异时相Cyclin B1的表达与细胞凋亡的关系及其机制的探讨
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摘要
第一部分：四环素可控表达Cyclin B1的载体构建和单克隆细胞株的筛选
目的：筛选四环素诱导细胞周期素B1(Cyclin B1)可控表达的293单克隆细胞株(Tetracycline-Regulated Expression 293 cells，T-REx~(TM)-293)。
方法：用多聚酶链反应(polymerase chain reactions，PCR)的方法从人胎肝cDNA文库中调出带有酶切位点的Cyclin B1基因全长，在37℃经过BamH1和X-bal1双酶切过夜后，与经过同样酶切后的pcDNA4／TO／myc-HisB载体在16度连接过夜，转化BL21细菌感受态，然后涂在带有氨苄抗性的LB培养板上，37度培养过夜，挑细菌克隆，摇菌，测序鉴定。接着大提测序正确的质粒，用脂质体Lipofectamine~(TM)2000转染T-REx~(TM)-293细胞，用含有杀稻瘟菌素(Blasticidin，5μg／ml)和腐草霉素(Zeocin，200μg／ml)双药物的DMEM培养两周后，将细胞传96孔板，等单个细胞扩增出单细胞克隆。最后，用Western blot和流式细胞仪检测单克隆细胞株在加入四环素诱导后目的基因的表达情况。
结果：构建的pcDNA4／TO／myc-HisB-Cyclin B1载体，经英骏公司测序鉴定序列正确。筛选出的单克隆细胞株，在未加四环素时没有外源性的Cyclin B1表达，在加入四环素后，3小时就有表达，随时间的增加，Cyclin B1表达量也增加，48小时最多。
结论：筛选出的T-REx~(TM)-293单克隆细胞株能经四环素可控表达Cyclin B1。
第二部分：G_0／G_1期异时相Cyclin B1的表达促进细胞凋亡
目的：利用四环素诱导Cyclin B1可控表达的293单克隆细胞株来证明G_0／G_1期异时相Cyclin B1的表达促进细胞凋亡
方法：挑选出一个最优的Thymidine的浓度，其能最大程度的阻滞细胞于G_0／G_1期。接着，将细胞大量阻滞在G_0／G_1期，然后在阻滞好的细胞中加入微量的四环素诱导Cyclin B1表达48小时，最后用Annexin V-PI双标法和API法检测细胞凋亡的量和凋亡的周期特异性，同时用Western blot检测细胞周期素依赖蛋白激酶1(Cdk1)的活性情况。
结果：用Thymidine处理细胞后，绝大部分细胞都同步化在G_0／G_1期。在四环素诱导Cyclin B1表达48小时后，诱导组比非诱导组的Cyclin B1蛋白总量明显增加，细胞凋亡明显增加。此时，活性的Cdk1(Thr~(161)磷酸化)蛋白量较未诱导组也明显增加。
结论：G_0／G_1期异时相Cyclin B1的表达能促进细胞凋亡。
第三部分：Cyclin B1导致的G_0／G_1期特异性细胞凋亡机制的探索
目的：探索在由Cyclin B1导致的G_0／G_1期特异的细胞凋亡中，除了激活Cdk1外，Cyclin B1还可能和哪些蛋白相互作用，或者Cyclin B1／Cdk1能通过磷酸化哪些蛋白质起作用。
方法：我们筛选好的单克隆细胞株在加入四环素后能表达带有Myc和His标签的外源性Cyclin B1。将未诱导的和用四环素诱导48小时的细胞裂解后，分别加入10μgMyc的单克隆抗体，用免疫沉淀(immune precipitation，IP)的方法将可能和Cyclin B1结合的蛋白沉淀下来，接着将蛋白混合物进行SDS PAGE电泳，用考马斯亮蓝染色法和银染法找出未诱导组和诱导组之间的差异蛋白，然后用质谱(mass spectrometry，MASS)分析出差异蛋白的名称。
结果：用质谱鉴定出的蛋白有细胞周期素依赖蛋白激酶一(Cyclin-dependentkinase 1，Cdk1)，细胞周期素依赖蛋白激酶二(Cyclin-dependent kinase 2，Cdk2)，有丝分裂阻滞缺失样蛋白一(Mitotic arrest deficient-like protein 1，MAD1-like 1，MAD1L1)和热休克蛋白八(heat shock 70kDa protein 8 isoform 1)。
结论：在由Cyclin B1导致的G_0／G_1期特异的细胞凋亡中，除了磷酸化Thr~(161)残基激活Cdk1后导致细胞凋亡外，Cyclin B1(Cyclin B1／Cdk1)与Cdk2、MAD1L1和Hsp70的结合都有可能参与其中。
Objective:A monoclone cell line was needed for expressing Cyclin B1 induced byTetracycline.
Methods:The full sequence of Cyclin B1 was got from fetal liver cDNA library byPCR.After both cutted by BamH1 and X-ball at 37℃overnight,the product of Cyclin B1and empty vector pcDNA4/TO/myc-HisB were ligased at 16℃overnight by T4 DNAligase.The constructed pcDNA4/TO/myc-HisB-Cyclin B1 was sequenced,and the rightone was used to transfect T-REx~(TM)-293 cells using Lipofectamine~(TM)2000.The cells werecultured in DMEM containing Blasticidin (5μg/ml)and Zeocin (200μg/ml)for two weeks.Then they were seeded into 96 well plates.So,different clones were got,which amplifyingfrom a single cell.After cyclin B1 induced for different time by Tetracycline,cells wereharvested,and Cyclin B 1 expression was tested by Western blot and flow cytometry.
Results:The pcDNA4/TO/myc-HisB-Cyclin B1 vector was successfully constructed,and the sequence was right.The monoclone cells could express Cyclin B 1-myc-his as earlyas 3 hours after Tetracycline added to DMEM,and the highest amount of CyclinB 1-myc-his was expressed 48 hours after Tetracycline added.
Conclusions:The monoclone T-REx~(TM)-293 cell line could express Cyclin B1 inducedby Tetracycline under control.
