ATR-Chk1-Cdc25 involved in the regulation of Dictyostelium discoideum during G2/M cell cycle
-
摘要: 显微镜观察盘基网柄菌野生型KAx-3细胞和突变型RNAi-allC细胞,计数结果表明后者的单细胞繁殖速度约为前者的8倍. 为探究该突变型盘基网柄菌细胞周期缩短的原因,用荧光定量PCR和western blot研究了ATR-Chk1-Cdc25信号通路在其中的可能作用. 实验结果表明:RNAi-allC细胞中cdc25基因相对表达量约为KAx-3细胞的8倍,而其Chk1与ATR基因的相对表达量却明显低于KAx-3细胞. 突变细胞中Cdc25蛋白含量高于KAx-3细胞,但其Chk1蛋白含量却显著低于KAx-3细胞. 这些数据表明,两种类型细胞之间的ATR、Chk1、Cdc25在mRNA水平和蛋白表达上均存在差异,特别是ATR基因表达量的不同明显影响ChK1和Cdc25的表达量,提示ATR-Chk1-Cdc25信号通路应该在一定程度上参与了盘基网柄菌细胞周期G2/M期的调控.
-
关键词:
- ATR-Chk1-Cdc25信号通路 /
- G2/M细胞周期 /
- Q-PCR /
- Western blot /
- 盘基网柄菌
Abstract: The cell proliferation of wild type KAx-3 and mutant type RNAi-allC observed by light microscope and cell counting, The latter was divided 8 time faste than the former. To evaluate the reason why RNAi-allC cell cycle shortened, the function of ATR-Chk1-Cdc25 signaling pathway were explored by quantitative PCR and western blot techniques. The results showed the differences of ATR, Chk1, Cdc25 in mRNA contents and protein level existed in KAx-3 and RNAi-allC cells, that is, the expression of ATR and Chk1 ratio of RNAi-allC to KAx-3 was 0.69〖DK〗∶1 and 0.1〖DK〗∶1 respectively; the expression of Cdc25 in RNAi-allC cells was 8 times that of KAx-3 cells. The data suggested that once the expression of ATR had little change, Chk1 and Cdc25 expression changed greatly. Western blotting results were consistent with Q-PCR reports. The Chk1 protein contents were significantly less in mutant type RNAi-allC than that in KAx-3cells; the Cdc25 protein contents were higher in RNAi-allC cells. The above mentioned results suggest that ATR-Chk1-Cdc25 signaling pathway involved in the regulation of the G2/M phase in Dictyostelium discoideum.-
Key words:
- ATR-Chk1-Cdc25 signaling pathway /
- G2/M cell cycle /
- Q-PCR /
- Western blot /
- Dictyostelium discoideum
-
[1] [1] MAEDA Y. Cell-cycle checkpoint for transition from cell division to differentiation[J]. Develop Growth Differ, 2011,53:463-481.[2] KAKIZUKA A, SEBASTIAN B, BORGMEYER U, et al. A mouse Cdc25 homolog is differentially and developmentally expressed [J]. Gene s Dev, 1992, 6(4):578-590. [3] WICKRAMASINGHE D, BECKER S, ERNST M K, et al. Two Cdc25 homologues are differentially expressed during mouse development [J]. Development,1995 121(7):2047-2056. [4] HOUTPRAAF J H, VERSMISSEN J, VAN DERGIESSEN W J. A concise review of DNA damage checkpoints and repair in mammalian cells[J].Cardiovasc RevascMed, 20067(3):165-172.[5] LEULLIOT N, QUEVILLON-CHERUEL S, Sorel I, et al. Crystal structure of yeast allantoicase reveals a repeated jelly roll motif [J]. Biological Chemistry, 2004, 279:23447-23452.[6] 季宇彬,高鹏, 邹翔.G2/M检验点调控机制研究概述[C]. 2008 年中国药学会学术年会暨第八届中国药师周论文集, 2008.[7] XU B, KIM S T, LIM D S, et al. Two molecularly distinctXu B, Kim ST, Lim DS, G2/M checkpoints are induced by ionizing irradiation[J]. Mol Cell Biol, 2002, 22(4):1049-1059.[8] MITRA J, ENDERS G H. CyclinA/Cdk2 com p lexes regulate activation of Cdk1 and Cdc25 phosphatases in hum an cells[J]. Oncogene, 2004, 23:3361-3367.[9] SHECHTER D, COSTANZO V, GAUTIER J. ATR and ATM regulate the timing of DNA replication origin firing[J].Nature cell biology, 2004, 648-655.[10] FRAZER C, YOUNG P G. Redundant Mechanisms Prevent Mitotic Entry FollowingReplication Arrest in the Absence of Cdc25 Hyper-Phosphorylation in Fission Yeast[J]. PLoS ONE, 2011(6):1-11.[11] 朱虹,缪泽鸿,丁健.ATM、ATR和DNA损伤介导的细胞周期阻滞[J]. 生命科学, 2007,19(2):139-148.[12] TIMOFEEV O, CIZMECIOGLU O, SETTELE F, et al. Cdc25 Phosphatases Are Required for Timely Assembly of CDK1-Cyclin B at the G2/M Transition[J].Biological Chemistry, 2010,285(22):16978-16990.[13] 马云彤,齐浩.CDC25在细胞周期运行和细胞周期检验点应答中的作用和调控机制[J]. 西安文理学院学报, 2006,9(4):12-17.[14] SEVIOUR E G, LIN S Y. The DNA damage response: Balancing the scale between cancer and ageing[J].AGING, 2010,12(2):900-907.[15] 张晖,陈秋生.细胞周期调控因子Cdc25的研究进展[J]. 中国兽医科学, 2008,38(05):447-450.[16] 叶平,宋金辉,侯连生.细胞色素C在盘基网柄菌细胞凋亡中的作用[J]. 动物学杂志, 2011, 46(6): 73-79.[17] 代卉,候连生.盘基网柄菌发育中尿囊酸酶表达的定量定位研究[J]. 华东师范大学学报:自然科学版,2012(4):1-6.
点击查看大图
计量
- 文章访问数: 1732
- HTML全文浏览量: 17
- PDF下载量: 1439
- 被引次数: 0