膀胱移行细胞癌中c-FLIP mRNA表达及临床相关性
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摘要
前言
膀胱癌在中国是最常见的泌尿系统恶性肿瘤之一。其组织病理类型以移行细胞癌(transitional cell carcinoma,TCC)为主,约占90%以上。对膀胱肿瘤的病因研究很多,但病因仍不清楚。目前学者们普遍认为膀胱肿瘤的发生、发展是一个复杂的多因素、多步骤、多变化的过程,是细胞凋亡增殖失衡的结果。
细胞型Fas相关死亡区域蛋白样白介素-1β转换酶抑制蛋白(cellularFADD-like interleukin-1β-converting enzyme inhibitory protein c-FLIP)是近年来发现的一类含有死亡效应结构域(death effect domains,DED)的凋亡抑制蛋白,已证实c-FLIP与炎症、肿瘤、自身免疫性疾病的发生发展有关。因此对c-FLIP的进一步研究将有助于深化对这些疾病的认识,并为临床治疗这些疾病提供新的方法和思路。为进一步明确膀胱移行细胞癌发生发展过程中c-FLIP所起到的作用,并且进一步探讨其对膀胱肿瘤预后的影响,我们采用免疫组化及半定量RT-PCR方法探讨c-FLIP mRNA及其蛋白在膀胱移行细胞癌中的表达情况。探讨c-FLIP mRNA的表达与膀胱移行细胞癌的临床分期、病理组织分级及预后之间的关系,现将结果报导如下。
材料与方法
一、临床资料
(一)一般资料
收集2002年4月至2005年10月,我科手术治疗膀胱移行细胞癌患者100例。从中随机选出30例,男23例,女7例,年龄在20岁—80岁。按临床分期标准:T_a期6例,T_1期8例,T_2期9例,T_3期7例。按病理分级标准:Ⅰ级4例、Ⅰ-Ⅱ级12例、Ⅱ级5例、Ⅱ-Ⅲ级5例、Ⅲ级4例,同时采集10例正常膀胱移行上皮组织,年龄46岁—61岁。取材后,置入-80℃低温冰箱保存备检。
(二)实验试剂和仪器
免疫组化试剂盒购于北京博奥森生物技术公司、c-FLIP兔抗人抗体(一抗)购于北京博奥森生物技术有限公司。其中试剂盒中包括:3%H_2O_2去离子水、封闭用正常山羊血清工作液、生物素化二抗工作液,为生物素标记的山羊抗兔LgG、辣根酶标记链霉卵白素工作液。RT—PCR试剂盒购于Takara Shuzo Co.Ltd.;c-FLIP—F,c—FLIP—R及β-actin引物购于Takara公司;TRIZOL试剂购于GIBCOBRL公司;超净工作台、石蜡切片机(上海);聚乙烯探子(北京);TG328B光电分析天平(上海);低温冰箱及超低温冰箱(日本SANYO公司);自动电泳凝胶成像分析仪为美国syngene公司生产;PCR扩增仪PTC—100~(TM)为美国公司生产。
二、方法
免疫组化方法采用链霉菌抗生物素蛋白-过氧化物酶连结(SP)法:取石蜡切片,烤片,68℃,20分钟,常规二甲苯脱蜡,梯度酒精脱水;二甲苯Ⅰ20min—二甲苯Ⅱ20 min—100%酒精Ⅰ10min—100%Ⅱ10min—95%5min—80%5min—70%5min;阻断灭活内源性过氧化物酶:3%H2O2 37℃孵育10min,PBS冲洗3X5min;抗原修复:置0.01M枸橼酸缓冲液(PH 6.0)中用煮沸(95℃,15—20min),自然冷却20min以上,再用冷水冲洗缸子,加快冷却至室温,PBS冲洗3X5min。正常羊血清工作液封闭,37℃10 min,倾去勿洗;滴加一抗4℃冰箱孵育过夜,PBS冲洗3X5min(用PBS缓冲液代替一抗作阴性对照);滴加生物素标记二抗,37℃孵育30min,PBS冲洗3X5min:滴加辣根过氧化物酶标记的链霉素卵白素工作液,37℃孵育30min,PBS冲洗3X5min;DAB/H2O2反应染色,自来水充分冲洗后苏木素复染,常规脱水,透明,干燥,封片。
RT-PCR方法:提取组织总RNA,按TROZOL抽提试剂盒说明进行,紫外分光光度仪检测RNA纯度及浓度。逆转录反应:在20ul逆转录反应体系中含总RNA(1ug/ul)1ul,Oligo(dT)_(15),引物0.5ul,25mM MgSo_42ul,10mMdNTPs 0.5ul,2×buffer 5ul,RNA Inhibitor 0.25ul,反转录酶(22U/ul)0.5ul,dd.H_2O 0.25ul。反应条件:65℃1min,30℃5min,15—30min匀速升温至65℃,65℃30min,98℃5min终止反应,5℃5min灭活反转录酶。扩增c-FLIP cDNA:取cDNA3ul,上下游引物各0.1ul,Taq DNA聚合酶0.2ul,加入PCR反应体系中扩增,反应条件:94℃变性3min,94℃45s,54.2℃1min,72℃1min 35cycls,72℃7min延伸。PCR产物分析:PCR产物经凝胶成像及分析系统扫描后,以β-actin为参照求出其相对值。基因表达值(%)=(c-FLIP/β-actin)×100。引物序列:c-FLIP F,5′—GTT CTT CGG GAC ACC TTC AGT—3′,R,5′—TAT CCA CGC CAAACA CGC TCT—3。β-actin F,5′—GTG GGG CGC CCC AGG CAC CA—3′R,5′—CTC CTT AAT GTC ACG CAC GAT TTC—3′
三、结果判定及统计学处理
c-FLIP蛋白染色为棕黄色颗粒,定位于细胞浆内。每张切片随机选择观察10个高倍视野,每个高倍视野计数100个细胞并计算平均数,c-FLIP的表达以着色肿瘤细胞平均数大于10%为阳性引入标准。
RT-PCR结果采用t检验,P＜0.05有统计学意义。在预后的研究过程中主要探讨c-FLIP mRNA表达与患者术后无病生存率DFS(disease free survival)之间的关系。DFS定义为自随访之日起至患者出现复发灶,或因该病发生死亡的时间。采用Kaplan—meier进行单因素分析,COX风险模型进行多因素分析。所有的统计计算应用spss13.0软件。
结果
一、c-FLIP蛋白在膀胱移行细胞癌中的表达。
c-FLIP蛋白呈棕黄色颗粒,定位于胞浆。膀胱癌组织中FLIP蛋白的阳性率为76.77%(23/30),正常膀胱移行上皮c-FLIP蛋白表达为阴性。
二、c-FLIP mRNA在膀胱移行细胞癌中的表达。
RT-PCR产物经2%琼脂糖凝胶电泳后,紫外透射仪下可见到扩增的特异产物条带,目的基因片段产物与引物设计目的片段大小完全吻合。30例癌组织标本中19例呈阳性表达,阳性率63.3%,10例对照均呈阴性表达。
三、c-FLIP mRNA在膀胱移行细胞癌组织及正常膀胱上皮组织中的检测结果。
显示膀胱移行细胞癌组织及正常膀胱上皮组织中c-FLIP mRNA表达结果,两者比较,差异显著(P＜0.05)。
四、c-FLIP mRNA的表达与临床分期、病理组织分级之间的关系。
显示膀胱移行细胞癌组织中c-FLIP mRNA表达与其临床分期、病理分级之间差异不显著(P＞0.05)。
五、c-FLIP mRNA的表达与膀胱移行细胞癌预后之间关系。
