Yuko Okai et al.: in Human Deciduous Tooth Pulp Cells Journal of Hard Tissue Biology 21[4] (2012) p413-420 © 2012 The Hard Tissue Biology Network Association Printed in Japan, All rights reserved. CODEN-JHTBFF, ISSN 1341-7649 Original Study on Apoptosis in Human Deciduous Tooth Pulp Cells

Yuko Okai1), Kyoko Harada1), Kiyoshi Ohura2) and Kenji Arita1)

1)Department of Pediatric Dentistry, Osaka Dental University,Osaka, Japan 2)Department of Pharmacology, Osaka Dental University, Osaka, Japan (Accepted for publication, July 5, 2012) Abstract: Dental pulp plays an important role in tooth vitality. The pulp of deciduous tooth thus shows vigorous metabolism and repeated, vigorous cell regeneration and proliferation. Furthermore, natural, programmed cell death (apoptosis) frequently occurs as part of this cell regeneration and proliferation process. Apoptosis is defined as cell death governed by intracellular signals (active death of the cell triggered by changes in physiological or external conditions), and the birth of new cells occurs simultaneously with cell death. Apoptosis thus functions as a driving force for morphological and other changes in organisms, and is essential for phenomena such as ontogenetic processes or aging. However, In order to clarify the mechanism of apoptosis in the deciduous tooth pulp cells, we used SuperArray analysis and immunostaining to examine both deciduous and permanent tooth pulp cells. Of the 84 major involved in apoptosis, seven showed greater expression in the deciduous tooth pulp cells than in the permanent tooth pulp cells. expression of TP53BP2, CRADD, BAK1, BIRC3, CASP8, CASP2 and TP73 in the deciduous tooth pulp were 16.1, 9.0, 8.5, 8.1, 5.9, 5.8 and 3.2 times greater than those in the permanent tooth pulp cells, respectively.

Key words: Deciduous tooth, Pulp, Fibroblast, Apoptosis.

Introduction induce ossification when transplanted subcutaneously into Deciduous tooth formation begins in the eighth fetal week, immunodeficient mice, whereas permanent tooth pulp cells induce and the crowns are fully formed within one year post partum. dentin- or pulp-type structures 7,8). The pulp of deciduous tooth After a period of dental attrition, root resorption occurs from four thus shows vigorous metabolism and repeated, vigorous cell to eight years of age, and the deciduous tooth are lost by around regeneration and proliferation. Furthermore, natural, programmed 12 years. Because this is a short-term, dynamic process of cell death (apoptosis) frequently occurs as part of this cell development, the pulp of deciduous tooth has a greater number of regeneration and proliferation process. Apoptosis is defined as undifferentiated cells than that of permanent tooth, and is thus cell death governed by intracellular signals (active death of the more primitive and has different characteristics1-4). The pulp of cell triggered by changes in physiological or external conditions), deciduous tooth is also known to have higher cell iferation capacity and the renewal of cells occurs simultaneously with cell death. and vital activity than that of permanent tooth. The pulp of Apoptosis thus functions as a driving force for morphological and deciduous tooth has advanced reparative function; for example, other changes in organisms, and is essential for phenomena such caries that would result in dental pulp exposure in permanent tooth as ontogenetic processes or aging. Advanced apoptosis leads to the formation of secondary dentin in deciduous tooth. In mechanisms are thus highly likely to be present in the pulp of this situation, undifferentiated mesenchymal cells in the deciduous tooth, but it appears that no studies have been reported perivascular region are believed to differentiate into odontoblasts, in relation to apoptosis in deciduous tooth pulp. fibroblasts, histiocytes, and other cells in response to various In order to clarify the mechanism of apoptosis in deciduous stimuli5). In addition, from the point of view of , tooth pulp cells, we used SuperArray analysis and immunostaining vascular endothelial growth factor and growth factors have been to examine both deciduous and permanent tooth pulp cells. reported to exhibit greater preferential expression in deciduous than permanent tooth 6), reported difference between deciduous Materials and Methods and permanent tooth pulp cells is that deciduous tooth pulp cells Cell culture Deciduous tooth pulp was collected from tooth removed from Corresponcenced to: Dr.Yuko Okai, Department of Pediatric Dentistry, Osaka Dental University, 8-1 KuzuhaHanazono-cho, Hirakata-city, patients at the Department of Pediatric Dentistry, Osaka Dental Osaka573-1121, Japan, E-mail [email protected] University, with patients consenting to the provision of tooth for

