929 cells were seeded in 96-well plates at 2×104 Recently, cDNA microarray-based methods have cells/cm2 and used for experiments after 24 hours of been introduced for high-throughput monitoring of incubation. Culture medium was removed from each gene expression. This technology has revolutionized well, and monomer solution was added at the start gene expression studies by providing a means to si- of the experiment. Monomer solutions were prepared multaneously measure mRNA levels in thousands of by diluting MMA （Wako Pure Chemical Industries genes in both simple and complex biological sam- Ltd., Japan）with culture medium to the concentra- ples16－19）. tion specified for each experiment, and then applied The present study was designed to further inves- to the cells. As a negative control, cells were grown tigate the pleiotropic effects of MMA by systemati- only with culture medium. cally identifying the genes differentially expressed To determine MMA IC50（concentration causing a after exposure of the mouse fibroblast cell line L929, 50％ decrease in cell number）－ the cell culture to be which is routinely used for testing the cytotoxic used for cDNA microarray experiment, cell viability properties of dental materials due to their reproduci- tests were performed. Each well was preliminarily ble growth rates and biological responses20,21）. Re- exposed to monomer solutions after 24 hours, and sults of this study may provide insight into the mo- concentration was adjusted to 10, 20, 50, 100, 150, or lecular biological effects of MMA. 200 mmol/l. Cell number in each well was counted 206 CHANGES IN GENE EXPRESSION BY MMA using a hemocytometer after 24 hours of incubation. Using the dose-response curve of relative cell viabil- ity, IC50 was determined. The MMA exposure period for cDNA microarray was determined by time-course experiments. After 24 hours of preliminary incubation, each well was ex- posed to monomer solution（10 mmol/l）and continu- ous incubation was carried out for 2, 24, 48, or 72 hours. To precisely estimate the logarithmic growth phase, a growth curve was plotted. DNA microarray analysis A set of 38,467 sequence-verified mouse cDNA clones （Microarrays Inc., Nashville, TN） was used. L929 cells harvested after 24 hours of preliminary incuba- tion were cultured with （＋MMA） or without （－MMA）10 mmol/l MMA for 12 hours, and total RNA was extracted from the cells. Cultured cells were washed twice with PBS and lysed with TRIzol ReagentTM （Invitrogen, Carlsbad, CA）. Cell lysates were cryopreserved at －80℃ and sent to Genomic Inc., Alliance Biosystems, Japan, where DNA microarray analysis, including RNA purification, la- beling, hybridization, data acquisition and the analy- sis itself, was performed. cDNA targets were syn- thesized from total RNA samples in the presence of either Cy3-dUTP or Cy5-dUTP and applied to the cDNA microarray. RNA from the -MMA sample was used as a reference（Cy3）, and RNA from the ＋MMA sample constituted the experimental （Cy5） sample. Microarray scanning and data normalization were performed using GenePix Pro 4.0 （Molecular Devices Corporation, Sunnyvale, CA）. Gene expres- sion values（GEVs: median ratio in Genepix pro 4.0） were calculated for each hybridized spot. Normaliza- tion of raw data was performed using the LOWESS method, and data were filtered to eliminate the low signal data. Data were expressed as fold change in expression. cDNA microarray experiments were re- peated twice, and both fold expression change values for each gene were used for analysis. Real-time PCR analysis To verify the expression of four selected genes （Gsta1, Gsta2, Gsta4, Nqo1）detected by microarray analysis, real-time PCR was performed. Total cellu- lar RNAs extracted from control and MMA-exposed cells were reverse transcribed to cDNA. Each PCR mix （50 μl） contained between 10 and 100 ng of cDNA, forward and reverse oligonucleopeptide prim- ers, and probe in Taqman master mix. Assays for Gsta2, Gsta4 and Nqo1, as well as PCR experiments, were performed using assay-on-demand primers and probe kits. Custom-made primers and probes were used for Gsta1 because there was no assay-on- demand kit available for this gene（Table 1）. cDNA Fig. 1 Experimental schema. amplification was performed using a sequence detec- ISHIKAWA et al. 207 Table 1 Gene specific primer and probe sequences used for real-time PCR ABI Assay on demand primer & probe Gene name Assay ID Lot Number Gsta2 Mm008333353_mH 249563 Gsta4 Mm0049403_m1 194677 Nqo1 Mm00500821_m1 215862 GAPDH TaqMan Rodent GAPDH Cont TaqMan probe Gene name Probe Seqeonce（5’-3’） Primer Seqence（5’-3’） Gsta1 TTCCTTGCTTCTTGAATTTGTT Forward: CCCCTTTCCCTCTGCTGAAG TTGCATCCAT Reverce: TGAGCTTCACTGAATCTTGAAAG Abbreviations: Gsta2, glutathione-S-transferase, alpha type2; Gsta4, glutathione-S- transferase, alpha type 4; Nqo1, NAD（P）H dehydragenase, quinone 1; Gsta1, glutathione-S- transferase, alpha type1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. tor （Prism Model 7700 Sequence Detector, Applied Biosystems, Japan）, and the thermal cycling condi- tions consisted of initial denaturation at 50℃ for 2 minutes and 95℃ for 10 minutes, followed by 40 cy- cles of 95℃ for 15 seconds and 60℃ for 1 minute. Each assay was performed in triplicate. Relative value of each gene was calculated by the standard curve method. Correlations were verified between relative real-time PCR values and DNA microarray fold expression change values. RESULTS Fig. 2 Dose-response curve of relative cell viability, where Effects of MMA on L929 cell growth the concentration of MMA that decreases the We first considered whether MMA exposure had any number of cells by 50％, IC50, is calculated to be 10 effect on L929 cell growth. MMA exposure was mmol/l. found to result in concentration-dependent inhibition of L929 cell growth when compared with the control. tration of 10 mmol/l and the treatment time of 12 IC50 was calculated by the dose-response curve of hours were selected to investigate the effects of relative cell viability and found to be approximately MMA exposure on global gene expression in L929 10 mmol/l（Fig. 2）. This concentration was therefore cells by cDNA microarray. employed for microarray analysis. To determine the MMA exposure time for cells Gene expression changes induced by MMA in L929 in the microarray experiment, a time-course experi- cells ment was performed. As shown in Fig. 3A, a signifi- Of 38,467 genes screened, expression levels of 331 cant difference in cell number between control and genes were found to be affected by more than two- MMA-exposed cells（p＜0.01）was seen after 24 hours fold, and 53 genes were identified as significant by t- of incubation. The logarithmic growth phase ap- test（p＜0.05）in L929 cells treated with 10 mmol/l peared to begin between 24 and 72 hours. It should MMA for 12 hours － with an overlap of 44 genes in be highlighted that MMA-exposed cells underwent the Venn diagram（Fig. 4, Table 2）. morphological changes into an elongated shape, as One of the highly up-regulated genes was Gsta1 well as to bipolar or multipolar cells with long proc- （Glutathione S-transferase, alpha 1（Ya））, which was esses and beaded varicosity（Fig. 3B）. On the other up-regulated by 27.44-fold（median）. The expression hand, the control cells retained the original of Gsta1 was regulated by transcription factor Nrf2, fibroblastic morphology and tended to grow in clus- which is related to detoxification response.
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