Benzene is an established leukotoxin and leukemogen in humans. We have previously re- ported that exposure of workers to benzene and to benzene metabolite hydroquinone in cultured cells induced DNA-dependent protein k...Benzene is an established leukotoxin and leukemogen in humans. We have previously re- ported that exposure of workers to benzene and to benzene metabolite hydroquinone in cultured cells induced DNA-dependent protein kinase catalytic subunit （DNA-PKcs） to mediate the cellular response to DNA double strand break （DSB） caused by DNA-damaging metabolites. In this study, we used a new, small molecule, a selective inhibitor of DNA-PKcs, 2-（morpholin-4-yl）-benzo[h]chomen-4-one （NU7026）, as a probe to analyze the molecular events and pathways in hydroquinone-induced DNA DSB repair and apoptosis. Inhibition of DNA-PKcs by NU7026 markedly potentiated the apoptotic and growth inhibitory effects of hydroquinone in proerythroid leukemic K562 cells in a dose-dependent manner. Treatment with NU7026 did not alter the production of reactive oxygen species and oxidative stress by hydroquinone but repressed the protein level of DNA-PKcs and blocked the induction of the kinase mRNA and protein expression by hydroquinone. Moreover, hydroquinone increased the phos- phorylation of Akt to activate Akt, whereas co-treatment with NU7026 prevented the activation of Akt by hydroquinone. Lastly, hydroquinone and NU7026 exhibited synergistic effects on promoting apop- tosis by increasing the protein levels of pro-apoptotic proteins Bax and caspase-3 but decreasing the protein expression of anti-apoptotic protein Bcl-2. Taken together, the findings reveal a central role of DNA-PKcs in hydroquinone-induced hematotoxicity in which it coordinates DNA DSB repair, cell cycle progression, and apoptosis to regulate the response to hydroquinone-induced DNA damage.展开更多
Islets from RIP-PDE3B mice, exhibiting β-cell specific overexpression of the cAMP/cGMP-degrading enzyme phosphodiesterase 3B (PDE3B) and dysregulated insulin secretion, were subjected to microarray analysis. We show ...Islets from RIP-PDE3B mice, exhibiting β-cell specific overexpression of the cAMP/cGMP-degrading enzyme phosphodiesterase 3B (PDE3B) and dysregulated insulin secretion, were subjected to microarray analysis. We show that osteopontin (OPN) mRNA is increased in a dose-dependent manner in islets from RIP-PDE3B mice, as compared to wild-type islets. In addition, in silico analysis shows that PDE3B and OPN are interacting. Furthermore, OPN interacts with protein kinase CK2 ina distinct submodule of the protein-protein interaction network. We studied PDE3B and OPN proteins and, in some cases, also PDE1B and PDE4C, under conditions of relevance for insulin secretion. In the presence of forskolin, PDE inhibitors, insulin, or a protein kinase CK2 inhibitor, similar alterations in protein levels of PDE3B and OPN are shown. In summary, results from using a number of strategies demonstrate a connection between PDE3B and OPNas well as a role for protein kinase CK2 inpancreatic β-cells.展开更多
The endoplasmic reticulum is the central organelle within a eukaryotic cell where newly synthesized proteins are processed and properly folded. An excess of unfolded or mis-folded proteins induces ER stress signalling...The endoplasmic reticulum is the central organelle within a eukaryotic cell where newly synthesized proteins are processed and properly folded. An excess of unfolded or mis-folded proteins induces ER stress signalling pathways. Usually this means a pro-survival strategy for the cell, whereas under extended stress conditions the ER stress signalling pathways have a pro-apoptotic function. CK2 plays a key role in the regulation of the pro-survival as well as the proapoptotic ER stress signalling by directly modulating the activities of members of the ER stress signalling pathways by phosphorylation, regulating the expression of the key factors of the signalling pathways or binding to regulator proteins. The present review will summarize the state of the art in this new emerging field.展开更多