413 J.Hard Tissue Biology Vol. 21(4):413-420, 2012

Table1. The genes that work mainly in normal biological processes requiring cell elimination, such as differentiation and homeostasis, were examined using a profiler PCR array system (SuperArray Bioscience Corporation, Frederick, MD). 84 genes involved in apoptosis were extracted. Gene expression in each sample was measured by the ΔΔCT method using a TaqMan® Gene Expression Assay on the StepOne Plus system (Applied Biosystems), with GAPDH as an endogenous control.

Genebank GaneSymble Descripton

NM_005157 ABL1 C-abl oncogene 1, receptor tyrosine kinase NM_005163 AKT1 V-akt murine thymoma viral oncogene homolog 1 NM_001160 APAF1 Apoptotic peptidase activating factor 1 NM_004322 BAD BCL2-associated agonist of cell death NM_004323 BAG1 BCL2-associated athanogene NM_004281 BAG3 BCL2-associated athanogene 3 NM_004874 BAG4 BCL2-associated athanogene 4 NM_001188 BAK1 BCL2-antagonist/killer 1 NM_004324 BAX BCL2-associated X NM_003921 BCL10 B-cell CLL/lymphoma 10 NM_000633 BCL2 B-cell CLL/lymphoma 2 NM_004049 BCL2A1 BCL2-related protein A1 NM_138578 BCL2L1 BCL2-like 1 NM_020396 BCL2L10 BCL2-like 10 (apoptosis facilitator) NM_006538 BCL2L11 BCL2-like 11 (apoptosis facilitator) NM_004050 BCL2L2 BCL2-like 2 NM_014739 BCLAF1 BCL2-associated transcription factor 1 NM_016561 BFAR Bifunctional apoptosis regulator NM_001196 BID BH3 interacting domain death agonist NM_001197 BIK BCL2-interacting killer (apoptosis-inducing) NM_004536 NAIP NLR family, apoptosis inhibitory protein NM_001166 BIRC2 Baculoviral IAP repeat-containing 2 NM_001165 BIRC3 Baculoviral IAP repeat-containing 3 NM_001167 XIAP X-linked inhibitor of apoptosis NM_016252 BIRC6 Baculoviral IAP repeat-containing 6 NM_033341 BIRC8 Baculoviral IAP repeat-containing 8 NM_001205 BNIP1 BCL2/adenovirus E1B 19kDa interacting protein 1 NM_004330 BNIP2 BCL2/adenovirus E1B 19kDa interacting protein 2 NM_004052 BNIP3 BCL2/adenovirus E1B 19kDa interacting protein 3 NM_004331 BNIP3L BCL2/adenovirus E1B 19kDa interacting protein 3-like NM_004333 BRAF V-raf murine sarcoma viral oncogene homolog B1 NM_006092 NOD1 -binding oligomerization domain containing 1 NM_032587 CARD6 Caspase recruitment domain family, member 6 NM_014959 CARD8 Caspase recruitment domain family, member 8 NM_033292 CASP1 Caspase 1, apoptosis-related cysteine peptidase (interleukin 1, beta, convertase) NM_001230 CASP10 Caspase 10, apoptosis-related cysteine peptidase NM_012114 CASP14 Caspase 14, apoptosis-related cysteine peptidase NM_032982 CASP2 Caspase 2, apoptosis-related cysteine peptidase NM_004346 CASP3 Caspase 3, apoptosis-related cysteine peptidase NM_001225 CASP4 Caspase 4, apoptosis-related cysteine peptidase NM_004347 CASP5 Caspase 5, apoptosis-related cysteine peptidase NM_032992 CASP6 Caspase 6, apoptosis-related cysteine peptidase NM_001227 CASP7 Caspase 7, apoptosis-related cysteine peptidase NM_001228 CASP8 Caspase 8, apoptosis-related cysteine peptidase