在膀胱移行细胞癌的预后过程中,癌组织的临床分期与病理组织分级及单发或多发对患者的术后无病生存率DFS无显著性影响,(P＞0.05)。癌组织的c-FLIPmRNA的表达情况对患者的DFS存在显著性影响,RR=5.397,95%CI(1.148—25.377),P=0.033。提示c-FLIP mRNA呈阳性表达的患者术后无病生存率较低,预后不好。
结论
1、c-FLIP蛋白与c-FLIP mRNA在膀胱移行细胞癌中呈一致高表达。
2、c-FLIP mRNA在膀胱移行细胞癌中高表达,这一特征使之成为较有潜力的肿瘤标志物。
3、膀胱移行细胞癌c-FLIP mRNA的表达情况对患者的无病生存率存在显著性影响,提示c-FLIP mRNA呈阳性表达的患者术后无病生存率较低,预后不好。
Preface
Bladder cancer is a kind of malignant tumor frequently occurred in urinary system. In china,the occurrence of bladder cancer is 3-4 times more frequent in men than in women.Transitional cell carcinoma(TCC)is by far the most frequent histotype, although the distribution of histotypes varies in different populations.In Chinese people,90%of all bladder cancers were TCC.Most bladder carcinogens exert their action by direct contact with the bladder epithelium.Inhaled or ingested compounds, which are either directly carcinogenic or which can be transformed by the body in carcinogenic byproducts,are excreted via the urine,and in this way they reach the urinary bladder.There is also evidence that chronic inflammation,caused either by infections or stones,has a role in the promotion of bladder cancer.
A caspase-like protein termed c-FLIP(cellular FADD-like interleukin-1β-converting enzyme inhibitory protein)impairs the Fas-mediated cell death signal. c-FLIP is expressed mainly in a long(c-FLIPL)form and a short spliced form (c-FLIPS).The short form consists of exclusively two tandem repeats of death effector domain(DED),which inhibits procaspase-8 activation.The longer isoform contains two DED in its NH2-terminal region and shares extensive homology with the caspase-8 catalytic domain in its COOH-terminal region.However,c-FLIPL sequence lost the conserved Cys residue in the catalytic site and a closely positioned His residue,which are essential for the caspase-8 catalytic activity.
To explore the expression of c-FLIP in bladder transitional cell carcinoma and its'clinical significance,in this study,the expression levels of c-FLIP were evaluated by using RT-PCR in 30 bladder transitional cell carcinoma tissues,10 normal bladder tissues,Its correlations with the clinical stage,pathology type,prognosis were evaluated in positivecase.
Method
30 bladder cancer samples were Collected which are setected by operation in our university from 2002 to 2005,These samples were graded according to WHO/ISUP1998,which is 4gradeⅠ、12gradeⅠ-Ⅱ、5gradeⅡ、5stageⅡ-Ⅲ、4gradeⅢ.TNM staged were confirmed among these sample that is 6T_a、8T_1、9T_2, 7T_3.10Sampes from normal bladder tissues were collected after operation.All of samples are stored in -80℃after bathing by water.RT-RCR kit are obtained from Takara Shuzo Co.Ltd.c-FLIP-F,c-FLIP-R and B-actin primer are obtained from Takara;TRIZOL from GEBCOBRL.Expression levels of c-FLIP mRNA were determined by RT-PCR,Total RNA was refine according to kit directions,RNA concentration and purity were tested by Ultraviolet spectrum,the samples OD260/OD280 was larger than 1.575.2ul of RNA were used for each 20-ul RT-cDNA reaction.The reaction under the following condition:65℃for 1min,30℃for 5min,65℃for 30min,98℃for 5min,5℃for 5min.
