p120ctn对Kaiso的调控及两者在肺癌中作用的研究
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摘要
作为Armadillo蛋白亚家族中的重要成员之一的p120-catenin(p120ctn)一直被认为是E-cadherin/catenin黏附复合物的重要调节因子。同时,p120ctn也是小GTP酶的重要调节因子,它可以在细胞浆中通过调节小GTP酶的活性状态来调控细胞骨架的装配,它在细胞黏附以及肿瘤侵袭和转移方面的作用已经受到人们的广泛重视。大量研究表明,p120ctn的异常表达与肿瘤的发生、侵袭和转移密切相关。随着研究的深入,特别是核转录因子Kaiso的发现,人们发现p120ctn还可以作为一种信号分子穿梭于核/浆之间,通过影响核转录因子Kaiso调控基因表达。
Kaiso是BTB/POZ蛋白家族的重要成员,其氨基端有一个POZ/BTB结构域,羧基端有三个C-2H-2锌指结构。氨基端POZ/BTB结构域的功能是:(一)Kaiso分子之间形成同二聚体;(二)与其他蛋白分子(如CTCF、HDAC等)形成异二聚体。羧基端锌指结构的功能是:(一)与一些肿瘤相关基因的启动子区域(如matrilsin、CDKN2A等)结合,抑制这些基因的表达;(二)与p120ctn结合。p120ctn是Kaiso最重要的结合蛋白,在特定条件下,它能够阻断Kaiso对下游基因的抑制,从而调控这些基因的表达。
Kaiso在肿瘤中发挥的作用还很不清楚。不同研究小组得出的结论甚至严重冲突,有研究认为Kaiso可能是一个抑癌因子,还有研究则得出了完全相反的结论。故Kaiso是促癌因子还是抑癌因子目前还没有定论,有待进一步阐明。
与p120ctn类似,转录因子Kaiso也能够在核/浆之间穿梭,但它在细胞核、细胞浆中是否都发挥着同样的功能还有待研究。此外,Kaiso核浆穿梭的调控机制还不是十分清楚。有研究表明肿瘤微环境影响了Kaiso的核浆分布,而p120ctn并不参与Kaiso的核浆穿梭过程;而另有研究却给出了相反的实验证据,即p120ctn在细胞浆中过表达后,Kaiso也与p120ctn共定位于细胞浆。因此,p120ctn究竟能否调控Kaiso的核浆穿梭还有待进一步研究。
曾有学者利用免疫组织化学的方法检测了多种人类良恶性组织(均为小样本)中Kaiso的表达情况,结果发现:Kaiso在多种实体恶性肿瘤中以细胞浆为主,而在体外培养的多种细胞系(如MDCK、NIH3T3、HT29、SW48细胞等),Kaiso以细胞核表达为主。由于即使是处于进展期的肿瘤组织,也只是少数细胞处于增殖阶段(S期+G2期),肿瘤体积的增大是由于肿瘤细胞凋亡减少造成的,而体外培养的细胞在一段时间内会有大量的细胞处于增殖期。因此,我们推测Kaiso在核或浆的分布,很有可能还与细胞周期有关。当细胞处于增殖阶段时,Kaiso入核。
本研究目的旨在探讨肺癌组织中p120ctn和Kaiso的表达情况及与临床病理学参数意义、肺癌患者预后的关系。在细胞水平上,应用shRNA干扰技术研究Kaiso的核内功能,通过稳定干扰和过表达p120ctn的表达,分析p120ctn亚型蛋白1和3对Kaiso表达和定位的影响。运用细胞周期同步化技术探讨细胞周期对Kaiso核浆分布的影响。
方法
1、肺癌组织
原发性肺癌标本及配对的淋巴结转移癌均来自中国医科大学第一附属医院外科1998-2005年手术切除的标本。所有患者术前均未接受放、化疗。其中部分患者保存有完整、详实的预后资料。标本经4%中性甲醛固定,石蜡包埋,HE常规染色后明确诊断。其中部分癌旁肺组织(远离肿瘤至少5厘米)和淋巴结转移癌离体后立即放入液氮后-70℃冰箱保存备用。
2、免疫组织化学染色及结果判定