414 Yuko Okai et al.: Apoptosis in Human Deciduous Tooth Pulp Cells

NM_001229 CASP9 Caspase 9, apoptosis-related cysteine peptidase NM_001250 CD40 CD40 molecule, TNF receptor superfamily member 5 NM_000074 CD40LG CD40 ligand NM_003879 CFLAR CASP8 and FADD-like apoptosis regulator NM_001279 CIDEA Cell death-inducing DFFA-like effector a NM_014430 CIDEB Cell death-inducing DFFA-like effector b NM_003805 CRADD CASP2 and RIPK1 domain containing adaptor with death domain NM_004938 DAPK1 Death-associated protein kinase 1 NM_004401 DFFA DNA fragmentation factor, 45kDa, alpha polypeptide NM_003824 FADD Fas (TNFRSF6)-associated via death domain NM_000043 FAS Fas (TNF receptor superfamily, member 6) NM_000639 FASLG Fas ligand (TNF superfamily, member 6) NM_001924 GADD45A Growth arrest and DNA-damage-inducible, alpha NM_003806 HRK Harakiri, BCL2 interacting protein (contains only BH3 domain) NM_000875 IGF1R Insulin-like growth factor 1 receptor NM_000595 LTA Lymphotoxin alpha (TNF superfamily, member 1) NM_002342 LTBR Lymphotoxin beta receptor (TNFR superfamily, member 3) NM_021960 MCL1 Myeloid cell leukemia sequence 1 (BCL2-related) NM_003946 NOL3 Nucleolar protein 3 (apoptosis repressor with CARD domain) NM_013258 PYCARD PYD and CARD domain containing NM_003821 RIPK2 Receptor-interacting serine-threonine kinase 2 NM_000594 TNF Tumor necrosis factor (TNF superfamily, member 2) NM_003844 TNFRSF10A Tumor necrosis factor receptor superfamily, member 10a NM_003842 TNFRSF10B Tumor necrosis factor receptor superfamily, member 10b NM_002546 TNFRSF11B Tumor necrosis factor receptor superfamily, member 11b NM_001065 TNFRSF1A Tumor necrosis factor receptor superfamily, member 1A NM_014452 TNFRSF21 Tumor necrosis factor receptor superfamily, member 21 NM_003790 TNFRSF25 Tumor necrosis factor receptor superfamily, member 25 NM_001242 CD27 CD27 molecule NM_001561 TNFRSF9 Tumor necrosis factor receptor superfamily, member 9 NM_003810 TNFSF10 Tumor necrosis factor (ligand) superfamily, member 10 NM_001252 CD70 CD70 molecule NM_001244 TNFSF8 Tumor necrosis factor (ligand) superfamily, member 8 NM_000546 TP53 Tumor protein NM_005426 TP53BP2 Tumor protein p53 binding protein, 2 NM_005427 TP73 Tumor protein NM_003789 TRADD TNFRSF1A-associated via death domain NM_021138 TRAF2 TNF receptor-associated factor 2 NM_003300 TRAF3 TNF receptor-associated factor 3 NM_004295 TRAF4 TNF receptor-associated factor 4 NM_004048 B2M Beta-2-microglobulin NM_000194 HPRT1 Hypoxanthine phosphoribosyltransferase 1 NM_012423 RPL13A Ribosomal protein L13a NM_002046 GAPDH Glyceraldehyde-3-phosphate dehydrogenase NM_001101 ACTB Actin, beta research purposes. The pulp was collected from deciduous tooth 3 of the root resorped). Permanent tooth pulp was collected from extracted during deciduous tooth shedding; extracted tooth ranged non-carious tooth that had been extracted for the purpose of from the period of root completion to root resorption (less than 1/ orthodontic treatment (deciduous tooth pulp cells: DTPC, n=3; 415 J.Hard Tissue Biology Vol. 21(4):413-420, 2012 DTPC PTPC involved in apoptosis were extracted (Table 1). Gene expression 6 in each sample was measured by the △△ CT method using a TaqMan® Gene Expression Assay on the StepOne Plus system 5 (Applied Biosystems), with GAPDH as an endogenous control. 4 Immunostaining 3 In order to investigate expression of the four genes obtained by real-time PCR SuperArray analysis, the DTPC and the PTPC were