Objective:‘Unscheduled expression of Cyclin B1 in G_0/G_1 phase increasing cellapoptosis’was wanted to be confirmed by using a monoclone cell line,which couldexpress Cyclin B1 induced by Tetracycline.
Methods:Cells were caused to arrest in G_0/G_1 phase by Thymidine firstly.ThenTetracycline was added to induce Cyclin B1 expression for 48 hours,and thecontrol group was added no Tetracycline.At last,the cell apoptosis was detected byAnnexin V-PI and API methods.The activity of Cdk1 was also confirmed byWestern blot at the same time.
Results:Most cells were arrested in G_0/G_1 phase cause of Thymidine.CyclinB1 protein and cell apoptosis increased after Cyclin B1 expression for 48 hoursinduced by Tetracycline.And the activity of Cdk1 (Thr~(161)phosphorylation)was alsoincreased.
Conclusions:Unscheduled expression of Cyclin B1 in G_0/G_1 phase couldincrease cell apoptosis.
Objective:Study the proteins doing functions in specific apoptosis of G_0/G_1phase cause of Cyclin B1,which can interact with Cyclin B1 or be phosphorylatedby Cyclin B1/Cdk1 complex.
Methods:The monoclone cell line could express Cyclin B1 with a Myc and Histag after Tetracycline added.Cells uninduced or induced by Tetracycline for 48hwere both lysed,then 10pg Myc antibody was added to each.IP (immuneprecipitation)was used for detect proteins which might interact with Cyclin B1,thenthey were run in SDS-PAGE.The proteins were confirmed by mass spectrometry,after different bands cutted from SDS-PAGE by coomassie blue staining and silverstaining.
Results:The four proteins confirmed by MASS were Cdk1 (Cyclin-dependentkinase 1),Cdk2 (Cyclin-dependent kinase 2),MAD1L1 (Mitotic arrest deficient-likeprotein 1,MAD1-like 1)and heat shock 70kDa protein 8 isoform 1.
Conclusions:Cyclin B1 (or Cyclin B1/Cdk1)could interact with Cdk2,MAD1L1and Hsp70,which might had functions in specific apoptosis of G_0/G_1 phase cause ofCyclin B1,besides the activation of Cdk1.
目的：筛选四环素诱导细胞周期素B1(Cyclin B1)可控表达的293单克隆细胞株(Tetracycline-Regulated Expression 293 cells，T-REx~(TM)-293)。
方法：用多聚酶链反应(polymerase chain reactions，PCR)的方法从人胎肝cDNA文库中调出带有酶切位点的Cyclin B1基因全长，在37℃经过BamH1和X-bal1双酶切过夜后，与经过同样酶切后的pcDNA4／TO／myc-HisB载体在16度连接过夜，转化BL21细菌感受态，然后涂在带有氨苄抗性的LB培养板上，37度培养过夜，挑细菌克隆，摇菌，测序鉴定。接着大提测序正确的质粒，用脂质体Lipofectamine~(TM)2000转染T-REx~(TM)-293细胞，用含有杀稻瘟菌素(Blasticidin，5μg／ml)和腐草霉素(Zeocin，200μg／ml)双药物的DMEM培养两周后，将细胞传96孔板，等单个细胞扩增出单细胞克隆。最后，用Western blot和流式细胞仪检测单克隆细胞株在加入四环素诱导后目的基因的表达情况。
结果：构建的pcDNA4／TO／myc-HisB-Cyclin B1载体，经英骏公司测序鉴定序列正确。筛选出的单克隆细胞株，在未加四环素时没有外源性的Cyclin B1表达，在加入四环素后，3小时就有表达，随时间的增加，Cyclin B1表达量也增加，48小时最多。
结论：筛选出的T-REx~(TM)-293单克隆细胞株能经四环素可控表达Cyclin B1。
第二部分：G_0／G_1期异时相Cyclin B1的表达促进细胞凋亡
目的：利用四环素诱导Cyclin B1可控表达的293单克隆细胞株来证明G_0／G_1期异时相Cyclin B1的表达促进细胞凋亡
方法：挑选出一个最优的Thymidine的浓度，其能最大程度的阻滞细胞于G_0／G_1期。接着，将细胞大量阻滞在G_0／G_1期，然后在阻滞好的细胞中加入微量的四环素诱导Cyclin B1表达48小时，最后用Annexin V-PI双标法和API法检测细胞凋亡的量和凋亡的周期特异性，同时用Western blot检测细胞周期素依赖蛋白激酶1(Cdk1)的活性情况。
结果：用Thymidine处理细胞后，绝大部分细胞都同步化在G_0／G_1期。在四环素诱导Cyclin B1表达48小时后，诱导组比非诱导组的Cyclin B1蛋白总量明显增加，细胞凋亡明显增加。此时，活性的Cdk1(Thr~(161)磷酸化)蛋白量较未诱导组也明显增加。
结论：G_0／G_1期异时相Cyclin B1的表达能促进细胞凋亡。
第三部分：Cyclin B1导致的G_0／G_1期特异性细胞凋亡机制的探索
目的：探索在由Cyclin B1导致的G_0／G_1期特异的细胞凋亡中，除了激活Cdk1外，Cyclin B1还可能和哪些蛋白相互作用，或者Cyclin B1／Cdk1能通过磷酸化哪些蛋白质起作用。
方法：我们筛选好的单克隆细胞株在加入四环素后能表达带有Myc和His标签的外源性Cyclin B1。将未诱导的和用四环素诱导48小时的细胞裂解后，分别加入10μgMyc的单克隆抗体，用免疫沉淀(immune precipitation，IP)的方法将可能和Cyclin B1结合的蛋白沉淀下来，接着将蛋白混合物进行SDS PAGE电泳，用考马斯亮蓝染色法和银染法找出未诱导组和诱导组之间的差异蛋白，然后用质谱(mass spectrometry，MASS)分析出差异蛋白的名称。
结果：用质谱鉴定出的蛋白有细胞周期素依赖蛋白激酶一(Cyclin-dependentkinase 1，Cdk1)，细胞周期素依赖蛋白激酶二(Cyclin-dependent kinase 2，Cdk2)，有丝分裂阻滞缺失样蛋白一(Mitotic arrest deficient-like protein 1，MAD1-like 1，MAD1L1)和热休克蛋白八(heat shock 70kDa protein 8 isoform 1)。
结论：在由Cyclin B1导致的G_0／G_1期特异的细胞凋亡中，除了磷酸化Thr~(161)残基激活Cdk1后导致细胞凋亡外，Cyclin B1(Cyclin B1／Cdk1)与Cdk2、MAD1L1和Hsp70的结合都有可能参与其中。
Objective:A monoclone cell line was needed for expressing Cyclin B1 induced byTetracycline.
