Latent membrane protein 1 （LMP1）, an important protein encoded by Epstein Barr virus （EBV）, has been implied to link with the pathogenesis of nasopharyngeal carcinoma （NPC）. Its dual effects of increasing cell p...Latent membrane protein 1 （LMP1）, an important protein encoded by Epstein Barr virus （EBV）, has been implied to link with the pathogenesis of nasopharyngeal carcinoma （NPC）. Its dual effects of increasing cell proliferation and inhibiting cell apoptosis have been confirmed. In this study, we showed that the expression of Survivin and CDK4 protein in CNE-LMP1, a LMP1 positive NPC epithelial cell line, is higher than in LMP1 negative NPC epithelial cell line- CNE1, and the expression is LMP1 dosage-dependent. Although it was reported that Survivin specifically expressed in cell cycle G2/M phase, our studies suggested that LMP1 could promote the expression of Survivin in G0/G1, S and G2/ M phase. It also showed that Survivin and CDK4 could be accumulated more in the nuclei triggered by LMP1. More interestingly, Survivin and CDK4 could form a protein complex in the nuclei of CNE-LMP1 rather than in that of CNE1, which demonstrated that the interaction between these two proteins could be promoted by LMPI. These results strongly suggested that the role of LMP1 in the regulation of Survivin and CDK4 may also shed some light on the mechanism research of LMP1 in NPC.展开更多
Background Exposure to cigarette smoke stimulates the proliferation of human pulmonary artery smooth muscle cells （HPASMCs） in vivo and in vitro. However, the molecular mechanism remains unclear. This study aimed at...Background Exposure to cigarette smoke stimulates the proliferation of human pulmonary artery smooth muscle cells （HPASMCs） in vivo and in vitro. However, the molecular mechanism remains unclear. This study aimed at investigating the role of signaling pathways involving protein kinase C alpha （PKCα） and cyclin D1 in the cigarette smoke extract （CSE）-induced HPASMCs proliferation.Methods Synchronized HPASMCs were treated with different concentrations of CSE. Cell proliferation was evaluated by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyttetrazolium bromide （MTT） assay and cell counting. Cell cycle was analyzed by flow cytometry with propidium iodide staining. Activation of PKCα was measured by detecting the expression of PKCαprotein in the cytosolic and membrane fractions using Western blotting analysis. Small interfering RNA （siRNA） was used to knockdown PKCα and cyclin D1. The cyclin D1 mRNA was assessed by real-time RT-PCR. The PKCα and cyclin D1protein levels were detected by Western blotting.Results Low concentrations of CSE （1%-10%） stimulated proliferation of HPASMCs, with its maximal effect at 5%.CSE （5%） led to PKCα activation. Inhibition of PKCα activity using G（o） 6976 or siRNA-mediated knockdown of PKCα significantly attenuated CSE-induced cell proliferation and G1/S transition. Cyclin D1, one of key regulators of G1/S transition, was found to be upregulated by 5% CSE at both the mRNA and protein levels. CSE-stimulated cellproliferation and G1/S transition was abolished by cyclin D1 siRNA. Moreover, G（o） 6976 or PKCα siRNA significantlysuppressed CSE-induced upregulation of cyclin D1 at both the mRNA and protein levels.Conclusion PKCα-cyclin D1 pathway at least partially mediates the CSE-induced proliferation in HPASMCs.展开更多
In our recent studies, we found that LMP1 encoded by Epstein-Barr virus could accelerate the formation of active c-Jun/Jun B heterodimer. We studied the regulation of cyclinD1 by c-Jun/Jun B heterodimers by laser scan...In our recent studies, we found that LMP1 encoded by Epstein-Barr virus could accelerate the formation of active c-Jun/Jun B heterodimer. We studied the regulation of cyclinD1 by c-Jun/Jun B heterodimers by laser scanning confocal influorescence microscopy, Western blot, luciferase activity assay, super-EMSA and flow cytometry in the Tet-on-LMP1 HNE2 cell line, in which LMP1 expression was regulated by Tet-on system. c-Jun/Jun B heterodimers induced by LMP1 could up regulate cyclin D1 promoter activity and expression. Overexpression of cy-clinD1 accelerated the progression of cell cycle.展开更多