% of %cont. 2 sown in chamber slides at 5×105 cells/well and cultured at 37°C 1 in a humidified 5% CO2 atmosphere for 72 h. After removing DMEM, samples were washed in PBS (-), 4% paraformaldehyde 0 24 h 72 h 1 W in PBS (-) was added (2 mL/well), and were left at room *P<0.05 **p<0.01 temperature for 20-30 min. They were then washed in PBS (-), Figure1. Cell proliferation by MTS assay after incubation 37℃ 0.1% Triton X-100 in PBS (-) was added (2 mL/well), and samples for 24h, 72h，1w in the DMEM conditions. Three wells were then left at room temperature for 30 min, followed by washing containing only MTS and no cells were used as a blank. All with PBS (-). analysis was repeated in duplicate on three separate occasions. A solution of 1% BSA and 0.1% Tween 20 in PBS (-) was permanent tooth pulp cells: PTPC, n=5). Pulp tissue fragments added (2 mL/well), and samples were left at room temperature for 60 min. Next, samples were left to stand with primary antibody were cultured at 37°C in a humidified 5% CO2 atmosphere in Dulbecco’s modified Eagle’s medium (DMEM; Gibco Life for 60 min and then washed with 0.1% Tween 20 in PBS (-). Technologies Japan Ltd, Tokyo, Japan) supplemented with 10% Samples were then left to stand at room temperature with secondary fetal bovine serum (FBS; Gibco), 4 mM L-glutamine, 100 U/mL antibody shielded from light for 60 min, and were next washed penicillin and 100 mg/ml streptomycin. Outgrowth fibroblasts cells with 0.1% Tween 20 in PBS (-). Samples were embedded and were separated from tissue fragments using 0.25% trypsin-EDTA, fluorescence microscopic observation of stained cells was used followed by passage culture. Cells for experimental use were to investigate TP53BP2, Bak1, CASP2 and CASP8 gene cultured for three to five passages in DMEM with 10% FBS. The expression in the pulp cells. TP53BP2 monoclonal antibody was experiments were approved by the Ethical Committee of Osaka purchased from Abnova Corporation (Taipei, Taiwan), Bak1 Dental University (No.091129). monoclonal antibody was from Enzo Life Sciences Inc (UK), CASP2 monoclonal antibody was from Assay Biotechnology MTS assay Company (Changsha,China)and anti-CASP8 monoclonal antibody In order to investigate cell proliferation capacity, the DTPC was from Abnova Corporation (Taipei, Taiwan). Secondary and the PTPC were sown in 96-well plates at 5×104 cells/well and antibody was rabbit monoclonal antibody (DakoCytomat ion, Glostrup, Denmark). cultured at 37°C in a humidified 5% CO2 atmosphere for 24 h, 72 h or 1week.MTS [3-(4,5-dimcthylthiazol-2-yl) -5-(3- carboxymethoxypheny1)-2-(4-sulfophenyl)-2H-tetrazolium] Results reagent (200 µL/well) was added and after 2 h, optical density at MTS assay 490 nm was measured using a microplate reader to determine cell The proliferation in the DTPC showed notable increases from proliferation capacity. 72 h until 1 week after cultivation, and these increases were significantly greater than those in the PTPC (Fig.1). Real-time PCR superarray analysis The DTPC and the PTPC were sown in 6-cm dishes at 5×104 Real-time PCR superarray analysis Of the 84 major genes involved in apoptosis (Table 1), seven cells/well and cultured at 37°C in a humidified 5% CO2 atmosphere for 72 h. Total RNA was then isolated using a MagExtractor showed greater expression in deciduous tooth pulp cells than in (Toyobo Co., Ltd., Osaka, Japan), and following purification, it permanent tooth pulp cells(Table 2). Gene expression of the tumor was converted to cDNA using High Capacity RNA-to-cDNA protein p53 binding protein2 (TP53BP2), the death domain- Master Mix (Applied Biosystems, Foster City, CA, USA). The containing protein (CRADD), the BCL2-homologous antagonist/ genes that work mainly in normal biological processes requiring killer 1 (BAK1), the baculoviral IAP repeat-containing protein 3 cell elimination, such as differentiation and homeostasis, were (BIRC3), the caspase 8 (CASP8), the caspase 2 (CASP2), and the examined using a profiler PCR array system (SuperArray tumor protein 73 (TP73) were 16.10, 9.02, 8.46, 8.09, 5.89, 5.81, Bioscience Corporation, Frederick, MD). Eighty-four genes and 3.18 times greater than those in the PTPC, respectively.