For c-FLIP mRNA detection,3ul of RT-cDNA product were amplified using the primers c-FLIP -F 5'-GTT CTT CGG GAC ACC TTC AGT-3' and c-FLIP -R 5'- TAT CCA CGC CAA ACA CGC TCT -3'.Each 25-ul reaction contained dd.H_2O17.1ul 10×buffer 2.5ul dNTPs 2ul Taq-E 0.2ul hTERT-F 0.1ul hTERT-R 0.1ul.each cycling conditions were as follows:94℃for 3min,32cycles of 94℃45s,57.6℃1min,and 72℃1min,followed by one cycle of 72℃for 7min.The primers of B-actine as inside contrast take part in the reaction.
Result
There were over expression of c-FLIP mRNA in bladder urothelial carcinomas tissues.The positive rate in the bladder urothelial carcinomas tissues was distinctively different from the normal bladder urothelial tissues(P＜0.05).There was no correlation between positive case and clinical stage,pathology grade.The survival rate in c-FLIP mRNA overexpression case was significantly lower than that of the low expression case(p＜0.05).
Discussion
A caspase-like protein termed c-FLIP(cellular FADD-like interleukin-1β-converting enzyme inhibitory protein)impairs the Fas-mediated cell death signal. c-FLIP is expressed mainly in a long(c-FLIP_L)form and a short spliced form(c-FLIPs). The short form consists of exclusively two tandem repeats of death effector domain (DED),which inhibits procaspase-8 activation.The longer isoform contains two DED in its NH_2-terminal region and shares extensive homology with the caspase-8 catalytic domain in its COOH-terminal region.However,c-FLIP_L sequence lost the conserved Cys residue in the catalytic site and a closely positioned His residue,which are essential for the caspase-8 catalytic activity.
Fas is a transmembrane receptor that belongs to the tumor necrosis factor(TNF) receptor(TNFR)superfamily.Its cognate ligand,FasL,is a transmembrane protein found in a soluble trimeric form after cleavage by metalloproteinases.This soluble homotrimeric FasL complex is not able to induce cell death unless it is produced as a homohexameric complex.
Fas carries an intracellular conserved stretch of 80 amino acids called the death domain,which serves as a docking platform to trigger an intracellular signal.Indeed,on binding of FasL or multivalent agonistic antibodies,the Fas death domain recruits the adaptor molecule Fas-associated death domain protein(FADD),which in turn aggregates the initiator caspase-8 and caspase-10.This intracellular complex is called death-inducing signaling complex(DISC).The fact that caspases are brought together in close vicinity leads to their autocleavage and activation of the downstream apoptotic signal.Fas-mediated apoptosis can be determined by a critical threshold of Fas stimulation or by the activation of specific caspases.The caspases are synthesized as inactive proenzymes and are activated by proteolytic cleavage of aspartic residues. Cellular FLICE inhibitory protein(FLIP)inhibits caspase 8 activity,and,therefore, inhibits Fas-mediated apoptosis.Alternative splicing leads to long(55 kD)and short form(28 kD)variants of FLIPs,both of which can interact with FADD and caspase 8 via DED motifs,and can be recruited to the Fas DISC.Therefore,FLIP has been implicated in the regulation of the Fas pathway during the initial phase of apoptosis.
To explore the expression of c-FLIP in bladder transitional cell carcinoma and its' clinical significance,in this study,the expression levels of c-FLIP were evaluated by using RT-RCR in 30 bladder transitional cell.carcinoma tissues,10 normal bladder tissues.We analyzed the gene expression of c-FLIP in 30 bladder transitional cell carcinoma differing in stage and grade,and in 10 normal bladder tissues.There were over expression of c-FLIP mRNA in bladder transitional cell carcinoma whereas normal bladder tissues were all negative.The positive rate in the bladder transitional cell carcinoma was ditinctively different from the normal bladder tissues(p＜0.05).
Our data indicate that c-FLIP mRNA levels do not correlate with clinical stage、pathology grade in bladder transitional cell carcinoma.The reason is that a number of factors,including both cell mutation and gene abnormal expression、inactivation of suppressor gene and elimination of printing gene etc,can be invoke to explain the lack of relationship between them.
All the patients were followed up range 3.6 to 59.5 months.The survival rate in c-FLIP overexpression case was significantly lower than that of the lowexpression case (p＜0.05).The overexpression of c-FLIP mRNA was significantly higher in the bladder transitional cell carcinoma.It might serve as a prognostic marker for estimating the biologic characteristics of the bladder transitional cell carcinoma.
In conclusion,our findings indicate that bladder tumor c-FLIP mRNA expression level correlates with outcome in patients with bladder transitional cell carcinoma.A larger study will be necessary to determine whether c-FLIP mRNA expression is predictive of outcome independent of clinical stage、pathology grade in bladder transitional cell carcinoma.
Conclusion
The overexpression of c-FLIP mRNA was significantly higher in bladder urothelial carcinomas.It might serve as a prognostic marker for estimating the biologic characteristics of the bladder urothelial carcinomas.