Objective:T-LAK-cell-originated protein kinase(TOPK),a PSD95-Disc large-ZO1(PDZ)binding kinase(PBK),is a novel member of the mitogen-activated protein kinase(MAPK)family.Studies have shown that TOPK plays a critical r...Objective:T-LAK-cell-originated protein kinase(TOPK),a PSD95-Disc large-ZO1(PDZ)binding kinase(PBK),is a novel member of the mitogen-activated protein kinase(MAPK)family.Studies have shown that TOPK plays a critical role in the function of tumor cells,including apoptosis and mitosis.However,little is known on the effect of TOPK in cisplatin-induced acute kidney injury(CP-AKI).This study aimed to investigate the role and mechanism of TOPK in CPAKI.Methods:Cisplatin was administered to C57BL/6 mice and cultured kidney tubular epithelial cells(TECs)to establish the CP-AKI murine or cellular models.TECs were then stimulated with the specific inhibitor of TOPK OTS514 or transfected with the recombinant-activated plasmid TOPK-T9E to inhibit or activate TOPK.The TECs were treated with AKT inhibitorⅧfollowing stimulation with OTS514 or cisplatin.Western blotting and flow cytometry were used to evaluate the cell cycle and apoptosis of TECs.Results:The analysis revealed that the TOPK activity was significantly suppressed by cisplatin,both in vivo and in vitro.Furthermore,the pharmacological inhibition of TOPK by OTS514,a specific inhibitor of TOPK,exacerbated the cisplatin-induced cell cycle arrest in the G2/M phase and apoptosis of cultured TECs.Moreover,the TOPK activation via the TOPK-T9E plasmid transfection could partially reverse the cell cycle arrest at the G2/M phase and apoptosis of cisplatin-treated TECs.In addition,AKT/protein kinase B(PKB),as a TOPK target protein,was inhibited by cisplatin in cultured TECs.The pharmaceutical inhibition of AKT further aggravated the apoptosis of TECs induced by cisplatin or TOPK inhibition.TOPK systematically mediated the apoptosis via the AKT pathway in the CP-AKI cell model.Conclusion:These results indicate that TOPK activation protects against CP-AKI by ameliorating the G2/M cell cycle arrest and cell apoptosis.展开更多
Premature trypsinogen activation and production of oxygen free radicals (OFR) are early pathogenic events which occur within acinar cells and trigger acute pancreatitis (AP). OFR exert their harmful effects on various...Premature trypsinogen activation and production of oxygen free radicals (OFR) are early pathogenic events which occur within acinar cells and trigger acute pancreatitis (AP). OFR exert their harmful effects on various cell components causing lipid peroxidation, disturbances in calcium homeostasis and DNA damage, which lead to increased cell injury and eventually cell death. This review presents the most recent data concerning the effects of N-Acetylcysteine (NAC), in the treatment of AP. NAC is an antioxidant capable of restoring the levels of Glutathione, the most important cellular antioxidant. Studies show the benef icial effects of NAC treatment in preventing OFR production and therefore attenuating oxidative damage. Additionally, NAC treatment has been shown to prevent the increase in cytosolic Ca2+ concentration and reduce the accumulation of enzymes in acinar cells during AP. The prevention, by NAC, of these pathological events occurring within acinar would contribute to reducing the severity of AP. NAC is also capable of reducing the activation of transcription factors especially sensitive to the cellular redox state, such as Nuclear factor-κB, signal transducer and activator of transcription-3 and mitogenactivated protein kinase. This leads to a down-regulation of cytokines, adhesion molecules and chemokine expression in various cell types during AP. These f indingspoint to NAC as a powerful therapeutic treatment, attenuating oxidative-stress-induced cell injury and other pathological events at early stages of AP, and potentially contributing to reducion in the severity of disease.展开更多
基金National Nature Science Foundation for Distinguished Young Scholar of China (No.39525022)National Basic Research Program(No.2004CB518703) National Nature Science Foundation of China (No.30570085).