416 Yuko Okai et al.: Apoptosis in Human Deciduous Tooth Pulp Cells

TP53BP2 in the DTPC TP53BP2 in the PTPC

BAK1 in the DTPC BAK1 in the PTPC

CASP8 in the DTPC CASP8 in the PTPC

CASP2 in the DTPC CASP2 in the PTPC

Figure 2. In order to investigate expression of the four genes obtained by the real-time PCR SuperArray analysis, the 5 DTPC and the PTPC were sown in chamber slides at 5×10 cells/well and cultured at 37 C in a humidified 5% CO2 atmosphere for 72 h. The antibody uses TP53BP2, BAK1, CASP8, and CASP2. The cytoplasm is dyed to green, and the nucleus has been dyed to blue.

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Table2. Of the 84 major genes involved in apoptosis, seven showed greater expression in the DTPC and the PTPC.

Radio(DTPC/PTPC) Symbol Gene bank Description

16.10 TP53BP2 NM_005426 Tumor protein p53 binding protein, 2 9.02 CRADD NM_003805 CASP2 and RIPK1 domain containing adaptor with death domain 8.46 BAK1 NM_001188 BCL2-antagonist/killer 1 8.09 BIRC3 NM_001165 Baculoviral IAP repeat-containing 3 5.89 CASP8 NM_001228 Caspase 8, apoptosis-related cysteine peptidase 5.81 CASP2 NM_032982 Caspase 2, apoptosis-related cysteine peptidase 3.18 TP73 NM_005427 Tumor protein p73

Immunostaining cascades involving various adaptor molecules, phosphoinositide Immunostaining was carried out by focusing on TP53BP2, 3-kinase (PI3K)-protein kinase B (Akt) or mitogen-activated BAK1, CASP8 and CASP2. The DTPC showed greater protein protein (MAP) kinase15). expression than the PTPC in TP53BP2, which belongs to the The fate of the cell is believed to be determined further apoptosis-stimulating protein of p53 (ASPP) family of p53 binding downstream within the nucleus by control of major transcription that stimulate apoptosis; BAK1, which belongs to the factors, such as p53, that decide the life or death of the cell. Limited BCL2 family; CASP8, which activates the caspase group to which proteolysis by caspases of specific proteins (e.g., poly [ADP- CASP2 belongs; and CASP2, which is involved in the ribose] polymerase [PARP], inhibitor of caspase-3-activated fragmentation and activation of molecules that induce apoptosis. DNase [ICAD], apoptotic chromatin condensation inducer in the These were the only genes for which real-time PCR SuperArray nucleus [Acinus]) also plays a central role in the apoptosis analysis results were obtained (Fig 2). execution process. Caspases exist as inert procaspases and undergo limited proteolysis due to their own action or that of other caspases, Discussion and as a result, they are dimerized and converted to the active The capacity for proliferation of the cells used in the present form. study was compared by MTS assay in order to examine whether Caspases can be broadly classified into three groups in terms the results obtained would be the same as those previously of function. The first group comprises apoptosis-inducing factors, reported. Higher cellular proliferation capacity was observed in caspases 2, 8, 9 and 10, which have domains that bind directly to cells originating from deciduous tooth pulp than in cells originating adapter molecules and are activated first. The second group from permanent tooth pulp, which was consistent with prior comprises apoptosis execution factors, caspases 3, 7, and 6, which reports9-11). are influenced by the inducing factors. The third group comprises In particular, in the present study, both deciduous tooth and caspases 1 and 4, which cause activation of the cytokines permanent tooth pulp cells increased from the start of culture until interleukin-1 beta (IL-1b) and interleukin-18 (IL-18). Bcl-2 and