膀胱癌在中国是最常见的泌尿系统恶性肿瘤之一。其组织病理类型以移行细胞癌(transitional cell carcinoma,TCC)为主,约占90%以上。对膀胱肿瘤的病因研究很多,但病因仍不清楚。目前学者们普遍认为膀胱肿瘤的发生、发展是一个复杂的多因素、多步骤、多变化的过程,是细胞凋亡增殖失衡的结果。
细胞型Fas相关死亡区域蛋白样白介素-1β转换酶抑制蛋白(cellularFADD-like interleukin-1β-converting enzyme inhibitory protein c-FLIP)是近年来发现的一类含有死亡效应结构域(death effect domains,DED)的凋亡抑制蛋白,已证实c-FLIP与炎症、肿瘤、自身免疫性疾病的发生发展有关。因此对c-FLIP的进一步研究将有助于深化对这些疾病的认识,并为临床治疗这些疾病提供新的方法和思路。为进一步明确膀胱移行细胞癌发生发展过程中c-FLIP所起到的作用,并且进一步探讨其对膀胱肿瘤预后的影响,我们采用免疫组化及半定量RT-PCR方法探讨c-FLIP mRNA及其蛋白在膀胱移行细胞癌中的表达情况。探讨c-FLIP mRNA的表达与膀胱移行细胞癌的临床分期、病理组织分级及预后之间的关系,现将结果报导如下。
材料与方法
一、临床资料
(一)一般资料
收集2002年4月至2005年10月,我科手术治疗膀胱移行细胞癌患者100例。从中随机选出30例,男23例,女7例,年龄在20岁—80岁。按临床分期标准:T_a期6例,T_1期8例,T_2期9例,T_3期7例。按病理分级标准:Ⅰ级4例、Ⅰ-Ⅱ级12例、Ⅱ级5例、Ⅱ-Ⅲ级5例、Ⅲ级4例,同时采集10例正常膀胱移行上皮组织,年龄46岁—61岁。取材后,置入-80℃低温冰箱保存备检。
(二)实验试剂和仪器
免疫组化试剂盒购于北京博奥森生物技术公司、c-FLIP兔抗人抗体(一抗)购于北京博奥森生物技术有限公司。其中试剂盒中包括:3%H_2O_2去离子水、封闭用正常山羊血清工作液、生物素化二抗工作液,为生物素标记的山羊抗兔LgG、辣根酶标记链霉卵白素工作液。RT—PCR试剂盒购于Takara Shuzo Co.Ltd.;c-FLIP—F,c—FLIP—R及β-actin引物购于Takara公司;TRIZOL试剂购于GIBCOBRL公司;超净工作台、石蜡切片机(上海);聚乙烯探子(北京);TG328B光电分析天平(上海);低温冰箱及超低温冰箱(日本SANYO公司);自动电泳凝胶成像分析仪为美国syngene公司生产;PCR扩增仪PTC—100~(TM)为美国公司生产。
二、方法
免疫组化方法采用链霉菌抗生物素蛋白-过氧化物酶连结(SP)法:取石蜡切片,烤片,68℃,20分钟,常规二甲苯脱蜡,梯度酒精脱水;二甲苯Ⅰ20min—二甲苯Ⅱ20 min—100%酒精Ⅰ10min—100%Ⅱ10min—95%5min—80%5min—70%5min;阻断灭活内源性过氧化物酶:3%H2O2 37℃孵育10min,PBS冲洗3X5min;抗原修复:置0.01M枸橼酸缓冲液(PH 6.0)中用煮沸(95℃,15—20min),自然冷却20min以上,再用冷水冲洗缸子,加快冷却至室温,PBS冲洗3X5min。正常羊血清工作液封闭,37℃10 min,倾去勿洗;滴加一抗4℃冰箱孵育过夜,PBS冲洗3X5min(用PBS缓冲液代替一抗作阴性对照);滴加生物素标记二抗,37℃孵育30min,PBS冲洗3X5min:滴加辣根过氧化物酶标记的链霉素卵白素工作液,37℃孵育30min,PBS冲洗3X5min;DAB/H2O2反应染色,自来水充分冲洗后苏木素复染,常规脱水,透明,干燥,封片。
RT-PCR方法:提取组织总RNA,按TROZOL抽提试剂盒说明进行,紫外分光光度仪检测RNA纯度及浓度。逆转录反应:在20ul逆转录反应体系中含总RNA(1ug/ul)1ul,Oligo(dT)_(15),引物0.5ul,25mM MgSo_42ul,10mMdNTPs 0.5ul,2×buffer 5ul,RNA Inhibitor 0.25ul,反转录酶(22U/ul)0.5ul,dd.H_2O 0.25ul。反应条件:65℃1min,30℃5min,15—30min匀速升温至65℃,65℃30min,98℃5min终止反应,5℃5min灭活反转录酶。扩增c-FLIP cDNA:取cDNA3ul,上下游引物各0.1ul,Taq DNA聚合酶0.2ul,加入PCR反应体系中扩增,反应条件:94℃变性3min,94℃45s,54.2℃1min,72℃1min 35cycls,72℃7min延伸。PCR产物分析:PCR产物经凝胶成像及分析系统扫描后,以β-actin为参照求出其相对值。基因表达值(%)=(c-FLIP/β-actin)×100。引物序列:c-FLIP F,5′—GTT CTT CGG GAC ACC TTC AGT—3′,R,5′—TAT CCA CGC CAAACA CGC TCT—3。β-actin F,5′—GTG GGG CGC CCC AGG CAC CA—3′R,5′—CTC CTT AAT GTC ACG CAC GAT TTC—3′
三、结果判定及统计学处理
c-FLIP蛋白染色为棕黄色颗粒,定位于细胞浆内。每张切片随机选择观察10个高倍视野,每个高倍视野计数100个细胞并计算平均数,c-FLIP的表达以着色肿瘤细胞平均数大于10%为阳性引入标准。
RT-PCR结果采用t检验,P＜0.05有统计学意义。在预后的研究过程中主要探讨c-FLIP mRNA表达与患者术后无病生存率DFS(disease free survival)之间的关系。DFS定义为自随访之日起至患者出现复发灶,或因该病发生死亡的时间。采用Kaplan—meier进行单因素分析,COX风险模型进行多因素分析。所有的统计计算应用spss13.0软件。
结果
一、c-FLIP蛋白在膀胱移行细胞癌中的表达。
c-FLIP蛋白呈棕黄色颗粒,定位于胞浆。膀胱癌组织中FLIP蛋白的阳性率为76.77%(23/30),正常膀胱移行上皮c-FLIP蛋白表达为阴性。
二、c-FLIP mRNA在膀胱移行细胞癌中的表达。
RT-PCR产物经2%琼脂糖凝胶电泳后,紫外透射仪下可见到扩增的特异产物条带,目的基因片段产物与引物设计目的片段大小完全吻合。30例癌组织标本中19例呈阳性表达,阳性率63.3%,10例对照均呈阴性表达。
三、c-FLIP mRNA在膀胱移行细胞癌组织及正常膀胱上皮组织中的检测结果。
显示膀胱移行细胞癌组织及正常膀胱上皮组织中c-FLIP mRNA表达结果,两者比较,差异显著(P＜0.05)。
四、c-FLIP mRNA的表达与临床分期、病理组织分级之间的关系。
显示膀胱移行细胞癌组织中c-FLIP mRNA表达与其临床分期、病理分级之间差异不显著(P＞0.05)。
五、c-FLIP mRNA的表达与膀胱移行细胞癌预后之间关系。
在膀胱移行细胞癌的预后过程中,癌组织的临床分期与病理组织分级及单发或多发对患者的术后无病生存率DFS无显著性影响,(P＞0.05)。癌组织的c-FLIPmRNA的表达情况对患者的DFS存在显著性影响,RR=5.397,95%CI(1.148—25.377),P=0.033。提示c-FLIP mRNA呈阳性表达的患者术后无病生存率较低,预后不好。
结论
1、c-FLIP蛋白与c-FLIP mRNA在膀胱移行细胞癌中呈一致高表达。
2、c-FLIP mRNA在膀胱移行细胞癌中高表达,这一特征使之成为较有潜力的肿瘤标志物。
3、膀胱移行细胞癌c-FLIP mRNA的表达情况对患者的无病生存率存在显著性影响,提示c-FLIP mRNA呈阳性表达的患者术后无病生存率较低,预后不好。