】目的 :构建小鼠蛋白激酶 CK2 β亚基 c DNA重组表达质粒 ,以便深入进行 CK2结构与功能的研究。方法 :通过反转录 PCR从 NIH3T3小鼠成纤维细胞中获得小鼠蛋白激酶 CK2 β亚基编码区 c DNA,PCR扩增酶为高保真 DNA聚合酶。将 N de I/ H i...】目的 :构建小鼠蛋白激酶 CK2 β亚基 c DNA重组表达质粒 ,以便深入进行 CK2结构与功能的研究。方法 :通过反转录 PCR从 NIH3T3小鼠成纤维细胞中获得小鼠蛋白激酶 CK2 β亚基编码区 c DNA,PCR扩增酶为高保真 DNA聚合酶。将 N de I/ H ind 彻底双酶切 PCR产物定向连接到牛小肠碱性磷酸酶去 5′端磷酸的同样彻底 Nde I/ H ind 双酶切的表达载体 p T7- 7中 ,转化感受态大肠杆菌 DH5 α获得转化子 ,用 0 .8%琼脂糖凝胶电泳初步筛选转化子 ,将阳性克隆进行单和双酶切分析鉴定。随机挑选两个阳性克隆进行 DNA测序。结果 :转化子的阳性筛选率为 10 0 % ,限制性酶切分析结果表明插入片段和重组质粒的大小与理论推测值相符。 DNA测序结果表明 :两个重组小鼠 CK2 β亚基克隆中的插入片段序列均与两种已报道的小鼠 CK2 β亚基 c DNA编码区序列分别存在一个碱基差异 ,但我们报道的这 2个差异碱基与大鼠、兔、猪和人 CK2 β亚基 c DNA的编码区相应碱基序列一致。结论 :本实验克隆的小鼠蛋白激酶 CK2 β亚基 c展开更多
AIM:To evaluate the expression status of S-phase kinase-associated protein 2(Skp2)/cyclin-dependent kinases regulatory subunit 1(Cks1)and p27kip1,and assess the prognostic significance of Skp2/Cks1 expression with p27...AIM:To evaluate the expression status of S-phase kinase-associated protein 2(Skp2)/cyclin-dependent kinases regulatory subunit 1(Cks1)and p27kip1,and assess the prognostic significance of Skp2/Cks1 expression with p27kip1in patients with extrahepatic cholangiocarcinoma.METHODS:Seventy-six patients who underwent curative resection for histologically confirmed extrahepatic cholangiocarcinoma at our institution from December1994 to March 2008 were enrolled.Immunohistochemical staining for Skp2,Cks1,p27kip1,and Ki67,along with other relevant molecular biologic experiments,were performed.RESULTS:By Cox regression analyses,advanced age(>65 years),advanced AJCC tumor stage,poorly differentiated histology,and higher immunostaining intensity of Skp2 were identified as independent prognostic factors in patients with extrahepatic cholangiocarcinoma.Exogenous epidermal growth factor(EGF,especially 0.1-10 ng/mL)significantly increased the proliferation indices by MTT assay and the mRNA levels of Skp2/Cks1 and p27kip1in SNU-1196,SNU-1079,and SNU-245 cells.The protein levels of Skp2/Cks1(from nuclear lysates)and p27kip1(from cytosolic lysate)were also significantly increased in these cells.There were significant reductions in the protein levels of Skp2/Cks1and p27kip1(from nuclear lysate)after the treatment of LY294002.By chromatin immunoprecipitation assay,we found that E2F1 transcription factor directly binds to the promoter site of Skp2.CONCLUSION:Higher immunostaining intensity of Skp2/Cks1 was an independent prognostic factor for patients with extrahepatic cholangiocarcinoma.EGF upregulates the mRNA and protein levels of Skp2/Cks1and p27kip1via the PI3K/Akt pathway and direct binding of E2F1 transcription factor with the Skp2 promoter.展开更多
文摘Benzene is an established leukotoxin and leukemogen in humans. We have previously re- ported that exposure of workers to benzene and to benzene metabolite hydroquinone in cultured cells induced DNA-dependent protein kinase catalytic subunit （DNA-PKcs） to mediate the cellular response to DNA double strand break （DSB） caused by DNA-damaging metabolites. In this study, we used a new, small molecule, a selective inhibitor of DNA-PKcs, 2-（morpholin-4-yl）-benzo[h]chomen-4-one （NU7026）, as a probe to analyze the molecular events and pathways in hydroquinone-induced DNA DSB repair and apoptosis. Inhibition of DNA-PKcs by NU7026 markedly potentiated the apoptotic and growth inhibitory effects of hydroquinone in proerythroid leukemic K562 cells in a dose-dependent manner. Treatment with NU7026 did not alter the production of reactive oxygen species and oxidative stress by hydroquinone but repressed the protein level of DNA-PKcs and blocked the induction of the kinase mRNA and protein expression by hydroquinone. Moreover, hydroquinone increased the phos- phorylation of Akt to activate Akt, whereas co-treatment with NU7026 prevented the activation of Akt by hydroquinone. Lastly, hydroquinone and NU7026 exhibited synergistic effects on promoting apop- tosis by increasing the protein levels of pro-apoptotic proteins Bax and caspase-3 but decreasing the protein expression of anti-apoptotic protein Bcl-2. Taken together, the findings reveal a central role of DNA-PKcs in hydroquinone-induced hematotoxicity in which it coordinates DNA DSB repair, cell cycle progression, and apoptosis to regulate the response to hydroquinone-induced DNA damage.
