核膜在细胞周期中呈现高度的动态性:在细胞分裂的前中期,核膜崩解并分散到细胞质中;在细胞分裂的后期,核膜开始在染色体的表面重新装配,最终形成完整的核膜结构。近期的研究发现,Ran GTP酶、物质转运蛋白importinβ、内层核膜蛋白LBR(la...核膜在细胞周期中呈现高度的动态性:在细胞分裂的前中期,核膜崩解并分散到细胞质中;在细胞分裂的后期,核膜开始在染色体的表面重新装配,最终形成完整的核膜结构。近期的研究发现,Ran GTP酶、物质转运蛋白importinβ、内层核膜蛋白LBR(lamin B receptor)以及核孔复合体蛋白nucleoporins在核膜重建的过程中起关键性调控作用,并受到细胞周期调控因子p34cdc2激酶的调节。LBR是一个八次跨膜的膜蛋白,主要定位于内层核膜。在细胞分裂的早期,随着核膜崩解,LBR与核膜崩解而生成的小膜泡一起分散到细胞质中;在细胞分裂的后期,通过LBR与importinβ相互结合,含有LBR的膜泡被importinβ携带至染色质的表面参与核膜重建。目前已知p34cdc2激酶对LBR与importinβ介导的核膜重建起重要调控作用。Nucleoporins是核孔复合体主要组分。随核膜崩解,核孔复合体解聚成nucleoporins,分散到细胞质中,或结合到其他亚细胞成分上。细胞分裂后期,核孔复合体伴随核膜装配而组装。展开更多
Full grown oocytes derived from Bufo Bufo gargarizans rearing at high temperature environment (24℃),never underwent GVBD after progesterone treatment. No p34cdc2, H1 kinase activity was detected in the oocytes after ...Full grown oocytes derived from Bufo Bufo gargarizans rearing at high temperature environment (24℃),never underwent GVBD after progesterone treatment. No p34cdc2, H1 kinase activity was detected in the oocytes after progesterone stimulation or OA microinjection; Western blotting analysis showed that the level of p3cdc2, and p33 in the oocytes are significantly lower than those in the oocytes derived from the hibernating toads (below 10 ℃). 25S-Met incorporation analysis showed that when the oocytes were incubated at 6℃, synthesis of about thirty defferent polypeptides was promoted or induced, including p34cdc2 and some other p13suc1-binding proteins. All these results indicated that a low temperature environment is essential for the oocytes of Bufo Bufo gargarizans to express and store some cell cycle drivers and its regulators, and to gain the maturation competence. These results will also provide a new clue for explaining the molecular mechanisms why gametogenesis of some organisms depends on a relative low temperature and how to maintain the geographical distribution of some animals.展开更多
Silencing ATM gene gave rise to enhanced apoptotic response to irradiation and i rradiation like chemotherapy agents, this paper explored the crucial identities of the molecular elements responsible for the enhanced...Silencing ATM gene gave rise to enhanced apoptotic response to irradiation and i rradiation like chemotherapy agents, this paper explored the crucial identities of the molecular elements responsible for the enhanced apoptotic response in U937 cells mediated by silencing ATM gene. Two U937 cell mutants named U937 ASPI3K (ATM, negative) and U937 pZeosv2(+) (ATM, wild type) were used as a cell model system to identify the critical molecule(s) responsible for the varied apoptotic response in the absence or presence of ATM gene. Apoptosis was examined by measuring concentrations of free nucleosome in U937 cells. Western blot was employed to measure nuclear protein abundance of CDC25A, CDC25B, CDC25C, total p34cdc2, p34cdc2 (Thr 161) or p34cdc2 (Thr 14,Tyr 15). RT PCR was used to estimate CDC25 transcript levels. U937 ASPI3K exhibited an enhanced apoptotic response to lower dosage of irradiation, which could not be blocked by protein synthesis inhibitor. Protein serine threonine phosphatase inhibitor or cyclin dependent kinase (CDK) inhibitors, on the other hand, abolished the enhancement indicated that protein phosphorylation/dephosphorylation modification and CDK activity are required for the enhanced apoptotic response in the absence of ATM gene. Upon irradiation, p34cdc2 in U937 pZeosv2 (+) was maintained in an inactive state by phosphorylation on threonine 14 (Thr 14) and tyrosine 15 (Tyr 15), which was associated with a dramatic decrease of nuclear CDC25A, CDC25B and CDC25C proteins. In contrast, p34cdc2 in U937 ASPI3K maintained in an active state by dephosphorylation on threonine 14 (Thr 14) and tyrosine 15 (Tyr 15), which was associated with constant nuclear CDC25A, CDC25B and CDC25C protein abundance before and after irradiation. The responsive decrease of nuclear CDC25 proteins occurred at the post transcription level. Silencing ATM gene blocks the responsive decrease of nuclear CDC25 proteins, which is responsible for failure to inactivate p34cdc2 after irradiation. Active p34cdc2 and CDK2, in turn, acts as the death executors to trigger apoptosis. In abstract, aberrantly activated CDK activity is the critical molecular mechanism central to enhanced apoptotic responses in the absence of ATM gene.展开更多
