<strong>Introduction</strong>.<span><span><span style="font-family:;" "=""><span style="font-family:Verdana;"> The molecular biological mechanism ...<strong>Introduction</strong>.<span><span><span style="font-family:;" "=""><span style="font-family:Verdana;"> The molecular biological mechanism of the increased incidence of the various types of cancer in obesity or type 2 diabetes in rodents or humans has largely been resolved in recent years. By contrast, the molecular biological mechanism of the decreased, not increased, incidence of the various types of cancer in the homozygous long-lived Ames dwarf mice still remains unresolved. </span><b><span style="font-family:Verdana;">Objective.</span></b><span style="font-family:Verdana;"> The first objective of the present study was to investigate whether the decrease in the incidence of cancer in the homozygous long-lived Ames dwarf mice is due to the increase, not decrease, in the expression of p27Kip1, a cell cycle repressor protein. The second objective was to investigate whether the decrease in the incidence of cancer in the homozygous long-lived Ames dwarf mice is due to the decrease, not increase, in the levels of glucose or insulin. </span><b><span style="font-family:Verdana;">Methods.</span></b><span style="font-family:Verdana;"> To achieve these objectives, we first performed western immunoblot analysis of the hepatic expression of p27Kip1 protein. We then performed, using a human breast cancer cell line </span><i><span style="font-family:Verdana;">in</span></i> <i><span style="font-family:Verdana;">vitro</span></i><span style="font-family:Verdana;">, the luciferase reporter plasmid assay to determine whether the translation initiation activity of the p27Kip1 mRNA is increased when the concentrations of either glucose or insulin are decreased. </span><b><span style="font-family:Verdana;">Results and Conclusion. </span></b><span style="font-family:Verdana;">The results of the first objective indicated that the hepatic expression of p27Kip1 protein was up-regulated in the homozygous long-lived Ames dwarf mice as expected. We also found that the lower concentrations of glucose or insulin increased the translation initiation activity of the p27Kip1 mRNA.</span></span></span></span>展开更多
The p27Kip1 is a cell cycle repressor protein that regulates primarily the cell cycle transition from G1 to S phase and hence the DNA replication is in the S phase and cell division in the M phase. Expression of p27Ki...The p27Kip1 is a cell cycle repressor protein that regulates primarily the cell cycle transition from G1 to S phase and hence the DNA replication is in the S phase and cell division in the M phase. Expression of p27Kip1 protein has dual roles for both cancer prevention and promotion. For example, numerous nutritional and chemopreventive anti-cancer agents specifically increase the expression of p27Kip1 protein without directly affecting the expression of any other cell cycle regulatory proteins. On the other hand, pro-cancer agents (like glucose, insulin and other growth factors frequently seen in obesity and/or diabetes) specifically decrease the expression of p27Kip1 protein without directly affecting the expression of any other cell cycle regulatory proteins. Unlike expression of any other cell cycle regulatory proteins, expression of p27Kip1 protein is very unusual. The mRNA of p27Kip1 has a very long and unusual 5’-untranslated region (from -575 to -1 in human). It appears that the 5’-untranslated region of p27Kip1 mRNA forms two alternative secondary structures. One increases the expression of p27Kip1 protein when anti-cancer agents are added and another decrease the expression of p27K1p1 when pro-cancer agents are added. For this short concept proposal, Dr. Albert Einstein’s “visualized thought experiments (German: Gedanken experiment)” were used as a fundamental tool for understanding how either anti- or pro-cancer agents bring the primary structure of the 5’-untranslated region of p27Kip1 mRNA into two alternative secondary structures, thereby either increasing or decreasing, respectively, the translation initiation of p27Kip1 protein.展开更多