Methods:The full sequence of Cyclin B1 was got from fetal liver cDNA library byPCR.After both cutted by BamH1 and X-ball at 37℃overnight,the product of Cyclin B1and empty vector pcDNA4/TO/myc-HisB were ligased at 16℃overnight by T4 DNAligase.The constructed pcDNA4/TO/myc-HisB-Cyclin B1 was sequenced,and the rightone was used to transfect T-REx~(TM)-293 cells using Lipofectamine~(TM)2000.The cells werecultured in DMEM containing Blasticidin (5μg/ml)and Zeocin (200μg/ml)for two weeks.Then they were seeded into 96 well plates.So,different clones were got,which amplifyingfrom a single cell.After cyclin B1 induced for different time by Tetracycline,cells wereharvested,and Cyclin B 1 expression was tested by Western blot and flow cytometry.
Results:The pcDNA4/TO/myc-HisB-Cyclin B1 vector was successfully constructed,and the sequence was right.The monoclone cells could express Cyclin B 1-myc-his as earlyas 3 hours after Tetracycline added to DMEM,and the highest amount of CyclinB 1-myc-his was expressed 48 hours after Tetracycline added.
Conclusions:The monoclone T-REx~(TM)-293 cell line could express Cyclin B1 inducedby Tetracycline under control.
Objective:‘Unscheduled expression of Cyclin B1 in G_0/G_1 phase increasing cellapoptosis’was wanted to be confirmed by using a monoclone cell line,which couldexpress Cyclin B1 induced by Tetracycline.
Methods:Cells were caused to arrest in G_0/G_1 phase by Thymidine firstly.ThenTetracycline was added to induce Cyclin B1 expression for 48 hours,and thecontrol group was added no Tetracycline.At last,the cell apoptosis was detected byAnnexin V-PI and API methods.The activity of Cdk1 was also confirmed byWestern blot at the same time.
Results:Most cells were arrested in G_0/G_1 phase cause of Thymidine.CyclinB1 protein and cell apoptosis increased after Cyclin B1 expression for 48 hoursinduced by Tetracycline.And the activity of Cdk1 (Thr~(161)phosphorylation)was alsoincreased.
Conclusions:Unscheduled expression of Cyclin B1 in G_0/G_1 phase couldincrease cell apoptosis.
Objective:Study the proteins doing functions in specific apoptosis of G_0/G_1phase cause of Cyclin B1,which can interact with Cyclin B1 or be phosphorylatedby Cyclin B1/Cdk1 complex.
Methods:The monoclone cell line could express Cyclin B1 with a Myc and Histag after Tetracycline added.Cells uninduced or induced by Tetracycline for 48hwere both lysed,then 10pg Myc antibody was added to each.IP (immuneprecipitation)was used for detect proteins which might interact with Cyclin B1,thenthey were run in SDS-PAGE.The proteins were confirmed by mass spectrometry,after different bands cutted from SDS-PAGE by coomassie blue staining and silverstaining.
Results:The four proteins confirmed by MASS were Cdk1 (Cyclin-dependentkinase 1),Cdk2 (Cyclin-dependent kinase 2),MAD1L1 (Mitotic arrest deficient-likeprotein 1,MAD1-like 1)and heat shock 70kDa protein 8 isoform 1.
Conclusions:Cyclin B1 (or Cyclin B1/Cdk1)could interact with Cdk2,MAD1L1and Hsp70,which might had functions in specific apoptosis of G_0/G_1 phase cause ofCyclin B1,besides the activation of Cdk1.
引文
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7 M Shen, Y Feng, C Gao, et al. Detection of cyclin B1 expression in G_1-phase cancer cell lines and cancer tissues by postsorting Western blot analysis. Cancer Res, 64:1607-1610,2004
8 Roy M Golsteyn. Cdk1 and Cdk2 complexes (cyclin dependent kinases) in apoptosis: a role beyond the cell cycle. Cancer Letters, 217: 129-138, 2005
9 Vincent I, Jicha G, Rosado M, et al. Aberrant expression of mitotic cdc2/cyclin Bl kinase in degenerating neurons of Alzheimer's disease brain. J. Neurosci, 17:3588-3598, 1997
10 Vincent I, Bu B, Hudson K, et al. Constitutive Cdc25B tyrosine phosphatase activity in adult brain neurons with M phase-type alterations in Alzheimer's disease.Neuroscience, 105: 639-650, 2001,
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13 LA Porter, IH Cukier, JM Lee. Nuclear localization of cyclin B1 regulates DNA damage-induced apoptosis. Blood, 101: 1928-1933, 2003
14 J Wu, Y Feng, D Xie, et al. Unscheduled Cdk1 activity in G1 phase of the cell cycle triggers apoptosis in X-irradiated lymphocytic leukemia cells. Cell. Mol. Life Sci, 63:2538-45, 2006
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1. Porter LA, Donoghue DJ. Cyclin B1 and CDK1: nuclear localization and upstream regulators. Prog Cell Cycle Res, 5: 335-47,2003
2. van Leuken R, Clijsters L, Wolthuis R. To cell cycle, swing the APC/C, Biochim Biophys Acta, 1786(1): 49-59, 2008
3. Peters JM. The anaphase-promoting complex: proteolysis in mitosis and beyond. Mol.Cell, 9: 931-943, 2002
4. Douglas R Green. Apoptotic Pathways: Ten Minutes to Dead. Cell, 121: 671-674,2005
5. Hao Z, Duncan GS, Chang CC, et al. Cell, 121: 579-591, 2005