Preface
Bladder cancer is a kind of malignant tumor frequently occurred in urinary system. In china,the occurrence of bladder cancer is 3-4 times more frequent in men than in women.Transitional cell carcinoma(TCC)is by far the most frequent histotype, although the distribution of histotypes varies in different populations.In Chinese people,90%of all bladder cancers were TCC.Most bladder carcinogens exert their action by direct contact with the bladder epithelium.Inhaled or ingested compounds, which are either directly carcinogenic or which can be transformed by the body in carcinogenic byproducts,are excreted via the urine,and in this way they reach the urinary bladder.There is also evidence that chronic inflammation,caused either by infections or stones,has a role in the promotion of bladder cancer.
A caspase-like protein termed c-FLIP(cellular FADD-like interleukin-1β-converting enzyme inhibitory protein)impairs the Fas-mediated cell death signal. c-FLIP is expressed mainly in a long(c-FLIPL)form and a short spliced form (c-FLIPS).The short form consists of exclusively two tandem repeats of death effector domain(DED),which inhibits procaspase-8 activation.The longer isoform contains two DED in its NH2-terminal region and shares extensive homology with the caspase-8 catalytic domain in its COOH-terminal region.However,c-FLIPL sequence lost the conserved Cys residue in the catalytic site and a closely positioned His residue,which are essential for the caspase-8 catalytic activity.
To explore the expression of c-FLIP in bladder transitional cell carcinoma and its'clinical significance,in this study,the expression levels of c-FLIP were evaluated by using RT-PCR in 30 bladder transitional cell carcinoma tissues,10 normal bladder tissues,Its correlations with the clinical stage,pathology type,prognosis were evaluated in positivecase.
Method
30 bladder cancer samples were Collected which are setected by operation in our university from 2002 to 2005,These samples were graded according to WHO/ISUP1998,which is 4gradeⅠ、12gradeⅠ-Ⅱ、5gradeⅡ、5stageⅡ-Ⅲ、4gradeⅢ.TNM staged were confirmed among these sample that is 6T_a、8T_1、9T_2, 7T_3.10Sampes from normal bladder tissues were collected after operation.All of samples are stored in -80℃after bathing by water.RT-RCR kit are obtained from Takara Shuzo Co.Ltd.c-FLIP-F,c-FLIP-R and B-actin primer are obtained from Takara;TRIZOL from GEBCOBRL.Expression levels of c-FLIP mRNA were determined by RT-PCR,Total RNA was refine according to kit directions,RNA concentration and purity were tested by Ultraviolet spectrum,the samples OD260/OD280 was larger than 1.575.2ul of RNA were used for each 20-ul RT-cDNA reaction.The reaction under the following condition:65℃for 1min,30℃for 5min,65℃for 30min,98℃for 5min,5℃for 5min.
For c-FLIP mRNA detection,3ul of RT-cDNA product were amplified using the primers c-FLIP -F 5'-GTT CTT CGG GAC ACC TTC AGT-3' and c-FLIP -R 5'- TAT CCA CGC CAA ACA CGC TCT -3'.Each 25-ul reaction contained dd.H_2O17.1ul 10×buffer 2.5ul dNTPs 2ul Taq-E 0.2ul hTERT-F 0.1ul hTERT-R 0.1ul.each cycling conditions were as follows:94℃for 3min,32cycles of 94℃45s,57.6℃1min,and 72℃1min,followed by one cycle of 72℃for 7min.The primers of B-actine as inside contrast take part in the reaction.