文摘Islets from RIP-PDE3B mice, exhibiting β-cell specific overexpression of the cAMP/cGMP-degrading enzyme phosphodiesterase 3B (PDE3B) and dysregulated insulin secretion, were subjected to microarray analysis. We show that osteopontin (OPN) mRNA is increased in a dose-dependent manner in islets from RIP-PDE3B mice, as compared to wild-type islets. In addition, in silico analysis shows that PDE3B and OPN are interacting. Furthermore, OPN interacts with protein kinase CK2 ina distinct submodule of the protein-protein interaction network. We studied PDE3B and OPN proteins and, in some cases, also PDE1B and PDE4C, under conditions of relevance for insulin secretion. In the presence of forskolin, PDE inhibitors, insulin, or a protein kinase CK2 inhibitor, similar alterations in protein levels of PDE3B and OPN are shown. In summary, results from using a number of strategies demonstrate a connection between PDE3B and OPNas well as a role for protein kinase CK2 inpancreatic β-cells.
文摘The endoplasmic reticulum is the central organelle within a eukaryotic cell where newly synthesized proteins are processed and properly folded. An excess of unfolded or mis-folded proteins induces ER stress signalling pathways. Usually this means a pro-survival strategy for the cell, whereas under extended stress conditions the ER stress signalling pathways have a pro-apoptotic function. CK2 plays a key role in the regulation of the pro-survival as well as the proapoptotic ER stress signalling by directly modulating the activities of members of the ER stress signalling pathways by phosphorylation, regulating the expression of the key factors of the signalling pathways or binding to regulator proteins. The present review will summarize the state of the art in this new emerging field.
文摘】目的 :构建小鼠蛋白激酶 CK2 β亚基 c DNA重组表达质粒 ,以便深入进行 CK2结构与功能的研究。方法 :通过反转录 PCR从 NIH3T3小鼠成纤维细胞中获得小鼠蛋白激酶 CK2 β亚基编码区 c DNA,PCR扩增酶为高保真 DNA聚合酶。将 N de I/ H ind 彻底双酶切 PCR产物定向连接到牛小肠碱性磷酸酶去 5′端磷酸的同样彻底 Nde I/ H ind 双酶切的表达载体 p T7- 7中 ,转化感受态大肠杆菌 DH5 α获得转化子 ,用 0 .8%琼脂糖凝胶电泳初步筛选转化子 ,将阳性克隆进行单和双酶切分析鉴定。随机挑选两个阳性克隆进行 DNA测序。结果 :转化子的阳性筛选率为 10 0 % ,限制性酶切分析结果表明插入片段和重组质粒的大小与理论推测值相符。 DNA测序结果表明 :两个重组小鼠 CK2 β亚基克隆中的插入片段序列均与两种已报道的小鼠 CK2 β亚基 c DNA编码区序列分别存在一个碱基差异 ,但我们报道的这 2个差异碱基与大鼠、兔、猪和人 CK2 β亚基 c DNA的编码区相应碱基序列一致。结论 :本实验克隆的小鼠蛋白激酶 CK2 β亚基 c
基金Supported by A grant from Samsung Biomedical Research Institute,No.C-A9-210-1
文摘AIM:To evaluate the expression status of S-phase kinase-associated protein 2(Skp2)/cyclin-dependent kinases regulatory subunit 1(Cks1)and p27kip1,and assess the prognostic significance of Skp2/Cks1 expression with p27kip1in patients with extrahepatic cholangiocarcinoma.METHODS:Seventy-six patients who underwent curative resection for histologically confirmed extrahepatic cholangiocarcinoma at our institution from December1994 to March 2008 were enrolled.Immunohistochemical staining for Skp2,Cks1,p27kip1,and Ki67,along with other relevant molecular biologic experiments,were performed.RESULTS:By Cox regression analyses,advanced age(>65 years),advanced AJCC tumor stage,poorly differentiated histology,and higher immunostaining intensity of Skp2 were identified as independent prognostic factors in patients with extrahepatic cholangiocarcinoma.Exogenous epidermal growth factor(EGF,especially 0.1-10 ng/mL)significantly increased the proliferation indices by MTT assay and the mRNA levels of Skp2/Cks1 and p27kip1in SNU-1196,SNU-1079,and SNU-245 cells.The protein levels of Skp2/Cks1(from nuclear lysates)and p27kip1(from cytosolic lysate)were also significantly increased in these cells.There were significant reductions in the protein levels of Skp2/Cks1and p27kip1(from nuclear lysate)after the treatment of LY294002.By chromatin immunoprecipitation assay,we found that E2F1 transcription factor directly binds to the promoter site of Skp2.CONCLUSION:Higher immunostaining intensity of Skp2/Cks1 was an independent prognostic factor for patients with extrahepatic cholangiocarcinoma.EGF upregulates the mRNA and protein levels of Skp2/Cks1and p27kip1via the PI3K/Akt pathway and direct binding of E2F1 transcription factor with the Skp2 promoter.