6. J Wu, Y Feng, D Xie, et al. Unscheduled Cdkl activity in G1 phase of the cell cycle triggers apoptosis in X-irradiated lymphocytic leukemia cells. Cell. Mol. Life Sci, 63:2538-45, 2006
7. Vantieghem A, Xu Y, Assefa Z, et al. Phosphorylation of Bcl-2 in G_2/M phase-arrested cells following photodynamic therapy with hypericin involves a CDK1-mediated signal and delays the onset of apoptosis. J Biol Chem, 277(40): 37718-31, 2002
8. Konishi Y, Lehtinen M, Donovan N, et al. Cdc2 phosphorylation of BAD links the cell cycle to the cell death machinery. Mol. Cell, 9: 1005-1016, 2002
9. Lindsey Allan and Paul Clarke. Phosphorylation of Caspase-9 by Cdkl/Cyclin Bl Protects Mitotic Cells against Apoptosis. Molecular Cell, 26: 301-310, 2007
10. Juping Yuan, Ruilan Yan, Frank Eckerdt, et al. Cyclin Bl depletion inhibits proliferation and induces apoptosis in human tumor cells. Oncogene, 23: 5843-5852,2004
11. Sara EK, Ola Landgren, Mats Ehinger, et al. High expression of cyclin Bl predicts a favorable outcome in patients with follicular lymphoma. Blood, 105: 2908-2915, 2005
1. J Pines. Localization of cell cycle regulators by immunofluorescence. Methods Enzymol, 283:99-113, 1997
2. A Hagting, C Karlsson, P Clute, et al. MPF localisation is controlled by nuclear export. EMBO J, 17: 4127-4138,1998
3. LA Porter, IH Cukier, JM Lee. Nuclear localization of cyclin B1 regulates DNA damage-induced apoptosis. Blood, 101: 1928-1933, 2003
4. M Shen, Y Feng, C Gao, et al. Unscheduled expression of cyclin B1 in G1-phase among cultured and clinical tumor cells. Chin. J. Oncol, 24: 215-218,2002
5. M Shen, Y Feng, C Gao, et al. Detection of cyclin B1 expression in G(1)-phase cancer cell lines and cancer tissues by postsorting Western blot analysis.Cancer Res, 64: 1607-1610, 2004
6. Roy M Golsteyn. Cdk1 and Cdk2 complexes (cyclin dependent kinases) in apoptosis: a role beyond the cell cycle. Cancer Letters, 217: 129-138, 2005
7. Vincent I, Jicha G, Rosado M, et al. Aberrant expression of mitotic cdc2/cyclin B1 kinase in degenerating neurons of Alzheimer's disease brain. J. Neurosci,17:3588-3598, 1997
8. Vincent I, Bu B, Hudson K, et al. Constitutive Cdc25B tyrosine phosphatase activity in adult brain neurons with M phase-type alterations in Alzheimer's disease. Neuroscience, 105: 639-650, 2001,
9. J Wu, Y Feng, D Xie, et al. Unscheduled Cdk1 activity in G1 phase of the cell cycle triggers apoptosis in X-irradiated lymphocytic leukemia cells. Cell. Mol.Life Sci, 63: 2538-45, 2006
10. Vantieghem A, Xu Y, Assefa Z, et al. Phosphorylation of Bcl-2 in G2/M phase-arrested cells following photodynamic therapy with hypericin involves a CDK1-mediated signal and delays the onset of apoptosis. J Biol Chem, 277(40):37718-31,2002
11. Konishi Y, Lehtinen M, Donovan N, et al. Cdc2 phosphorylation of BAD links the cell cycle to the cell death machinery. Mol. Cell, 9: 1005-1016, 2002
12. Lindsey A Allan and Paul R Clarke. Phosphorylation of Caspase-9 by Cdk1/Cyclin B1 Protects Mitotic Cells against Apoptosis. Molecular Cell, 26:301-310,2007
13. Zengqiang Yuan, Esther BE Becker, Paola Merlo, et al. Activation of FOXO1 by Cdk1 in Cycling Cells and Postmitotic Neurons. Science, 319: 1665-68, 2008
14. Brown NR, Lowe ED, Petri E, et al. Cyclin B and cyclin A confer different substrate recognition properties on CDK2. Cell Cycle, 6(11): 1350-1359, 2007
15. Haojie Huang, Kevin M Regan, Zhenkun Lou, et al. CDK2-Dependent Phosphorylation of FOXO1 as an Apoptotic Response to DNA Damage.Science, 314: 294-297, 2006
1. Evan GI, Littlewood T. A matter of life and cell death. Science, 281: 1317-22,1998
2. King KL, Cidlowski JA. Cell cycle regulation and apoptosis. Annu. Rev.Physiol, 60: 601-16, 1998
3. Adams JM, Cory S. Life-or-death decisions by the Bcl-2 protein family. Trends Biochem. Sci, 26: 61-66, 2001
4. Green DR, Evan Gl. A matter of life and death. Cancer Cell, 1: 19-30,2002
5. Li C, Thompson CB. DNA damage, deamidation and death. Science, 298:1346-7,2002
6. Shi L, Nishioka WK, Th'ng J, et al. Premature p34cdc2 activation required for apoptosis. Science, 263: 1143-1145, 1994
7. Freeman RS, Estus S and Johnson Jr EM. Analysis of cell cycle-related gene expression in postmitotic neurons: selective induction of Cyclin D1 during programmed cell death. Neuron, 12: 343-355, 1994
8. Oberhammer FA, Hochegger K and Froschl G. Chromatin condensation during apoptosis is accompanied by degradation of laminA+B, without enhanced activation of cdc-2 kinase. J. Cell Biol, 126: 827-837, 1994
9. Norbury C, MacFarlande M, Fearnhead H, et al. CDC2 activation is not required for thymocyte apoptosis. Biochem. Biophys. Res. Comm, 202: 1400-1406,1994
10. AW Murray. Revisiting the cell cycle: cyclins revisited. Cell, 116: 221-234, 2004