Objective： To learn the effect of allitridi on inducing mitotic arrest in human gastric cell line SGC-7901 and its possible mechanisms. Methods： We treated SGC-7901 cells with allitridi, and observed the proliferati...Objective： To learn the effect of allitridi on inducing mitotic arrest in human gastric cell line SGC-7901 and its possible mechanisms. Methods： We treated SGC-7901 cells with allitridi, and observed the proliferation inhibitory rate with MTT colometric assay, changes of cell cycle using flow cytometry and Switzerland-Giemsa＇s staining, and morphologic changes of the microtubule structure and location changes of cyclin BI expression using immunofluorescence and confocal laser scanning microscope. Furthermore, the expression of cyclin B1 was analyzed quantitatively using Leica confocal software. Results： SGC-7901 cells were inhibited after exposure to allitridi and the IC50 was 7.2μg/ml for 24 h, 20μg/ml for 72 h. When the cells were treated with allitridi at concentrations of 3, 6, and 9μg/ml for 24 h respectively, there was a declining tendency in the percentage of G0/G1 cell but an increasing tendency in GE/M cell in the allitridi treated group compared with that of control （P〈0.01）. When cells were treated allitridi at concentration of 6 μg/ml for 24 h, its mitotic index was much higher （P〈0.01） than that of control, suggesting that allitridi caused arrest of gastric cancer cells in M phase. The cells were treated with allitridi became more shrunken and nepheloid, in which the microtubule networks disappeared, while the control cell exhibited an intact microtubule network. Contrasting with normal existence mainly in the cytoplasm, the cyclin B1 was expressed more significantly and concentrated in the nucleus after exposure to allitridi. Fluorescence intensity of cyclin B 1 protein in cells treated with allitridi was much more higher than that of control （P〈0.001）. Conclusion： Allitridican induce arrest of SGC-7901 cells in M phase, probably through enhancing microtubule depolymerization by elevating the expression of cyclin B1.展开更多
OBJECTIVE To investigate the influence of O-(4-ethoxyl-butyl)- berbamine(EBB)on the expression of cyclin B1 and cdc2-p34 in the human drug-resistant breast cancer MCF-7/ADR cell line. METHODS The MTT assay was used to...OBJECTIVE To investigate the influence of O-(4-ethoxyl-butyl)- berbamine(EBB)on the expression of cyclin B1 and cdc2-p34 in the human drug-resistant breast cancer MCF-7/ADR cell line. METHODS The MTT assay was used to assess the cytotoxicity of EBB.Different levels of EBB were added to different cell lines at series of time points solely or combined with doxorubicin(DOX) to detect the effect on the expression of cyclinB1 and cdc2-p34 by Western blots.cdc2-p34 tyrosine phosphorylation was detected by immunoprecipitation.In addition,apoptosis and cytoplastic Ca 2+ concentrations were systematically examined by laser scanning confocal microscopy(LSCM). RESULTS EBB showed little inhibitory activity on human umbilical vein endothelial cells(ECV304),whereas EBB inhibited cell growth(IC50 range,4.55~15.74μmol/L)in a variety of sensitive and drug-resistance cell lines.EBB also down-regulated the expression of cyclin B1 and cdc2-p34 in a concentration and time dependent manner,which was an important reason for the G2/M phase arrest.EBB was shown to induce apoptosis of MCF-7/ADR cells while increasing the level of cytoplastic Ca 2+ . CONCLUSION The low cytotoxicity of EBB suggests it may be useful as a rational reversal agent.The effect of EBB on cell cycle arrest and related proteins,apoptosis,and cytoplastic Ca 2+ concentration may be involved in reversing multidrug resistance.展开更多