AIM:To investigate a novel therapeutic strategy to target and suppress c-myc in human cancers using far up stream element(FUSE)-binding protein-interacting repressor(FIR).METHODS:Endogenous c-Myc suppression and apopt...AIM:To investigate a novel therapeutic strategy to target and suppress c-myc in human cancers using far up stream element(FUSE)-binding protein-interacting repressor(FIR).METHODS:Endogenous c-Myc suppression and apoptosis induction by a transient FIR-expressing vector was examined in vivo via a HA-tagged FIR(HA-FIR)expression vector.A fusion gene-deficient,non-transmissible,Sendai virus(SeV)vector encoding FIR cDNA,SeV/dF/FIR,was prepared.SeV/dF/FIR was examined for its gene transduction efficiency,viral dose dependency of antitumor effect and apoptosis induction in HeLa(cervical squamous cell carcinoma)cells and SW480(colon adenocarcinoma)cells.Antitumor efficacy in a mouse xenograft model was also examined.The molecular mechanism of the anti-tumor effect and c-Myc suppression by SeV/dF/FIR was examined using Spliceostatin A(SSA),a SAP155 inhibitor,or SAP155siRNA which induce c-Myc by increasing FIR△exon2 in HeLa cells.RESULTS:FIR was found to repress c-myc transcription and in turn the overexpression of FIR drove apoptosis through c-myc suppression.Thus,FIR expressing vectors are potentially applicable for cancer therapy.FIR is alternatively spliced by SAP155 in cancer cells lacking the transcriptional repression domain within exon 2(FIR△exon2),counteracting FIR for c-Myc protein expression.Furthermore,FIR forms a complex with SAP155 and inhibits mutual well-established functions.Thus,both the valuable effects and side effects of exogenous FIR stimuli should be tested for future clinical application.SeV/dF/FIR,a cytoplasmic RNA virus,was successfully prepared and showed highly efficient gene transduction in in vivo experiments.Furthermore,in nude mouse tumor xenograft models,SeV/dF/FIR displayed high antitumor efficiency against human cancer cells.SeV/dF/FIR suppressed SSA-activated c-Myc.SAP155 siRNA,potentially produces FIR△exon2,and led to c-Myc overexpression with phosphorylation at Ser62.HA-FIR suppressed endogenous c-Myc expression and induced apoptosis in HeLa and SW480 cells.A c-myc transcriptional suppressor FIR expressing SeV/dF/FIR showed high gene transduction efficiency with significant antitumor effects and apoptosis induction in HeLa and SW480 cells.CONCLUSION:SeV/dF/FIR showed strong tumor growth suppression with no significant side effects in an animal xenograft model,thus SeV/dF/FIR is potentially applicable for future clinical cancer treatment.展开更多
目的探究性别决定区Y框蛋白9(sex determining region Y box protein 9,SOX9)在角膜内皮损伤过程中的表达及功能。方法通过转录组数据库分析人角膜内皮损伤与SOX9表达的关联。冷冻法构建小鼠角膜内皮损伤模型,每只小鼠左眼造模、右眼空...目的探究性别决定区Y框蛋白9(sex determining region Y box protein 9,SOX9)在角膜内皮损伤过程中的表达及功能。方法通过转录组数据库分析人角膜内皮损伤与SOX9表达的关联。冷冻法构建小鼠角膜内皮损伤模型,每只小鼠左眼造模、右眼空白对照,采用裂隙灯照相、眼前节光学相干断层扫描技术(optical coherence tomography,OCT)、免疫荧光技术评估动物模型质量,角膜铺片免疫荧光染色观察损伤后内皮细胞中SOX9的表达变化。分别用低浓度(30μmol/L)和高浓度(50μmol/L)的甲萘醌对人角膜内皮细胞(B4G12)处理3 h(短时间组)、6 h(中时间组)、12 h(长时间组)、24 h(超长时间组),构建细胞模型。采用显微镜照相、细胞计数试剂盒(cell counting kit-8,CCK-8)实验评估细胞模型质量,实时荧光定量聚合酶链式反应(real-time quantitative polymerase chain reaction,qPCR)、Western blot检测SOX9和介导角膜内皮-间质转化(endothelium-mesenchymal transition,EndMT)关键转录因子Snail家族转录抑制因子2(snail family transcriptional repressor 2,SNAIL2)的表达变化。结果数据库资料显示在角膜内皮损伤代表性疾病Fuchs角膜内皮营养不良(Fuchs′endothelial corneal dystrophy,FECD)患者中,FECD3期以上患者角膜内皮SOX9表达量比2期患者明显升高(P<0.05),且SOX9的下游基因富含半胱氨酸的酸性分泌蛋白(secreted protein acidic and rich in cysteine,SPARC)表达量比正常人明显升高(P<0.05)。在动物模型中,对照组角膜内皮铺片免疫荧光染色显示无SOX9荧光,损伤后短期出现大量SOX9荧光,损伤后中期、长期SOX9荧光回归极低水平。在甲萘醌处理的细胞模型中,SOX9表达随处理时间的延长呈现先升高后降低的趋势;同时发现SNAIL2的表达变化也会随甲萘醌处理时间的延长呈现先升高后降低的趋势。结论SOX9的表达随角膜内皮损伤进程产生变化,并与损伤修复中的EndMT紧密相关。展开更多
文摘<strong>Introduction</strong>.<span><span><span style="font-family:;" "=""><span style="font-family:Verdana;"> The molecular biological mechanism of the increased incidence of the various types of cancer in obesity or type 2 diabetes in rodents or humans has largely been resolved in recent years. By contrast, the molecular biological mechanism of the decreased, not increased, incidence of the various types of cancer in the homozygous long-lived Ames dwarf mice still remains unresolved. </span><b><span style="font-family:Verdana;">Objective.</span></b><span style="font-family:Verdana;"> The first objective of the present study was to investigate whether the decrease in the incidence of cancer in the homozygous long-lived Ames dwarf mice is due to the increase, not decrease, in the expression of p27Kip1, a cell cycle repressor protein. The second objective was to investigate whether the decrease in the incidence of cancer in the homozygous long-lived Ames dwarf mice is due to the decrease, not increase, in the levels of glucose or insulin. </span><b><span style="font-family:Verdana;">Methods.</span></b><span style="font-family:Verdana;"> To achieve these objectives, we first performed western immunoblot analysis of the hepatic expression of p27Kip1 protein. We then performed, using a human breast cancer cell line </span><i><span style="font-family:Verdana;">in</span></i> <i><span style="font-family:Verdana;">vitro</span></i><span style="font-family:Verdana;">, the luciferase reporter plasmid assay to determine whether the translation initiation activity of the p27Kip1 mRNA is increased when the concentrations of either glucose or insulin are decreased. </span><b><span style="font-family:Verdana;">Results and Conclusion. </span></b><span style="font-family:Verdana;">The results of the first objective indicated that the hepatic expression of p27Kip1 protein was up-regulated in the homozygous long-lived Ames dwarf mice as expected. We also found that the lower concentrations of glucose or insulin increased the translation initiation activity of the p27Kip1 mRNA.</span></span></span></span>