11. T Evans, ET Rosenthal, J Youngblom, et.al. T Hunt. Cyclin: a protein specified by maternal mRNA in sea urchin eggs that is destroyed at each cleavage division. Cell, 33: 389-396, 1983
12. V Simanis, P Nurse. The cell cycle control gene cdc2C of fission yeast encodes a protein kinase potentially regulated by phosphorylation. Cell, 45: 261-268,1986
13. Nigg EA. Mitotic kinases as regulators of cell division and its checkpoints. Nat.Rev. Mol. Cell. Biol, 2: 21-32, 2001
14. Smits VA and Medema RH. Checking out the G2/M transition. Biochim. Biophys.Acta, 1519:1-12, 2001
15. Yun J, Chae HD, Choy HE, et al. p53 negatively regulates cdc2 transcription via the CCAAT-binding NF-Y transcription factor. J. Biol. Chem, 274: 29677-29682,1999
16. Taylor WR, DePrimo SE, Agarwal A, et al. Mechanisms of G2 arrest in response to overexpression of p53. Mol. Biol. Cell, 10: 3607-3622, 1999
17. Peters JM. The anaphase-promoting complex: proteolysis in mitosis and beyond. Mol. Cell, 9: 931-943, 2002
18. Ruchaud S, Korfali N, Villa P, et al. Caspase-6 gene disruption reveals a requirement for lamin A cleavage in apoptotic chromatin condensation. EMBO J,21: 1967-1977,2002
19. Melo J and Toczyski D. A unified view of the DNA-damage checkpoint. Curr.Opin. Cell. Biol, 14: 237-45, 2002
20. Ferri KF and Kroemer GK, Organelle-specific initiation of cell death pathways.Nature Cell Biol. 3: E255-E263, 2001
21. OngkekoW, Ferguson DJP, HarrisAL and NorburyC, Inactivation of Cdc2 increases the level of apoptosis induced by DNA damage. J. Cell Science 108:2897-2904,1995
22. Itzhaki JE, Gilbert CS and Porter AC. Construction by gene targeting in human cells of a 'conditional' CDC2 mutant that rereplicates its DNA. Nat. Genet, 15:258-265, 1997
23. Patel V, Senderowicz AM, Pinto Jr D, et al. Flavopiridol, a novel cyclindependent kinase inhibitor, suppresses the growth of head and neck squamous cell carcinomas by inducing apoptosis. J. Clin. Invest, 102:1674-1681,1998
24. Zhai S, Senderowicz AM, Sausville EA, et al. Flavopiridol, a novel cyclin-dependent kinase inhibitor, in clinical development. Ann. Pharmacother,36:905-911,2002
25. O'Connor DS, Grossman D, Plescia J, et al. Regulation of apoptosis at cell division by p34cdc2 phosphorylation of survivin. Proc. Natl. Acad. Sci, 97:13103-13107,2000
26. Vantieghem A, Xu Y, Assefa Z, et al. Phosphorylation of BCL-2 in G2/M phase arrested cells following photodynamic therapy with hypericin involves a Cdk1-mediated signal and delays the onset of apoptosis. J. Biol. Chem. 277:37718-31,2002
27. Zhou BB, Li H, Yuan J, et al. Caspase-dependent activation of cyclin-dependent kinases during Fas-induced apoptosis in Jurkat cells. Proc. Natl. Acad. Sci, 95:6785-6790, 1998
28. J Wu, Y Feng, D Xie, et al. Unscheduled Cdk1 activity in G1 phase of the cell cycle triggers apoptosis in X-irradiated lymphocytic leukemia cells. Cell. Mol.Life Sci, Vol. 63: 2538-45, 2006
29. L Shi, WK Niskioka, J Th'ng, EM Bradbury, et al. Premature p34cdc2 activation required for apoptosis. Nature, 263: 1143-1145, 1994
30. DO Morgan. Principles of Cdk regulation. Nature, 374: 131-134, 1995
31. L Shi, G Chen, D He, et al. Granzyme B induces apoptosis and cyclin A-associated cyclin-dependent kinase activity in all stages of the cell cycle. J.Immunol, 157: 2381-2385, 1996
32. Shen SC, Huang TS, Jee SH, et al. Taxol-induced p34cdc2 kinase activation and apoptosis inhibited by 12-O- tetradecanoylphorbol-13-acetate in human breast MCF-7 carcinoma cells. Cell Growth Differ, 9: 23-29,1998
33. Yu D, Jing T, Liu B, et al. Overexpression of ErbB2 blocks Taxol-induced apoptosis by upregulation of p21Cip1, which inhibits p34Cdc2 kinase. Mol. Cell,2:581-591,1998
34. Stewart ZA, Mays D and Pietenpol JA. Defective G1-S cell cycle checkpoint function sensitizes cells to microtubule inhibitor-induced apoptosis. Cancer Res,59:3831-3837,1999
35. Tan M, Jing T, Lan KH, et al. Phosphorylation on tyrosine-15 of p34 (Cdc2) by ErbB2 inhibit sp34 (Cdc2) activation and is involved in resistance totaxol-induced apoptosis. Mol. Cell, 9: 993-1004, 2002
36. Slamon DJ, Leyland-Jones B, Shak S, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N. Engl. J. Med, 344: 783-792, 2001
37. Ibrado AM, Kim CN and Bhalla K. Temporal relationship of Cdk1 activation and mitotic arrest to cytosolic accumulation of cytochrome C and caspase-3 activity during Taxol-induced apoptosis of human AML HL-60 cells. Leukemia, 12:1930-1936, 1998
38. Kroemer G and Reed JC. Mitochondrial control of cell death. Nat. Med, 6:513-519,2000
39. Konishi Y, Lehtinen M, Donovan N, et al. Cdc2 phosphorylation of BAD links the cell cycle to the cell death machinery. Mol. Cell, 9: 1005-1016, 2002
40. Debatin K-M, Poncet D and Kroemer G. Chemotherapy: targeting the mitochondrial cell death pathway. Oncogene, 21: 8786-8803, 2002
