title: The mechanism of Jurkat cells apoptosis induced by Aggregatibacter actinomycetemcomitans cytolethal distending toxin.
abstract: Cytolethal distending toxin (CDT) which is produced by Aggregatibacter actinomycetemcomitans causes apoptosis in lymphocytes. But the specific mechanism is not clear. The aim of our research was to investigate the effect and mechanism during this process. The wild-type CdtA, CdtB, CdtC (CdtAW, CdtBW, CdtCW) and mutant CdtB (CdtBM) were expressed and purified respectively and the purity of each subunit was examined by BandScan software. And the type I deoxyribonuclease and PI-3,4,5-triphosphate (PI-3,4,5-P3, PIP3) phosphatase activity were detected by DNA agarose gel electrophoresis and enzyme-linked immunosorbent assay respectively. The cell apoptosis rates were analyzed by flow cytometry. The morphological changes of apoptosis cells were observed by confocal laser scanning microscopy. The protein expression of Bax and Bcl-2 was examined by western blot. Differentially expressed apoptosis-related proteins were identified based on isobaric tags for relative and absolute quantitation technology. In the present study we found that: (i) recombinant wild-type CdtA, CdtB and CdtC (CdtAW, CdtBW, CdtCW) and mutant CdtB (CdtBM) were correctly expressed and the purity of each protein was higher than 80%, (ii) the average apoptosis rate in wild-type CDT (CDTW) treated groups was 50.54%, which was significantly higher than the controls (4.71%) and mutant CDT (CDTM) treated groups (5.58%) (p < 0.05), (iii) morphological changes of apoptosis were observed in CDTWtreated cells, (iv) the expression of Bax protein was significantly increased in CDTWtreated cells, while Bcl-2 protein expression was significantly decreased, (v) 17 apoptosis-related proteins were expressed differentially, among which 10 proteins (SMNDC1, TNFRSF10B, UBE2I, ITM2A, CASP3, P53, EIF1, TCF3, HMGN5, CASP8) were up-regulated and 7 proteins (RRM2, TPX2, KIF11, NUCKS1, TOP2A, XRCC1, PTPLAD1, RRM2) were down-regulated, (vi) one possible apoptotic pathway [Ubc9 (UBE2I)/P53/DR5 (TNFRSF10B)/Caspase-8 (CASP8)/ Caspase-3 (CASP3)] was selected and partially proved.
authors.affiliation: Jiangsu Key Laboratory of Oral Disease, Nanjing Medical University, Nanjing, Jiangsu, China.
authors.forename: Hui-Ping
authors.initials: HP
authors.lastname: Chen
authors.affiliation: Jiangsu Key Laboratory of Oral Disease, Nanjing Medical University, Nanjing, Jiangsu, China.|Department of Periodontics, Affiliated Hospital of Stomatology, Nanjing Medical University, 140 Hanzhong Road, Nanjing, 210029, Jiangsu, China.
authors.forename: Lu
authors.initials: L
authors.lastname: Li
authors.affiliation: Jiangsu Key Laboratory of Oral Disease, Nanjing Medical University, Nanjing, Jiangsu, China.
authors.forename: Xu
authors.initials: X
authors.lastname: Chen
authors.affiliation: Jiangsu Key Laboratory of Oral Disease, Nanjing Medical University, Nanjing, Jiangsu, China.
authors.forename: Mi-Fang
authors.initials: MF
authors.lastname: Yang
authors.affiliation: Jiangsu Key Laboratory of Oral Disease, Nanjing Medical University, Nanjing, Jiangsu, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Ye
authors.affiliation: Jiangsu Key Laboratory of Oral Disease, Nanjing Medical University, Nanjing, Jiangsu, China.
authors.forename: Xiao-Qian
authors.initials: XQ
authors.lastname: Wang
authors.affiliation: Jiangsu Key Laboratory of Oral Disease, Nanjing Medical University, Nanjing, Jiangsu, China. yanxu@njmu.edu.cn.|Department of Periodontics, Affiliated Hospital of Stomatology, Nanjing Medical University, 140 Hanzhong Road, Nanjing, 210029, Jiangsu, China. yanxu@njmu.edu.cn.
authors.forename: Yan
authors.initials: Y
authors.lastname: Xu
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D001427:Bacterial Toxins / Q000633:toxicity*; D003850:Deoxyribonuclease I / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling; D019169:Jurkat Cells; D008954:Models, Biological; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D021122:Protein Subunits / Q000378:metabolism; D040901:Proteomics; D015203:Reproducibility of Results; D051028:bcl-2-Associated X Protein / Q000378:metabolism
chemical_list: D001427:Bacterial Toxins; D021122:Protein Subunits; D051028:bcl-2-Associated X Protein; C447141:cytolethal distending toxin, Actinobacillus actinomycetemcomitans; D003850:Deoxyribonuclease I; D010749:Phosphoprotein Phosphatases
keywords: Aggregatibacter actinomycetemcomitans; Apoptosis; Cytolethal distending toxin; Jurkat cells; Proteomics; Signal transduction