文摘Latent membrane protein 1 （LMP1）, an important protein encoded by Epstein Barr virus （EBV）, has been implied to link with the pathogenesis of nasopharyngeal carcinoma （NPC）. Its dual effects of increasing cell proliferation and inhibiting cell apoptosis have been confirmed. In this study, we showed that the expression of Survivin and CDK4 protein in CNE-LMP1, a LMP1 positive NPC epithelial cell line, is higher than in LMP1 negative NPC epithelial cell line- CNE1, and the expression is LMP1 dosage-dependent. Although it was reported that Survivin specifically expressed in cell cycle G2/M phase, our studies suggested that LMP1 could promote the expression of Survivin in G0/G1, S and G2/ M phase. It also showed that Survivin and CDK4 could be accumulated more in the nuclei triggered by LMP1. More interestingly, Survivin and CDK4 could form a protein complex in the nuclei of CNE-LMP1 rather than in that of CNE1, which demonstrated that the interaction between these two proteins could be promoted by LMPI. These results strongly suggested that the role of LMP1 in the regulation of Survivin and CDK4 may also shed some light on the mechanism research of LMP1 in NPC.
基金This work was supported by a grant from National Natural Science Foundation of China （No. 30871128）.
文摘Background Exposure to cigarette smoke stimulates the proliferation of human pulmonary artery smooth muscle cells （HPASMCs） in vivo and in vitro. However, the molecular mechanism remains unclear. This study aimed at investigating the role of signaling pathways involving protein kinase C alpha （PKCα） and cyclin D1 in the cigarette smoke extract （CSE）-induced HPASMCs proliferation.Methods Synchronized HPASMCs were treated with different concentrations of CSE. Cell proliferation was evaluated by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyttetrazolium bromide （MTT） assay and cell counting. Cell cycle was analyzed by flow cytometry with propidium iodide staining. Activation of PKCα was measured by detecting the expression of PKCαprotein in the cytosolic and membrane fractions using Western blotting analysis. Small interfering RNA （siRNA） was used to knockdown PKCα and cyclin D1. The cyclin D1 mRNA was assessed by real-time RT-PCR. The PKCα and cyclin D1protein levels were detected by Western blotting.Results Low concentrations of CSE （1%-10%） stimulated proliferation of HPASMCs, with its maximal effect at 5%.CSE （5%） led to PKCα activation. Inhibition of PKCα activity using G（o） 6976 or siRNA-mediated knockdown of PKCα significantly attenuated CSE-induced cell proliferation and G1/S transition. Cyclin D1, one of key regulators of G1/S transition, was found to be upregulated by 5% CSE at both the mRNA and protein levels. CSE-stimulated cellproliferation and G1/S transition was abolished by cyclin D1 siRNA. Moreover, G（o） 6976 or PKCα siRNA significantlysuppressed CSE-induced upregulation of cyclin D1 at both the mRNA and protein levels.Conclusion PKCα-cyclin D1 pathway at least partially mediates the CSE-induced proliferation in HPASMCs.
基金This work was supported by the StateKey Basic Research Program,Foundational Investigation on Human Carcinogenes is of China(Grant No.G1998051201)National Natural Science Foundat ion for Distinguished Young Scholars of China(Grant No.39525022)National Natural Science Foundation of China(Grant Nos.30300403,30100005&30000087).