文摘核膜在细胞周期中呈现高度的动态性:在细胞分裂的前中期,核膜崩解并分散到细胞质中;在细胞分裂的后期,核膜开始在染色体的表面重新装配,最终形成完整的核膜结构。近期的研究发现,Ran GTP酶、物质转运蛋白importinβ、内层核膜蛋白LBR(lamin B receptor)以及核孔复合体蛋白nucleoporins在核膜重建的过程中起关键性调控作用,并受到细胞周期调控因子p34cdc2激酶的调节。LBR是一个八次跨膜的膜蛋白,主要定位于内层核膜。在细胞分裂的早期,随着核膜崩解,LBR与核膜崩解而生成的小膜泡一起分散到细胞质中;在细胞分裂的后期,通过LBR与importinβ相互结合,含有LBR的膜泡被importinβ携带至染色质的表面参与核膜重建。目前已知p34cdc2激酶对LBR与importinβ介导的核膜重建起重要调控作用。Nucleoporins是核孔复合体主要组分。随核膜崩解,核孔复合体解聚成nucleoporins,分散到细胞质中,或结合到其他亚细胞成分上。细胞分裂后期,核孔复合体伴随核膜装配而组装。
文摘Full grown oocytes derived from Bufo Bufo gargarizans rearing at high temperature environment (24℃),never underwent GVBD after progesterone treatment. No p34cdc2, H1 kinase activity was detected in the oocytes after progesterone stimulation or OA microinjection; Western blotting analysis showed that the level of p3cdc2, and p33 in the oocytes are significantly lower than those in the oocytes derived from the hibernating toads (below 10 ℃). 25S-Met incorporation analysis showed that when the oocytes were incubated at 6℃, synthesis of about thirty defferent polypeptides was promoted or induced, including p34cdc2 and some other p13suc1-binding proteins. All these results indicated that a low temperature environment is essential for the oocytes of Bufo Bufo gargarizans to express and store some cell cycle drivers and its regulators, and to gain the maturation competence. These results will also provide a new clue for explaining the molecular mechanisms why gametogenesis of some organisms depends on a relative low temperature and how to maintain the geographical distribution of some animals.
基金the National NaturalScience Foundation of China (Serial No.3 980 0 1 49)
文摘Silencing ATM gene gave rise to enhanced apoptotic response to irradiation and i rradiation like chemotherapy agents, this paper explored the crucial identities of the molecular elements responsible for the enhanced apoptotic response in U937 cells mediated by silencing ATM gene. Two U937 cell mutants named U937 ASPI3K (ATM, negative) and U937 pZeosv2(+) (ATM, wild type) were used as a cell model system to identify the critical molecule(s) responsible for the varied apoptotic response in the absence or presence of ATM gene. Apoptosis was examined by measuring concentrations of free nucleosome in U937 cells. Western blot was employed to measure nuclear protein abundance of CDC25A, CDC25B, CDC25C, total p34cdc2, p34cdc2 (Thr 161) or p34cdc2 (Thr 14,Tyr 15). RT PCR was used to estimate CDC25 transcript levels. U937 ASPI3K exhibited an enhanced apoptotic response to lower dosage of irradiation, which could not be blocked by protein synthesis inhibitor. Protein serine threonine phosphatase inhibitor or cyclin dependent kinase (CDK) inhibitors, on the other hand, abolished the enhancement indicated that protein phosphorylation/dephosphorylation modification and CDK activity are required for the enhanced apoptotic response in the absence of ATM gene. Upon irradiation, p34cdc2 in U937 pZeosv2 (+) was maintained in an inactive state by phosphorylation on threonine 14 (Thr 14) and tyrosine 15 (Tyr 15), which was associated with a dramatic decrease of nuclear CDC25A, CDC25B and CDC25C proteins. In contrast, p34cdc2 in U937 ASPI3K maintained in an active state by dephosphorylation on threonine 14 (Thr 14) and tyrosine 15 (Tyr 15), which was associated with constant nuclear CDC25A, CDC25B and CDC25C protein abundance before and after irradiation. The responsive decrease of nuclear CDC25 proteins occurred at the post transcription level. Silencing ATM gene blocks the responsive decrease of nuclear CDC25 proteins, which is responsible for failure to inactivate p34cdc2 after irradiation. Active p34cdc2 and CDK2, in turn, acts as the death executors to trigger apoptosis. In abstract, aberrantly activated CDK activity is the critical molecular mechanism central to enhanced apoptotic responses in the absence of ATM gene.