41. Pathan N, Aime-Sempe C, Kitada S, et al. Microtubule targeting drugs induce Bcl-2 phosphorylation and association with Pin1. Neoplasia, 3: 70-79, 2001
42. Scatena CD, Stewart ZA, Mays D, et al. Mitotic phosphorylation of Bcl-2 during normal cell cycle progression and Taxol-induced growth arrest. J. Biol. Chem, 273:30777-30784,1998
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44. Zengqiang Yuan, Esther BE Becker, Paola Merlo, et al. Activation of FOXO1 by Cdk1 in Cycling Cells and Postmitotic Neurons. Science, 319: 1665-68, 2008
45. Lindsey A Allan and Paul R Clarke. Phosphorylation of Caspase-9 by Cdk1/Cyclin B1 Protects Mitotic Cells against Apoptosis. Molecular Cell, 26:301-310,2007
46. Vincent I, Jicha G, Rosado M, et al. Aberrant expression of mitotic cdc2/cyclin B1 kinase in degenerating neurons of Alzheimer's disease brain. J. Neurosci,17:3588-3598, 1997
47. Vincent I, Bu B, Hudson K, et al. Constitutive Cdc25B tyrosine phosphatase activity in adult brain neurons with M phase-type alterations in Alzheimer's disease. Neuroscience, 105: 639-650, 2001
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55. M Shen, Y Feng, C Gao, et al. Unscheduled expression of cyclin B1 in G1-phase among cultured and clinical tumor cells. Chin. J. Oncol, 24: 215-218,2002
56. M Shen, Y Feng, C Gao, et al. Detection of cyclin B1 expression in G(1)-phase cancer cell lines and cancer tissues by postsorting Western blot analysis.Cancer Res, 64: 1607-1610, 2004
57. J Pines. Localization of cell cycle regulators by immunofluorescence. Methods Enzymol, 283:99-113, 1997
58. Hagting C Karlsson, P Clute, M Jackman, et al. MPF localisation is controlled by nuclear export. EMBO J, 17:4127-4138, 1998
59. LA Porter, IH Cukier, JM Lee. Nuclear localization of cyclin B1 regulates DNA damage-induced apoptosis. Blood, 101: 1928-1933, 2003
60. S Ortega, I Prieto, J Odajima, et al. Cyclin-dependent kinase 2 is essential for meiosis but not for mitotic cell division in mice. Nat. Genet, 35: 25-31, 2003
61. EB Becker, A Bonni. Cell cycle regulation of neuronal apoptosis in development and disease. Prog. Neurobiol, 72: 1-25, 2004
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2 V Simanis, P Nurse. The cell cycle control gene cdc2C of fission yeast encodes a protein kinase potentially regulated by phosphorylation. Cell, 45: 261-268,1986
3 Peters JM. The anaphase-promoting complex: proteolysis in mitosis and beyond. Mol.Cell, 9:931-943,2002
4 Nigg EA. Mitotic kinases as regulators of cell division and its checkpoints. Nat. Rev.Mol. Cell Biol, 2: 21-32, 2001
5 Smits VA and Medema RH. Checking out the G_2/M transition. Biochim. Biophys.Acta, 1519:1-12,2001
6 M Shen, Y Feng, C Gao, et al. Unscheduled expression of cyclin B1 in G_1-phase among cultured and clinical tumor cells. Chin. J. Oncol, 24: 215-218, 2002
7 M Shen, Y Feng, C Gao, et al. Detection of cyclin B1 expression in G_1-phase cancer cell lines and cancer tissues by postsorting Western blot analysis. Cancer Res, 64:1607-1610,2004
8 Roy M Golsteyn. Cdk1 and Cdk2 complexes (cyclin dependent kinases) in apoptosis: a role beyond the cell cycle. Cancer Letters, 217: 129-138, 2005
9 Vincent I, Jicha G, Rosado M, et al. Aberrant expression of mitotic cdc2/cyclin Bl kinase in degenerating neurons of Alzheimer's disease brain. J. Neurosci, 17:3588-3598, 1997
10 Vincent I, Bu B, Hudson K, et al. Constitutive Cdc25B tyrosine phosphatase activity in adult brain neurons with M phase-type alterations in Alzheimer's disease.Neuroscience, 105: 639-650, 2001,
11 J Pines. Localization of cell cycle regulators by immunofluorescence. Methods Enzymol, 283: 99-113, 1997
12 A Hagting, C Karlsson, P Clute, et al. MPF localisation is controlled by nuclear export.EMBO J, 17: 4127-4138, 1998
13 LA Porter, IH Cukier, JM Lee. Nuclear localization of cyclin B1 regulates DNA damage-induced apoptosis. Blood, 101: 1928-1933, 2003
14 J Wu, Y Feng, D Xie, et al. Unscheduled Cdk1 activity in G1 phase of the cell cycle triggers apoptosis in X-irradiated lymphocytic leukemia cells. Cell. Mol. Life Sci, 63:2538-45, 2006
15 Douglas R Green. Apoptotic Pathways: Ten Minutes to Dead. Cell, 121: 671-674,2005
16 Hao Z, Duncan GS, Chang CC, et al. Cell, 121: 579-591,2005
1.Nasmyth K.Viewpoint:putting the cell cycle in order.Science,274:1643,1996
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1. Porter LA, Donoghue DJ. Cyclin B1 and CDK1: nuclear localization and upstream regulators. Prog Cell Cycle Res, 5: 335-47,2003
2. van Leuken R, Clijsters L, Wolthuis R. To cell cycle, swing the APC/C, Biochim Biophys Acta, 1786(1): 49-59, 2008
3. Peters JM. The anaphase-promoting complex: proteolysis in mitosis and beyond. Mol.Cell, 9: 931-943, 2002
4. Douglas R Green. Apoptotic Pathways: Ten Minutes to Dead. Cell, 121: 671-674,2005
5. Hao Z, Duncan GS, Chang CC, et al. Cell, 121: 579-591, 2005