72 h with no great difference between the two. However, from 72 anti-apoptotic regulator Bcl-xL (Bcl-XL), which are from the Bcl- h until 1 week; deciduous tooth pulp cells showed significantly 2 family of apoptosis regulator proteins, inhibit apoptosis by greater increases than cells from permanent tooth pulp. Because partially blocking the release of cytochrome c from mitochondria. the mechanism of apoptosis is the same in all animal cells12-14).The Conversely, the Bcl-2-associated death (BAD) is a pro- 84 genes believed to be involved in apoptosis in deciduous and apoptotic member of the Bcl-2 protein family. BAD binds to permanent tooth pulp (Shown in Table 1) were searched using a proteins that inhibit apoptosis to halt their activity, thus activating SuperArray. Deciduous tooth pulp cells showed gene expression procaspases and facilitating apoptosis. The Bcl-2-associated X of TP53BP roughly 16 times greater, expression of CRADD protein (Bax) and the BCL-2-interacting killer (Bak) function as roughly 9 times greater, expression of BAK1 roughly 9 times stimulating factors, facilitating the release of cytochrome c from greater, expression of BIRC3 roughly 8 times greater, expression the mitochondria. In addition, cell stress increases the activity of of CASP8 roughly 6 times greater, expression of CASP2 roughly the tumor suppressor tumor protein p53 (TP53), resulting in 6 times greater, and expression of TP73 roughly 3 times greater activation of Bax and Bak by Bcl-2 pro-apoptosis factors such as than per anent tooth pulp cells. BH3 interacting domain death agonist (BID). Immunostaining of The main factor acting as a signal to trigger apoptosis is believed deciduous and permanent tooth pulp cells for TP53BP2, BAK1, to be stress due to withdrawal of growth factor or neurotrophic CASP8 and CASP2 was carried out. Of the seven genes for which factor, active enzymes, DNA damage, cytokine or drugs. Directly SuperArray results were obtained, TP53BP2, BAK1, CASP8 and below the receptors for these apoptosis signals are phosphate CASP2 showed the same differences in gene staining as observed