文摘The p27Kip1 is a cell cycle repressor protein that regulates primarily the cell cycle transition from G1 to S phase and hence the DNA replication is in the S phase and cell division in the M phase. Expression of p27Kip1 protein has dual roles for both cancer prevention and promotion. For example, numerous nutritional and chemopreventive anti-cancer agents specifically increase the expression of p27Kip1 protein without directly affecting the expression of any other cell cycle regulatory proteins. On the other hand, pro-cancer agents (like glucose, insulin and other growth factors frequently seen in obesity and/or diabetes) specifically decrease the expression of p27Kip1 protein without directly affecting the expression of any other cell cycle regulatory proteins. Unlike expression of any other cell cycle regulatory proteins, expression of p27Kip1 protein is very unusual. The mRNA of p27Kip1 has a very long and unusual 5’-untranslated region (from -575 to -1 in human). It appears that the 5’-untranslated region of p27Kip1 mRNA forms two alternative secondary structures. One increases the expression of p27Kip1 protein when anti-cancer agents are added and another decrease the expression of p27K1p1 when pro-cancer agents are added. For this short concept proposal, Dr. Albert Einstein’s “visualized thought experiments (German: Gedanken experiment)” were used as a fundamental tool for understanding how either anti- or pro-cancer agents bring the primary structure of the 5’-untranslated region of p27Kip1 mRNA into two alternative secondary structures, thereby either increasing or decreasing, respectively, the translation initiation of p27Kip1 protein.
基金Supported by In part by the 21st Century COE(Center Of Ex-cellence)Programs to Dr.Takenori Ochiaiby a Grant-in-Aid 18591453 to K.M from the Ministry of Education,Science,Sports and Culture of Japan
文摘AIM:To investigate a novel therapeutic strategy to target and suppress c-myc in human cancers using far up stream element(FUSE)-binding protein-interacting repressor(FIR).METHODS:Endogenous c-Myc suppression and apoptosis induction by a transient FIR-expressing vector was examined in vivo via a HA-tagged FIR(HA-FIR)expression vector.A fusion gene-deficient,non-transmissible,Sendai virus(SeV)vector encoding FIR cDNA,SeV/dF/FIR,was prepared.SeV/dF/FIR was examined for its gene transduction efficiency,viral dose dependency of antitumor effect and apoptosis induction in HeLa(cervical squamous cell carcinoma)cells and SW480(colon adenocarcinoma)cells.Antitumor efficacy in a mouse xenograft model was also examined.The molecular mechanism of the anti-tumor effect and c-Myc suppression by SeV/dF/FIR was examined using Spliceostatin A(SSA),a SAP155 inhibitor,or SAP155siRNA which induce c-Myc by increasing FIR△exon2 in HeLa cells.RESULTS:FIR was found to repress c-myc transcription and in turn the overexpression of FIR drove apoptosis through c-myc suppression.Thus,FIR expressing vectors are potentially applicable for cancer therapy.FIR is alternatively spliced by SAP155 in cancer cells lacking the transcriptional repression domain within exon 2(FIR△exon2),counteracting FIR for c-Myc protein expression.Furthermore,FIR forms a complex with SAP155 and inhibits mutual well-established functions.Thus,both the valuable effects and side effects of exogenous FIR stimuli should be tested for future clinical application.SeV/dF/FIR,a cytoplasmic RNA virus,was successfully prepared and showed highly efficient gene transduction in in vivo experiments.Furthermore,in nude mouse tumor xenograft models,SeV/dF/FIR displayed high antitumor efficiency against human cancer cells.SeV/dF/FIR suppressed SSA-activated c-Myc.SAP155 siRNA,potentially produces FIR△exon2,and led to c-Myc overexpression with phosphorylation at Ser62.HA-FIR suppressed endogenous c-Myc expression and induced apoptosis in HeLa and SW480 cells.A c-myc transcriptional suppressor FIR expressing SeV/dF/FIR showed high gene transduction efficiency with significant antitumor effects and apoptosis induction in HeLa and SW480 cells.CONCLUSION:SeV/dF/FIR showed strong tumor growth suppression with no significant side effects in an animal xenograft model,thus SeV/dF/FIR is potentially applicable for future clinical cancer treatment.