6. J Wu, Y Feng, D Xie, et al. Unscheduled Cdkl activity in G1 phase of the cell cycle triggers apoptosis in X-irradiated lymphocytic leukemia cells. Cell. Mol. Life Sci, 63:2538-45, 2006
7. Vantieghem A, Xu Y, Assefa Z, et al. Phosphorylation of Bcl-2 in G_2/M phase-arrested cells following photodynamic therapy with hypericin involves a CDK1-mediated signal and delays the onset of apoptosis. J Biol Chem, 277(40): 37718-31, 2002
8. Konishi Y, Lehtinen M, Donovan N, et al. Cdc2 phosphorylation of BAD links the cell cycle to the cell death machinery. Mol. Cell, 9: 1005-1016, 2002
9. Lindsey Allan and Paul Clarke. Phosphorylation of Caspase-9 by Cdkl/Cyclin Bl Protects Mitotic Cells against Apoptosis. Molecular Cell, 26: 301-310, 2007
10. Juping Yuan, Ruilan Yan, Frank Eckerdt, et al. Cyclin Bl depletion inhibits proliferation and induces apoptosis in human tumor cells. Oncogene, 23: 5843-5852,2004
11. Sara EK, Ola Landgren, Mats Ehinger, et al. High expression of cyclin Bl predicts a favorable outcome in patients with follicular lymphoma. Blood, 105: 2908-2915, 2005
1. J Pines. Localization of cell cycle regulators by immunofluorescence. Methods Enzymol, 283:99-113, 1997
2. A Hagting, C Karlsson, P Clute, et al. MPF localisation is controlled by nuclear export. EMBO J, 17: 4127-4138,1998
3. LA Porter, IH Cukier, JM Lee. Nuclear localization of cyclin B1 regulates DNA damage-induced apoptosis. Blood, 101: 1928-1933, 2003
4. M Shen, Y Feng, C Gao, et al. Unscheduled expression of cyclin B1 in G1-phase among cultured and clinical tumor cells. Chin. J. Oncol, 24: 215-218,2002
5. M Shen, Y Feng, C Gao, et al. Detection of cyclin B1 expression in G(1)-phase cancer cell lines and cancer tissues by postsorting Western blot analysis.Cancer Res, 64: 1607-1610, 2004
6. Roy M Golsteyn. Cdk1 and Cdk2 complexes (cyclin dependent kinases) in apoptosis: a role beyond the cell cycle. Cancer Letters, 217: 129-138, 2005
7. Vincent I, Jicha G, Rosado M, et al. Aberrant expression of mitotic cdc2/cyclin B1 kinase in degenerating neurons of Alzheimer's disease brain. J. Neurosci,17:3588-3598, 1997
8. Vincent I, Bu B, Hudson K, et al. Constitutive Cdc25B tyrosine phosphatase activity in adult brain neurons with M phase-type alterations in Alzheimer's disease. Neuroscience, 105: 639-650, 2001,
9. J Wu, Y Feng, D Xie, et al. Unscheduled Cdk1 activity in G1 phase of the cell cycle triggers apoptosis in X-irradiated lymphocytic leukemia cells. Cell. Mol.Life Sci, 63: 2538-45, 2006
10. Vantieghem A, Xu Y, Assefa Z, et al. Phosphorylation of Bcl-2 in G2/M phase-arrested cells following photodynamic therapy with hypericin involves a CDK1-mediated signal and delays the onset of apoptosis. J Biol Chem, 277(40):37718-31,2002
11. Konishi Y, Lehtinen M, Donovan N, et al. Cdc2 phosphorylation of BAD links the cell cycle to the cell death machinery. Mol. Cell, 9: 1005-1016, 2002
12. Lindsey A Allan and Paul R Clarke. Phosphorylation of Caspase-9 by Cdk1/Cyclin B1 Protects Mitotic Cells against Apoptosis. Molecular Cell, 26:301-310,2007
13. Zengqiang Yuan, Esther BE Becker, Paola Merlo, et al. Activation of FOXO1 by Cdk1 in Cycling Cells and Postmitotic Neurons. Science, 319: 1665-68, 2008
14. Brown NR, Lowe ED, Petri E, et al. Cyclin B and cyclin A confer different substrate recognition properties on CDK2. Cell Cycle, 6(11): 1350-1359, 2007
15. Haojie Huang, Kevin M Regan, Zhenkun Lou, et al. CDK2-Dependent Phosphorylation of FOXO1 as an Apoptotic Response to DNA Damage.Science, 314: 294-297, 2006
1. Evan GI, Littlewood T. A matter of life and cell death. Science, 281: 1317-22,1998
2. King KL, Cidlowski JA. Cell cycle regulation and apoptosis. Annu. Rev.Physiol, 60: 601-16, 1998
3. Adams JM, Cory S. Life-or-death decisions by the Bcl-2 protein family. Trends Biochem. Sci, 26: 61-66, 2001
4. Green DR, Evan Gl. A matter of life and death. Cancer Cell, 1: 19-30,2002
5. Li C, Thompson CB. DNA damage, deamidation and death. Science, 298:1346-7,2002
6. Shi L, Nishioka WK, Th'ng J, et al. Premature p34cdc2 activation required for apoptosis. Science, 263: 1143-1145, 1994
7. Freeman RS, Estus S and Johnson Jr EM. Analysis of cell cycle-related gene expression in postmitotic neurons: selective induction of Cyclin D1 during programmed cell death. Neuron, 12: 343-355, 1994
8. Oberhammer FA, Hochegger K and Froschl G. Chromatin condensation during apoptosis is accompanied by degradation of laminA+B, without enhanced activation of cdc-2 kinase. J. Cell Biol, 126: 827-837, 1994
9. Norbury C, MacFarlande M, Fearnhead H, et al. CDC2 activation is not required for thymocyte apoptosis. Biochem. Biophys. Res. Comm, 202: 1400-1406,1994
10. AW Murray. Revisiting the cell cycle: cyclins revisited. Cell, 116: 221-234, 2004