文摘In our recent studies, we found that LMP1 encoded by Epstein-Barr virus could accelerate the formation of active c-Jun/Jun B heterodimer. We studied the regulation of cyclinD1 by c-Jun/Jun B heterodimers by laser scanning confocal influorescence microscopy, Western blot, luciferase activity assay, super-EMSA and flow cytometry in the Tet-on-LMP1 HNE2 cell line, in which LMP1 expression was regulated by Tet-on system. c-Jun/Jun B heterodimers induced by LMP1 could up regulate cyclin D1 promoter activity and expression. Overexpression of cy-clinD1 accelerated the progression of cell cycle.
基金supported by the National Nature Science Foundation of China(No.81974102).
文摘Objective:T-LAK-cell-originated protein kinase(TOPK),a PSD95-Disc large-ZO1(PDZ)binding kinase(PBK),is a novel member of the mitogen-activated protein kinase(MAPK)family.Studies have shown that TOPK plays a critical role in the function of tumor cells,including apoptosis and mitosis.However,little is known on the effect of TOPK in cisplatin-induced acute kidney injury(CP-AKI).This study aimed to investigate the role and mechanism of TOPK in CPAKI.Methods:Cisplatin was administered to C57BL/6 mice and cultured kidney tubular epithelial cells(TECs)to establish the CP-AKI murine or cellular models.TECs were then stimulated with the specific inhibitor of TOPK OTS514 or transfected with the recombinant-activated plasmid TOPK-T9E to inhibit or activate TOPK.The TECs were treated with AKT inhibitorⅧfollowing stimulation with OTS514 or cisplatin.Western blotting and flow cytometry were used to evaluate the cell cycle and apoptosis of TECs.Results:The analysis revealed that the TOPK activity was significantly suppressed by cisplatin,both in vivo and in vitro.Furthermore,the pharmacological inhibition of TOPK by OTS514,a specific inhibitor of TOPK,exacerbated the cisplatin-induced cell cycle arrest in the G2/M phase and apoptosis of cultured TECs.Moreover,the TOPK activation via the TOPK-T9E plasmid transfection could partially reverse the cell cycle arrest at the G2/M phase and apoptosis of cisplatin-treated TECs.In addition,AKT/protein kinase B(PKB),as a TOPK target protein,was inhibited by cisplatin in cultured TECs.The pharmaceutical inhibition of AKT further aggravated the apoptosis of TECs induced by cisplatin or TOPK inhibition.TOPK systematically mediated the apoptosis via the AKT pathway in the CP-AKI cell model.Conclusion:These results indicate that TOPK activation protects against CP-AKI by ameliorating the G2/M cell cycle arrest and cell apoptosis.
文摘Premature trypsinogen activation and production of oxygen free radicals (OFR) are early pathogenic events which occur within acinar cells and trigger acute pancreatitis (AP). OFR exert their harmful effects on various cell components causing lipid peroxidation, disturbances in calcium homeostasis and DNA damage, which lead to increased cell injury and eventually cell death. This review presents the most recent data concerning the effects of N-Acetylcysteine (NAC), in the treatment of AP. NAC is an antioxidant capable of restoring the levels of Glutathione, the most important cellular antioxidant. Studies show the benef icial effects of NAC treatment in preventing OFR production and therefore attenuating oxidative damage. Additionally, NAC treatment has been shown to prevent the increase in cytosolic Ca2+ concentration and reduce the accumulation of enzymes in acinar cells during AP. The prevention, by NAC, of these pathological events occurring within acinar would contribute to reducing the severity of AP. NAC is also capable of reducing the activation of transcription factors especially sensitive to the cellular redox state, such as Nuclear factor-κB, signal transducer and activator of transcription-3 and mitogenactivated protein kinase. This leads to a down-regulation of cytokines, adhesion molecules and chemokine expression in various cell types during AP. These f indingspoint to NAC as a powerful therapeutic treatment, attenuating oxidative-stress-induced cell injury and other pathological events at early stages of AP, and potentially contributing to reducion in the severity of disease.