418 Yuko Okai et al.: Apoptosis in Human Deciduous Tooth Pulp Cells in gene expression. binds to the complex through mediation of the FADD molecule TP53 is believed to have two main types of action. The first is and undergoes limited proteolysis and activation. It then activates to protect against . When damage is sustained through UV group A caspases, inducing apoptosis 26). rays, chemical substances or other agents, TP53 is activated Thus, it appears that TP53BP2 and BAK1 are activated via the through the mediation of protein kinases and activates WNT receptors present on the cell membrane that receive all the different inhibitory factor 1 (WIF1) downstream. The activated WIF1 cell death signals, and TP53BP2 activation is increased. In the suppresses cyclins D and E, thus arresting the at G1 and present study, simultaneous expression was observed in CASP8, suppressing cell proliferation until the DNA is repaired. The which is an apoptosis-inducing factor, and CASP2, which is second action is to induce apoptosis, which is mediated by the activated by CASP8. With regard to apoptosis in deciduous tooth, p53-inducible gene 3 (PIG3) or by Bax, a pro-apoptotic Bcl-2 the present study indicates that apoptosis can be induced either protein containing BH1, BH2 and BH3 domains. TP53 is thus via a mechanism involving TP53BP2 and BAK1 or via a believed to make the signal branch into the first action, arresting mechanism involving CASP8 and CASP2, which is activated by the cell cycle at G1, and the second action, apoptosis. TP73 is CASP8. The present study also suggests that deciduous tooth pulp believed to suppress cell proliferation and induce apoptosis in the cells are characterized by readier induction of apoptosis than same way as TP53. TP53BP2 belongs to the ASPP (apoptosis permanent tooth, as CASP8, the first stage of apoptosis, and stimulating protein of TP53) family of TP53 binding proteins. CASP2, the second stage which is activated by CASP8, are TP53BP2 includes an SH3 domain, which is involved in expressed simultaneously; thus, deciduous tooth pulp apoptosis interaction between proteins. It is localized around the nucleus in is rapidly triggered by the presence of the appropriate factor. the cytoplasm and is believed to regulate apoptosis and cell proliferation through interaction with other control molecules, Acknowledgments including TP53 family members16-24). This work was supported by a Grant-in-Aid for Young Scientists BAK1 belongs to the BCL2 family, which brings about (B) from the Japan Society for the Promotion of Science apoptosis by forming oligomers or heterodimers to start voltage- (K.Harada, 23792459), a “High-Tech Research Center” Project dependent anion channels in the mitochondrial membrane. BAK1 for Private Universities: matching fund subsidy from MEXT is also reported to increase activation of the tumor suppressor (Ministry of Education, Culture, Sports, Science and Technology), P53 by causing cell stress25). Caspases are proteases that generally 2007-2011 and a “Osaka Dental University Research Funds” (No carry out limited proteolysis of substrates, and by fragmenting 11-9,2012). We are most grateful to Dr. Yoshiya Hashimoto for specific proteins (caspases themselves; BH3 interacting domain helpful advice on this study. This study was performed in the death agonist [Bid]; DNA fragmentation factor, 45 kDa, alpha institute of Dental Research, Osaka Dental University. polypeptide [DFFA]; ICAD/DFF45 including cell death-inducing DFFA-like effector a [CIDEA]), cause them to act. ICAD/DFF45, Reference which was discovered by the research team of Wang and Nagata 1. Eslaminejad MB, Vahabi S, Shariati M, NazaLee S, An S, et al. is a structural component of the caspase-dependent DNA Kang TH, Kim KH, Chang NH, Kang S, Kwak CK and Park fragmentation observed at apoptosis and undergoes limited HS. Compar Regen ison of mesenchymal-like stem/ proteolysis by caspase. The cleaved ICAD/DFF45 causes progenitor cells derived from supernumerary teeth with stem activation of the nucleases carbamoyl-phosphate synthetase 2, cells from human exfoliated deciduous teeth. Med 6(6): 689- aspartate transcarbamylase, and dihydroorotase (CAD)/DNA 699, 2011 fragmentation factor, 40 kDa, beta polypeptide (caspase-activated 2. rian H. In vitro Growth and Characterization of Stem Cells DNase) (DFF40), another component of the complex, which from Human Dental Pulp of Deciduous Versus Permanent fragment DNA. The research team of Nicholson et al. from the Teeth. J Dent (Tehran) 7(4):185-195, 2010 Merck Frosst Centre for Therapeutic Research reported that 3. Hirata T, Ishkitiev N, Yaegaki K, Calenic B, Ishikawa H, caspases can be broadly classified into two types according to Nakahara T, Mitev V, Tanaka T and Haapasalo M. Expression their substrate specificity. CASP2 belongs to group A, which is of multiple stem cell markers in dental pulp cells cultured in involved in proteolysis and activation of molecules that cause serum-free media. J Endod 36(7):1139-1144, 2010 apoptosis. CASP8 belongs to group B and is believed to activate 4. Coppe C, Zhang Y and Den Besten PK. Characterization of group A caspases. CASP8 contains in its prodomain region an N- primary dental pulp cells in vitro. Pediatr Dent 31(7): 467- terminal domain with to the FADD molecule, 71, 2009 which forms an association with the apoptosis-inducing Fas 5. Takiguchi R. Textbook of Oral Embryology, Gakkenshoin, molecule, indicating the presence of CASP8 in the Fas molecule 17, 1991 complex. As a result of activation of the Fas molecule, CASP8 6. Harada R, Watanabe K, Shirasu S, Kato M and Daito M.