Result
There were over expression of c-FLIP mRNA in bladder urothelial carcinomas tissues.The positive rate in the bladder urothelial carcinomas tissues was distinctively different from the normal bladder urothelial tissues(P＜0.05).There was no correlation between positive case and clinical stage,pathology grade.The survival rate in c-FLIP mRNA overexpression case was significantly lower than that of the low expression case(p＜0.05).
Discussion
A caspase-like protein termed c-FLIP(cellular FADD-like interleukin-1β-converting enzyme inhibitory protein)impairs the Fas-mediated cell death signal. c-FLIP is expressed mainly in a long(c-FLIP_L)form and a short spliced form(c-FLIPs). The short form consists of exclusively two tandem repeats of death effector domain (DED),which inhibits procaspase-8 activation.The longer isoform contains two DED in its NH_2-terminal region and shares extensive homology with the caspase-8 catalytic domain in its COOH-terminal region.However,c-FLIP_L sequence lost the conserved Cys residue in the catalytic site and a closely positioned His residue,which are essential for the caspase-8 catalytic activity.
Fas is a transmembrane receptor that belongs to the tumor necrosis factor(TNF) receptor(TNFR)superfamily.Its cognate ligand,FasL,is a transmembrane protein found in a soluble trimeric form after cleavage by metalloproteinases.This soluble homotrimeric FasL complex is not able to induce cell death unless it is produced as a homohexameric complex.
Fas carries an intracellular conserved stretch of 80 amino acids called the death domain,which serves as a docking platform to trigger an intracellular signal.Indeed,on binding of FasL or multivalent agonistic antibodies,the Fas death domain recruits the adaptor molecule Fas-associated death domain protein(FADD),which in turn aggregates the initiator caspase-8 and caspase-10.This intracellular complex is called death-inducing signaling complex(DISC).The fact that caspases are brought together in close vicinity leads to their autocleavage and activation of the downstream apoptotic signal.Fas-mediated apoptosis can be determined by a critical threshold of Fas stimulation or by the activation of specific caspases.The caspases are synthesized as inactive proenzymes and are activated by proteolytic cleavage of aspartic residues. Cellular FLICE inhibitory protein(FLIP)inhibits caspase 8 activity,and,therefore, inhibits Fas-mediated apoptosis.Alternative splicing leads to long(55 kD)and short form(28 kD)variants of FLIPs,both of which can interact with FADD and caspase 8 via DED motifs,and can be recruited to the Fas DISC.Therefore,FLIP has been implicated in the regulation of the Fas pathway during the initial phase of apoptosis.
To explore the expression of c-FLIP in bladder transitional cell carcinoma and its' clinical significance,in this study,the expression levels of c-FLIP were evaluated by using RT-RCR in 30 bladder transitional cell.carcinoma tissues,10 normal bladder tissues.We analyzed the gene expression of c-FLIP in 30 bladder transitional cell carcinoma differing in stage and grade,and in 10 normal bladder tissues.There were over expression of c-FLIP mRNA in bladder transitional cell carcinoma whereas normal bladder tissues were all negative.The positive rate in the bladder transitional cell carcinoma was ditinctively different from the normal bladder tissues(p＜0.05).
Our data indicate that c-FLIP mRNA levels do not correlate with clinical stage、pathology grade in bladder transitional cell carcinoma.The reason is that a number of factors,including both cell mutation and gene abnormal expression、inactivation of suppressor gene and elimination of printing gene etc,can be invoke to explain the lack of relationship between them.
All the patients were followed up range 3.6 to 59.5 months.The survival rate in c-FLIP overexpression case was significantly lower than that of the lowexpression case (p＜0.05).The overexpression of c-FLIP mRNA was significantly higher in the bladder transitional cell carcinoma.It might serve as a prognostic marker for estimating the biologic characteristics of the bladder transitional cell carcinoma.
In conclusion,our findings indicate that bladder tumor c-FLIP mRNA expression level correlates with outcome in patients with bladder transitional cell carcinoma.A larger study will be necessary to determine whether c-FLIP mRNA expression is predictive of outcome independent of clinical stage、pathology grade in bladder transitional cell carcinoma.
Conclusion
The overexpression of c-FLIP mRNA was significantly higher in bladder urothelial carcinomas.It might serve as a prognostic marker for estimating the biologic characteristics of the bladder urothelial carcinomas.
引文
1 Thorburn A. Death receptor-induced cell killing. Cell Signal. 2004 Feb;16 (2):139-44.
2 Elmore S. Apoptosis: a review of programmed cell death. Toxicol Pathol. 2007;35 (4):495-516.
3 Mouzakiti A, Packham G. Regulation of tumour necrosis factor-related apoptosis -inducing ligand (TRAIL)-induced apoptosis in Burkitt's lymphoma cell lines._Br J Haematol. 2003 Jul;122(1):61-9.
4 Irmler M, Thome M, Hahne M, et al. Inhibition of death receptor signals by cellular FLIP.Nature. 1997 Jul 10;388(6638):190-5.
5 Kataoka T. The caspase-8 modulator c-FLIP. Crit Rev Immunol. 2005;25(1):31-58.
6 Peter ME, Krammer PH. The CD95(APO/Fas) DISC and beyond. Cell Death Differ.2003; 10(1):26-35.
7 Lavrik I, Golks A, Krammer PH.Death receptor signalingJ Cell Sci.2005;118 (Pt2): 265-267.
8 Igney FH,Krammer PH.Death and anti-death:tumor resistance to apoptosis. Nat Rev Cancer.2002 ;2(4):277-288.
9 Thome M,Tschopp J.Regulation of lymphocyte proliferation and death by FLIP. Nat Rev Immunol.2001;1(1):50-58.
10 Tourneur L,Mistou S,Michiels FM, et al.Loss of FADD protein expression results in a biased Fas signaling pathway and correlates with the development of tumoral status in thyroid follicular cells. Oncogene.2003;22(18):2795-2804.