11. T Evans, ET Rosenthal, J Youngblom, et.al. T Hunt. Cyclin: a protein specified by maternal mRNA in sea urchin eggs that is destroyed at each cleavage division. Cell, 33: 389-396, 1983
12. V Simanis, P Nurse. The cell cycle control gene cdc2C of fission yeast encodes a protein kinase potentially regulated by phosphorylation. Cell, 45: 261-268,1986
13. Nigg EA. Mitotic kinases as regulators of cell division and its checkpoints. Nat.Rev. Mol. Cell. Biol, 2: 21-32, 2001
14. Smits VA and Medema RH. Checking out the G2/M transition. Biochim. Biophys.Acta, 1519:1-12, 2001
15. Yun J, Chae HD, Choy HE, et al. p53 negatively regulates cdc2 transcription via the CCAAT-binding NF-Y transcription factor. J. Biol. Chem, 274: 29677-29682,1999
16. Taylor WR, DePrimo SE, Agarwal A, et al. Mechanisms of G2 arrest in response to overexpression of p53. Mol. Biol. Cell, 10: 3607-3622, 1999
17. Peters JM. The anaphase-promoting complex: proteolysis in mitosis and beyond. Mol. Cell, 9: 931-943, 2002
18. Ruchaud S, Korfali N, Villa P, et al. Caspase-6 gene disruption reveals a requirement for lamin A cleavage in apoptotic chromatin condensation. EMBO J,21: 1967-1977,2002
19. Melo J and Toczyski D. A unified view of the DNA-damage checkpoint. Curr.Opin. Cell. Biol, 14: 237-45, 2002
20. Ferri KF and Kroemer GK, Organelle-specific initiation of cell death pathways.Nature Cell Biol. 3: E255-E263, 2001
21. OngkekoW, Ferguson DJP, HarrisAL and NorburyC, Inactivation of Cdc2 increases the level of apoptosis induced by DNA damage. J. Cell Science 108:2897-2904,1995
22. Itzhaki JE, Gilbert CS and Porter AC. Construction by gene targeting in human cells of a 'conditional' CDC2 mutant that rereplicates its DNA. Nat. Genet, 15:258-265, 1997
23. Patel V, Senderowicz AM, Pinto Jr D, et al. Flavopiridol, a novel cyclindependent kinase inhibitor, suppresses the growth of head and neck squamous cell carcinomas by inducing apoptosis. J. Clin. Invest, 102:1674-1681,1998
24. Zhai S, Senderowicz AM, Sausville EA, et al. Flavopiridol, a novel cyclin-dependent kinase inhibitor, in clinical development. Ann. Pharmacother,36:905-911,2002
25. O'Connor DS, Grossman D, Plescia J, et al. Regulation of apoptosis at cell division by p34cdc2 phosphorylation of survivin. Proc. Natl. Acad. Sci, 97:13103-13107,2000
26. Vantieghem A, Xu Y, Assefa Z, et al. Phosphorylation of BCL-2 in G2/M phase arrested cells following photodynamic therapy with hypericin involves a Cdk1-mediated signal and delays the onset of apoptosis. J. Biol. Chem. 277:37718-31,2002
27. Zhou BB, Li H, Yuan J, et al. Caspase-dependent activation of cyclin-dependent kinases during Fas-induced apoptosis in Jurkat cells. Proc. Natl. Acad. Sci, 95:6785-6790, 1998
28. J Wu, Y Feng, D Xie, et al. Unscheduled Cdk1 activity in G1 phase of the cell cycle triggers apoptosis in X-irradiated lymphocytic leukemia cells. Cell. Mol.Life Sci, Vol. 63: 2538-45, 2006
29. L Shi, WK Niskioka, J Th'ng, EM Bradbury, et al. Premature p34cdc2 activation required for apoptosis. Nature, 263: 1143-1145, 1994
30. DO Morgan. Principles of Cdk regulation. Nature, 374: 131-134, 1995
31. L Shi, G Chen, D He, et al. Granzyme B induces apoptosis and cyclin A-associated cyclin-dependent kinase activity in all stages of the cell cycle. J.Immunol, 157: 2381-2385, 1996
32. Shen SC, Huang TS, Jee SH, et al. Taxol-induced p34cdc2 kinase activation and apoptosis inhibited by 12-O- tetradecanoylphorbol-13-acetate in human breast MCF-7 carcinoma cells. Cell Growth Differ, 9: 23-29,1998
33. Yu D, Jing T, Liu B, et al. Overexpression of ErbB2 blocks Taxol-induced apoptosis by upregulation of p21Cip1, which inhibits p34Cdc2 kinase. Mol. Cell,2:581-591,1998
34. Stewart ZA, Mays D and Pietenpol JA. Defective G1-S cell cycle checkpoint function sensitizes cells to microtubule inhibitor-induced apoptosis. Cancer Res,59:3831-3837,1999
35. Tan M, Jing T, Lan KH, et al. Phosphorylation on tyrosine-15 of p34 (Cdc2) by ErbB2 inhibit sp34 (Cdc2) activation and is involved in resistance totaxol-induced apoptosis. Mol. Cell, 9: 993-1004, 2002
36. Slamon DJ, Leyland-Jones B, Shak S, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N. Engl. J. Med, 344: 783-792, 2001
37. Ibrado AM, Kim CN and Bhalla K. Temporal relationship of Cdk1 activation and mitotic arrest to cytosolic accumulation of cytochrome C and caspase-3 activity during Taxol-induced apoptosis of human AML HL-60 cells. Leukemia, 12:1930-1936, 1998
38. Kroemer G and Reed JC. Mitochondrial control of cell death. Nat. Med, 6:513-519,2000
39. Konishi Y, Lehtinen M, Donovan N, et al. Cdc2 phosphorylation of BAD links the cell cycle to the cell death machinery. Mol. Cell, 9: 1005-1016, 2002
40. Debatin K-M, Poncet D and Kroemer G. Chemotherapy: targeting the mitochondrial cell death pathway. Oncogene, 21: 8786-8803, 2002
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