419 J.Hard Tissue Biology Vol. 21(4):413-420, 2012 DNA microarray analysis of dental pulp fibroblasts exfoliated 17. Ahn J, Byeon IJ, Byeon CH and Gronenborn AM. Insight from deciduous teeth. Pediatric Dental Journal 19(1) 38-45, into the structural basis of pro- and antiapoptotic p53 2009 modulation by ASPP proteins. J Biol Chem 284(20): 13812- 7. Gronthos S, Mankani M, Brahim J, Robey PG and Shi S. 13822, 2009 Postnatal human dental pulp stem cells (DPSCs) in vitro 18. Chen G, DenBoer L and Shin J. Design of a single plasmid- and in vivo. Proc Natl Acad Sci U S A 97(25): 13625-13630, based modified yeast one-hybrid system for investigation of 2000 in vivo protein-protein and protein-DNA interactions. 8. Seo B, Sonoyama W and Yamaza T, Coppe C, Kikuiri T, Biotechniques 45(3): 295-304, 2008 Akiyama K, Lee JS and Shi S. SHED repair critical-size 19. Rotem S, Katz C, Benyamini H, Lebendiker M, Veprintsev calvarial defects in mice. Oral Dis 14(5): 428-434, 2008 D, Rüdiger S, Danieli T and Friedler A. The structure and 9. Soleimani M, Taghipour Z, Karbalaie K, Mousavi SB, Talebi interactions of the proline-rich domain of ASPP2. J Biol A, Nadali F, Tanhaei S, Kiyani GA, Nematollahi M, Rabiei Chem 283(27): 18990-18999, 2008 F, Mardani M, Bahramiyan H, Torabinejad M, Nasr-Esfahani 20. Braithwaite AW, Del Sal G and Lu X. Some p53-binding MH and Baharvand H . Induced in vitro differentiation of proteins that can function as arbiters of life and death. Cell neural-like cells from human exfoliated deciduous teeth- Death Differ 13(6): 984-93, 2006 derived stem cells. Int J Dev Biol 55(2): 189-195, 2011 21. Zhu Z, Ramos J, Kampa K, Adimoolam S, Sirisawad M, Yu 10. Suchánek J, Soukup T, Ivancaková R, Karbanová J, Hubková Z, Chen D, Naumovski L and Lopez CD. Control of ASPP2/ V, Pytlík R and Kucerová L. Human dental pulp stem cells— (53BP2L) protein levels by proteasomal degradation isolation and long term cultivation. Acta Medica 50(3): 195- modulates p53 apoptotic function. J Biol Chem 280(41): 201, 2007 34473-34480, 2005 11. Lee S, An S, Kang TH, Kim KH, Chang NH, Kang S, Kwak 22. Bergamaschi D, Samuels Y, Jin B, Duraisingham S, Crook CK and Park HS. Comparison of mesenchymal-like stem/ T and Lu X. ASPP1 and ASPP2: common activators of p53 progenitor cells derived from supernumerary teeth with stem family members. Mol Cell Biol 24(3): 1341-1350, 2004 cells from human exfoliated deciduous teeth. Regen Med 23. Ao Y, Rohde LH and Naumovski L. p53-interacting protein 6(6): 689-699, 2011 53BP2 inhibits clonogenic survival and sensitizes cells to 12. Ranger AM, Malynn BA and Korsmeyer SJ. Mouse models doxorubicin but not paclitaxel-induced apoptosis. Oncogene of cell death. Nat Genet 28(2): 113-118, 2001 20(21): 2720-2725, 2001 13. Vaux DL and Korsmeyer SJ. Cell death in development. Cell 24. Espanel X and Sudol M. Yes-associated protein and p53- 96(2): 245-254, 1999 binding protein-2 interact through their WW and SH3 14. Jacobson MD, Weil M and Raff MC. Programmed cell death domains. J Biol Chem 2276(17): 14514-14523, 2001 in animal development. Cell 88(3): 347-354, 1997 25. Sawai H and Domae N. Differential roles for Bak in Triton 15. Peter ME, Heufelder AE and Hengartner MO. Advances in X-100- and deoxycholate-induced apoptosis. Biochem apoptosis research. Proc Natl Acad Sci U S A 94(24): 12736- Biophys Res Commun 378(3): 529-533, 2009 7, 1997 26. Ritter N, Mussig E, Steinberg T, Kohl A, Komposch G and 16. Kampa KM, Bonin M and Lopez CD. New insights into the Tomakidi P. Elevated expression of genes assigned to NF- expanding complexity of the tumor suppressor ASPP2. Cell kappaB and apoptotic pathways in human periodontal Cycle. 8(18): 2871-2876, 2009 ligament fibroblasts following mechanical stretch. Cell Tissue Res 328(3): 537-548, 2007

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