11 Bullani RR, Huard B, Viard Leveugle I, et al. Selective expression of FLIP in malignant melanocytic skin lesions. J Invest Dermatol.2001;117(2):360-364.
12 Xiao C, Yang BF, Song JH, et al. Inhibition of CaMKII-mediated c-FLIP expression sensitizes malignant melanoma cells to TRAIL-induced apoptosis. Exp Cell Res. 2005 Mar 10;304 (1):244-55. Epub 2004 Nov 26.
13 Zhou XD, Yu JP, Liu J, et al. Overexpression of cellular FLICE-inhibitory protein (FLIP) in gastric adenocarcinoma. Clin Sci (Lond). 2004 Apr; 106(4) :3 97-405.
14 Chen HX, Liu YJ, Zhou XD, et al. Expression of cellular FLICE/caspase-8 inhibitory protein is associated with malignant potential in endometrial carcinoma. Int J Gynecol Cancer. 2005 Jul-Aug;15(4):663-70.
15 Korkolopoulou P, Saetta AA, Levidou G, et al. c-FLIP expression in colorectal carcinomas: association with Fas/FasL expression and prognostic implications. Histopathology. 2007 Aug;51(2): 150-6. Epub 2007 Jun 8.
16 Korkolopoulou P, Goudopoulou A, Voutsinas G, et al. c-FLIP expression in bladder urothelial carcinomas: its role in resistance to Fas-mediated apoptosis and clinicopathologic correlations. Urology. 2004 Jun;63(6): 1198-204.
1 Irmler M, Thome M, Hahne M, et al. Inhibition of death receptor signals by cellular FLIP. Nature, 1997 ,388 (6638):190-195.
2 Peter ME, Krammer PH. The CD95(APO-1 /Fas) DISC and bevond. Cell Death Differ,2003, 10(1);26-35.
3 Rasper DM, Vaillancourt JP, HadanoS,et al.Cell death attenuation by'Usurpin', a mammalian DED-caspase homologue that precludes caspase-8 recruitment and activation by the CD95 (Fas,APO-1) receptor complex[J]. Cell Death Differ, 1998, 5(4): 271-288.
4 Golks A, Brenner D, Fritsch C,et al. C-FLIPR-a new regulator of death receptor-induced apoptosis. J Biol Chem, 2005, 280(15):14507-14513.
5 Lavrik 1, Golks A, Krammer PH. Death receptor signaling. J Cell Sci,2005,l 18(Pt2):265-267.
6 Sharp DA, Lawrence DA, Ashkenazi A. Selective knockdown of the long variant of cellular FLICE-inhibitory protein augments death receptor-mediated caspase-8 activation and apoptosis. J Biol Chem,2005,280(19):19401-19409.
7 Lacour S, Micheau 0, Hammann A, et al. Chemotherapy enhances TNF-related apoptosis-inducing ligand DISC assembly in HT29 human colon cancer cells. Oncogene, 2003,22( 12):1807—1816.
8 Thome M, Tschopp J. Regulation of lymphocyte proliferation and death, by FLIP. Nat Rev Immunol,2001, l(I);50-58.
9 Igney FH, Krammer PH. Death and anti-death: tumour resistance to apoptosis. Nat Rev Cancer,2002,2 (4) :277-288.
10 Muzio M, Chinnaiyan AM, Kischkel FC,et al. FLICE,a novel FADD-homologous ICE/CED-3-like protease,is recruited to the CD95(Fas/Apo-l)death inducing signalliing complex(DISC)[J] .Cell, 1996,85:817.
11 MacFarlane M. TRAIL-induced signalling and apoptosis. Toxicol Let, 139:89-97,2003.
12 MacFarlane M, Merrison W, Dinsdale D, Cohen GM. Active caspases andcleaved cytokeratins are sequestered into cytoplasmic inclusions in TRAIL-induced apoptosis. J Cell Biol, 148: 1239-1254,2000.
13 Hussein MR, Haemel AK, Wood GS. Apoptosis and melanoma: molecular mechanisms. J Pathol, 199:275-288, 2003.
14 KimY, SuhN, SpornM,et al.An inducible pathway for degradation of FLIP protein sensitizestumor cellstoTRAIL-induced apoptosis[J]. J Bi-ol Chem,2002,277(25):22320-22329.
15 Yen WC, [tie A, Elia AJ, et al. Requirement for Casper(。-FLIP)in regulation of death receptor-induced apoptosis and embryonic development. Immunity, 2000 ,12 (6) :633-642.
16 Micheau O, Lens S, Gaide O, et al. NF-KB signals induce the expression of c-FLIP. Mol CeB Biol,2001,21(16):5299-5305.
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36 Zhou XD, Yu JP, Liu J, et al. Overexpression of cellular FLICE-inhibitory protein (FLIP) in gastric adenocarcinoma. Clin Sci (Lond). 2004 Apr;106(4):397-405.
37 Chen HX, Liu YJ, Zhou XD, et al. Expression of cellular FLICE/caspase-8 inhibitory protein is associated with malignant potential in endometrial carcinoma. Int J Gynecol Cancer. 2005 Jul-Aug;15(4):663-70.
38 Korkolopoulou P, Saetta AA, Levidou G, et al. c-FLIP expression in colorectal carcinomas: association with Fas/FasL expression and prognostic implications. Histopathology. 2007 Aug;51(2): 150-6. Epub 2007 Jun 8.
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2 Elmore S. Apoptosis: a review of programmed cell death. Toxicol Pathol. 2007;35 (4):495-516.
