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
目的:探讨小鼠卵母细胞不同细胞周期时相中,由于p34cdc2不同磷酸化状态所引起的电泳迁移率变化。方法:用免疫印迹(W estern b lot)技术观察G2期和MⅡ期小鼠卵母细胞中p34cdc2电泳迁移率的变化。结果:p34cdc2在G2期只表现两条带即上、中...目的:探讨小鼠卵母细胞不同细胞周期时相中,由于p34cdc2不同磷酸化状态所引起的电泳迁移率变化。方法:用免疫印迹(W estern b lot)技术观察G2期和MⅡ期小鼠卵母细胞中p34cdc2电泳迁移率的变化。结果:p34cdc2在G2期只表现两条带即上、中带;而MⅡ期表现为中、下带。结论:可以用电泳迁移率的变化作为观察p34cdc2磷酸化状态的一种方法。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
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
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.
文摘目的:探讨小鼠卵母细胞不同细胞周期时相中,由于p34cdc2不同磷酸化状态所引起的电泳迁移率变化。方法:用免疫印迹(W estern b lot)技术观察G2期和MⅡ期小鼠卵母细胞中p34cdc2电泳迁移率的变化。结果:p34cdc2在G2期只表现两条带即上、中带;而MⅡ期表现为中、下带。结论:可以用电泳迁移率的变化作为观察p34cdc2磷酸化状态的一种方法。
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 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.
