ATR-Chk1-Cdc25信号通路参与盘基网柄菌G2/M转变期的调控

华东师范大学学报(自然科学版) ›› 2014, Vol. 2014 ›› Issue (1): 116-122.

ATR-Chk1-Cdc25信号通路参与盘基网柄菌G2/M转变期的调控

程小凤, 侯连生

华东师范大学生命科学学院,上海 200062

收稿日期:2013-03-01 修回日期:2013-06-01 出版日期:2014-01-25 发布日期:2015-09-25

ATR-Chk1-Cdc25 involved in the regulation of Dictyostelium discoideum during G2/M cell cycle

CHENG Xiao-feng, HOU Lian-sheng

School of Life Science, East China Normal University, Shanghai 200062, China

Received:2013-03-01 Revised:2013-06-01 Online:2014-01-25 Published:2015-09-25

摘要/Abstract

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

中图分类号:

Q952.6

程小凤, 侯连生. ATR-Chk1-Cdc25信号通路参与盘基网柄菌G2/M转变期的调控[J]. 华东师范大学学报(自然科学版), 2014, 2014(1): 116-122.

CHENG Xiao-feng, HOU Lian-sheng. ATR-Chk1-Cdc25 involved in the regulation of Dictyostelium discoideum during G2/M cell cycle[J]. Journal of East China Normal University(Natural Sc, 2014, 2014(1): 116-122.

参考文献

［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.

[1]	梁静静，田莉，李丽，侯连生. 盘基网柄菌KAx-3中类免疫细胞的初步研究[J]. 华东师范大学学报(自然科学版), 20120, 2012(6): 96-102.
[2]	李丽;王大磊;侯连生. 盘基网柄菌发育期间蛞蝓体的形态学研究[J]. 华东师范大学学报(自然科学版), 2010, 2010(6): 109-115.
[3]	陈能星;魏晓静;刘伟;侯连生. 盘基网柄菌allC的克隆、表达和多克隆抗体的制备[J]. 华东师范大学学报(自然科学版), 2010, 2010(4): 77-84.
[4]	张树任;施佳乐;刘伟;陈能星;侯连生. 盘基网柄菌细胞发育中gp150分子与凋亡蛋白作用分析[J]. 华东师范大学学报(自然科学版), 2008, 2008(6): 110-115.
[5]	王一铮;张敏;侯连生. 盘基网柄菌野生型KAx-3和突变型AK127细胞mRNA差异显示分析[J]. 华东师范大学学报(自然科学版), 2008, 2008(2): 53-58,9.
[6]	韩轶;陈颖盈;侯连生. gp150蛋白对盘基网柄菌发育阶段膜蛋白的影响[J]. 华东师范大学学报(自然科学版), 2007, 2007(4): 101-106.
[7]	侯连生. 盘基网柄菌柄细胞分化过程中gp150分子的作用[J]. 华东师范大学学报(自然科学版), 2006, 2006(2): 63-67.
[8]	侯连生;付卓敏. KAx-3细胞和AK127细胞发育期间细胞骨架蛋白组分比较研究 [J]. 华东师范大学学报(自然科学版), 2005, 2005(4): 73-77.
[9]	侯连生;马宁莎;王明;韩轶;付卓敏. 盘基网柄菌(Dictyostelium discoideum ) KAX-3和AK127细胞形态发生和同工酶的比较研究[J]. 华东师范大学学报(自然科学版), 2004, 2004(4): 103-110.