3 Mouzakiti A, Packham G. Regulation of tumour necrosis factor-related apoptosis -inducing ligand (TRAIL)-induced apoptosis in Burkitt's lymphoma cell lines._Br J Haematol. 2003 Jul;122(1):61-9.
4 Irmler M, Thome M, Hahne M, et al. Inhibition of death receptor signals by cellular FLIP.Nature. 1997 Jul 10;388(6638):190-5.
5 Kataoka T. The caspase-8 modulator c-FLIP. Crit Rev Immunol. 2005;25(1):31-58.
6 Peter ME, Krammer PH. The CD95(APO/Fas) DISC and beyond. Cell Death Differ.2003; 10(1):26-35.
7 Lavrik I, Golks A, Krammer PH.Death receptor signalingJ Cell Sci.2005;118 (Pt2): 265-267.
8 Igney FH,Krammer PH.Death and anti-death:tumor resistance to apoptosis. Nat Rev Cancer.2002 ;2(4):277-288.
9 Thome M,Tschopp J.Regulation of lymphocyte proliferation and death by FLIP. Nat Rev Immunol.2001;1(1):50-58.
10 Tourneur L,Mistou S,Michiels FM, et al.Loss of FADD protein expression results in a biased Fas signaling pathway and correlates with the development of tumoral status in thyroid follicular cells. Oncogene.2003;22(18):2795-2804.
11 Bullani RR, Huard B, Viard Leveugle I, et al. Selective expression of FLIP in malignant melanocytic skin lesions. J Invest Dermatol.2001;117(2):360-364.
12 Xiao C, Yang BF, Song JH, et al. Inhibition of CaMKII-mediated c-FLIP expression sensitizes malignant melanoma cells to TRAIL-induced apoptosis. Exp Cell Res. 2005 Mar 10;304 (1):244-55. Epub 2004 Nov 26.
13 Zhou XD, Yu JP, Liu J, et al. Overexpression of cellular FLICE-inhibitory protein (FLIP) in gastric adenocarcinoma. Clin Sci (Lond). 2004 Apr; 106(4) :3 97-405.
14 Chen HX, Liu YJ, Zhou XD, et al. Expression of cellular FLICE/caspase-8 inhibitory protein is associated with malignant potential in endometrial carcinoma. Int J Gynecol Cancer. 2005 Jul-Aug;15(4):663-70.
15 Korkolopoulou P, Saetta AA, Levidou G, et al. c-FLIP expression in colorectal carcinomas: association with Fas/FasL expression and prognostic implications. Histopathology. 2007 Aug;51(2): 150-6. Epub 2007 Jun 8.
16 Korkolopoulou P, Goudopoulou A, Voutsinas G, et al. c-FLIP expression in bladder urothelial carcinomas: its role in resistance to Fas-mediated apoptosis and clinicopathologic correlations. Urology. 2004 Jun;63(6): 1198-204.
1 Irmler M, Thome M, Hahne M, et al. Inhibition of death receptor signals by cellular FLIP. Nature, 1997 ,388 (6638):190-195.
2 Peter ME, Krammer PH. The CD95(APO-1 /Fas) DISC and bevond. Cell Death Differ,2003, 10(1);26-35.
3 Rasper DM, Vaillancourt JP, HadanoS,et al.Cell death attenuation by'Usurpin', a mammalian DED-caspase homologue that precludes caspase-8 recruitment and activation by the CD95 (Fas,APO-1) receptor complex[J]. Cell Death Differ, 1998, 5(4): 271-288.
4 Golks A, Brenner D, Fritsch C,et al. C-FLIPR-a new regulator of death receptor-induced apoptosis. J Biol Chem, 2005, 280(15):14507-14513.
5 Lavrik 1, Golks A, Krammer PH. Death receptor signaling. J Cell Sci,2005,l 18(Pt2):265-267.
6 Sharp DA, Lawrence DA, Ashkenazi A. Selective knockdown of the long variant of cellular FLICE-inhibitory protein augments death receptor-mediated caspase-8 activation and apoptosis. J Biol Chem,2005,280(19):19401-19409.
7 Lacour S, Micheau 0, Hammann A, et al. Chemotherapy enhances TNF-related apoptosis-inducing ligand DISC assembly in HT29 human colon cancer cells. Oncogene, 2003,22( 12):1807—1816.
8 Thome M, Tschopp J. Regulation of lymphocyte proliferation and death, by FLIP. Nat Rev Immunol,2001, l(I);50-58.
9 Igney FH, Krammer PH. Death and anti-death: tumour resistance to apoptosis. Nat Rev Cancer,2002,2 (4) :277-288.
10 Muzio M, Chinnaiyan AM, Kischkel FC,et al. FLICE,a novel FADD-homologous ICE/CED-3-like protease,is recruited to the CD95(Fas/Apo-l)death inducing signalliing complex(DISC)[J] .Cell, 1996,85:817.
11 MacFarlane M. TRAIL-induced signalling and apoptosis. Toxicol Let, 139:89-97,2003.
12 MacFarlane M, Merrison W, Dinsdale D, Cohen GM. Active caspases andcleaved cytokeratins are sequestered into cytoplasmic inclusions in TRAIL-induced apoptosis. J Cell Biol, 148: 1239-1254,2000.
13 Hussein MR, Haemel AK, Wood GS. Apoptosis and melanoma: molecular mechanisms. J Pathol, 199:275-288, 2003.
14 KimY, SuhN, SpornM,et al.An inducible pathway for degradation of FLIP protein sensitizestumor cellstoTRAIL-induced apoptosis[J]. J Bi-ol Chem,2002,277(25):22320-22329.
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