文摘目的探究性别决定区Y框蛋白9(sex determining region Y box protein 9,SOX9)在角膜内皮损伤过程中的表达及功能。方法通过转录组数据库分析人角膜内皮损伤与SOX9表达的关联。冷冻法构建小鼠角膜内皮损伤模型,每只小鼠左眼造模、右眼空白对照,采用裂隙灯照相、眼前节光学相干断层扫描技术(optical coherence tomography,OCT)、免疫荧光技术评估动物模型质量,角膜铺片免疫荧光染色观察损伤后内皮细胞中SOX9的表达变化。分别用低浓度(30μmol/L)和高浓度(50μmol/L)的甲萘醌对人角膜内皮细胞(B4G12)处理3 h(短时间组)、6 h(中时间组)、12 h(长时间组)、24 h(超长时间组),构建细胞模型。采用显微镜照相、细胞计数试剂盒(cell counting kit-8,CCK-8)实验评估细胞模型质量,实时荧光定量聚合酶链式反应(real-time quantitative polymerase chain reaction,qPCR)、Western blot检测SOX9和介导角膜内皮-间质转化(endothelium-mesenchymal transition,EndMT)关键转录因子Snail家族转录抑制因子2(snail family transcriptional repressor 2,SNAIL2)的表达变化。结果数据库资料显示在角膜内皮损伤代表性疾病Fuchs角膜内皮营养不良(Fuchs′endothelial corneal dystrophy,FECD)患者中,FECD3期以上患者角膜内皮SOX9表达量比2期患者明显升高(P<0.05),且SOX9的下游基因富含半胱氨酸的酸性分泌蛋白(secreted protein acidic and rich in cysteine,SPARC)表达量比正常人明显升高(P<0.05)。在动物模型中,对照组角膜内皮铺片免疫荧光染色显示无SOX9荧光,损伤后短期出现大量SOX9荧光,损伤后中期、长期SOX9荧光回归极低水平。在甲萘醌处理的细胞模型中,SOX9表达随处理时间的延长呈现先升高后降低的趋势;同时发现SNAIL2的表达变化也会随甲萘醌处理时间的延长呈现先升高后降低的趋势。结论SOX9的表达随角膜内皮损伤进程产生变化,并与损伤修复中的EndMT紧密相关。
文摘目的:探讨逆转录病毒介导的E1A激活基因阻遇子(CREG)过表达对球囊损伤后大鼠颈动脉新生内膜形成的影响。方法:以球囊拉伤法制备大鼠颈动脉损伤模型,应用Pluronic F127胶分别包裹含有pLNCX/CREG、pLNCX/GFP载体的逆转录病毒,并于损伤即刻涂抹于血管外壁。随后在损伤不同时点取出损伤动脉,测量内膜面积及内膜/中膜面积比。用免疫组织化学法观察CREG、SMα-actin及Ki-67变化,Western blotting检测CREG蛋白表达。结果:血管损伤后2 d pLNCX/GFP组血管中膜平滑肌层GFP表达。pLNCX/CREG组大鼠血管CREG表达明显多于单纯损伤组。感染CREG组大鼠球囊损伤后血管新生内膜的形成明显受抑,Ki-67蛋白表达明显少于、而SMα-actin表达则明显多于单纯损伤组。结论:CREG过表达能抑制血管损伤后平滑肌细胞的增殖并促进其分化,提示调控CREG基因的表达可能为预防PCI术后再狭窄提供有效手段。