基金the National 10th Five-year Plan Key Technologies R & D Program of China(No.2004BA703B04-02)
文摘Objective： To learn the effect of allitridi on inducing mitotic arrest in human gastric cell line SGC-7901 and its possible mechanisms. Methods： We treated SGC-7901 cells with allitridi, and observed the proliferation inhibitory rate with MTT colometric assay, changes of cell cycle using flow cytometry and Switzerland-Giemsa＇s staining, and morphologic changes of the microtubule structure and location changes of cyclin BI expression using immunofluorescence and confocal laser scanning microscope. Furthermore, the expression of cyclin B1 was analyzed quantitatively using Leica confocal software. Results： SGC-7901 cells were inhibited after exposure to allitridi and the IC50 was 7.2μg/ml for 24 h, 20μg/ml for 72 h. When the cells were treated with allitridi at concentrations of 3, 6, and 9μg/ml for 24 h respectively, there was a declining tendency in the percentage of G0/G1 cell but an increasing tendency in GE/M cell in the allitridi treated group compared with that of control （P〈0.01）. When cells were treated allitridi at concentration of 6 μg/ml for 24 h, its mitotic index was much higher （P〈0.01） than that of control, suggesting that allitridi caused arrest of gastric cancer cells in M phase. The cells were treated with allitridi became more shrunken and nepheloid, in which the microtubule networks disappeared, while the control cell exhibited an intact microtubule network. Contrasting with normal existence mainly in the cytoplasm, the cyclin B1 was expressed more significantly and concentrated in the nucleus after exposure to allitridi. Fluorescence intensity of cyclin B 1 protein in cells treated with allitridi was much more higher than that of control （P〈0.001）. Conclusion： Allitridican induce arrest of SGC-7901 cells in M phase, probably through enhancing microtubule depolymerization by elevating the expression of cyclin B1.
基金This work was supported by grants from the National Nature Science Foundation of China(No.30572203,30570772)the Key Fund of Science of TianJin Municipal Government(No.07JCZDJC04900)
文摘OBJECTIVE To investigate the influence of O-(4-ethoxyl-butyl)- berbamine(EBB)on the expression of cyclin B1 and cdc2-p34 in the human drug-resistant breast cancer MCF-7/ADR cell line. METHODS The MTT assay was used to assess the cytotoxicity of EBB.Different levels of EBB were added to different cell lines at series of time points solely or combined with doxorubicin(DOX) to detect the effect on the expression of cyclinB1 and cdc2-p34 by Western blots.cdc2-p34 tyrosine phosphorylation was detected by immunoprecipitation.In addition,apoptosis and cytoplastic Ca 2+ concentrations were systematically examined by laser scanning confocal microscopy(LSCM). RESULTS EBB showed little inhibitory activity on human umbilical vein endothelial cells(ECV304),whereas EBB inhibited cell growth(IC50 range,4.55~15.74μmol/L)in a variety of sensitive and drug-resistance cell lines.EBB also down-regulated the expression of cyclin B1 and cdc2-p34 in a concentration and time dependent manner,which was an important reason for the G2/M phase arrest.EBB was shown to induce apoptosis of MCF-7/ADR cells while increasing the level of cytoplastic Ca 2+ . CONCLUSION The low cytotoxicity of EBB suggests it may be useful as a rational reversal agent.The effect of EBB on cell cycle arrest and related proteins,apoptosis,and cytoplastic Ca 2+ concentration may be involved in reversing multidrug resistance.