标本用中性福尔马林溶液固定,石蜡包埋,制成4μm切片,经脱蜡,脱苯,水化后,采用链菌素抗生物素蛋白-过氧化物酶免疫组化法(S-P法)检测Kaiso和p120ctn蛋白表达。单克隆抗体Kaiso和p120ctn 4℃孵育过夜。以癌旁肺支气管粘膜上皮细胞着色强度和定位为内对照,PBS缓冲液代替一抗为阴性对照。结果判定:高倍视野(×400)下选阳性信号最强区域计数200个肿瘤细胞中阳性细胞数,按Kaiso表达百分率分为以下四个等级:<25%为0分,26%-50%为1分,51%-75%为2分,>75%为3分。根据免疫组化染色强度分为三个等级:浅黄色计为1分,棕黄色计为2分,黄褐色计为3分。以阳性细胞率和染色强度的分值乘积作为每一例的积分,积分为0和1分者判定为阴性,积分≥2为阳性。当Kaiso核阳性信号≥5%时定为核阳性表达。p120ctn正常表达的阳性信号定位于细胞膜,表现为膜着色清楚、连续且阳性细胞数≥90%;膜表达下降表现为膜着色淡且不连续,阳性细胞数<90%;而超过10%的肿瘤细胞胞浆出现阳性信号定为异位表达。p120ctn的异位表达和膜表达下降统归为p120ctn异常表达。
3、间接免疫荧光检测
固定后的冰冻组织切片和细胞爬片用0.2%Triton X-100处理15分钟,非免疫动物血清封闭,一抗为单克隆抗体小鼠抗人Kaiso和小鼠抗人p120ctn 4℃孵育过夜;二抗为异硫氰酸荧光素(FITC)标记或罗丹明标记(TRITC)标记的山羊抗小鼠IgG,碘化丙啶或DAPI进行核复染。50%缓冲甘油封片,荧光显微镜Olympus IX51于波长为527nm处观察罗丹明荧光强度,于372nm波长处观察DAPI的荧光染色观察并照相。阴性对照实验:用等量的0.01mol/LPBS代替一抗。
4、Western BlOt
将含80μg总蛋白的裂解产物进行SDS-PAGE电泳后,转印至PVDF膜,单克隆抗体Kaiso或p120ctn4~C孵育过夜,辣根过氧化物酶标记二抗37℃孵育2小时后DAB或ECL显色。
5、RT-PCR
采用Trizol~TM试剂提取肺癌组织或培养细胞的总RNA,利用RNA PCR Kit(AMV)Ver.3.0试剂盒进行反转录。分别扩增Kaiso、p120ctn和matrilysin,以β-actin为内参。扩增产物经1.5%琼脂糖凝胶电泳后,成像分析。
6、细胞培养
在37℃,含5%CO_2的培养箱内培养人类肺癌细胞系BE1、SPC-A-1、LTEP-A-2和A549细胞系,培养基分别为含10%灭活胎牛血清的RPMI 1640或高糖DMEM,贴壁生长,每2-3天胰酶消化传代一次。
7、质粒构建与转染
shRNA干扰质粒导入感受态宿主细菌E.coli中扩增、纯化,使用Arrest-In~(TM)Transfection Reagent转染试剂将质粒分别转染至肺癌细胞系LTEP-A-2、SPC-A-1和BE1中,通过Western Blot和RT-PCR鉴定转染效果。
将含有目的基因的载体导入感受态宿主细菌DH5a中扩增、纯化,测序验证后,使用Lipofect 2000转染试剂将质粒分别转染至肺癌细胞系A549中,并以G418筛选出阳性细胞克隆,通过Western Blot和RT-PCR鉴定转染效果后,挑取转染成功的单克隆细胞在含有G418的RPMI 1640或高糖DMEM培养液中继续培养。
8、体外基质胶侵袭实验检测细胞侵袭能力
按照BD公司说明操作,取100μl细胞悬液(5×10~6个/m1)滴入上室内,下室加入含有10%胎牛血清的RPMI 1640培养液,上下室之间用孔径为8μm的聚碳酸酯微孔滤膜分开,将小室置37℃,5%CO_2条件下放置后,弃去上室内液体,擦尽膜上Matrigel胶,100%甲醇固定30分钟,常规苏木素染色,光镜下计数细胞数。
9、四甲基偶氮唑盐(tetrazolium,MTT)法检测细胞增殖能力
取对数生长期的肺癌细胞悬液(细胞密度3×10~5个/ml)分别接种于4个96孔板培养板,每板分6组:空白组(A)(B),干扰对照组(C)、干扰组(D)、抗体抑制对照组(E)和抗体移植组(F)。培养第24、48、72小时分别加入MTT(5mg/m1)20μl,继续孵育4小时后,每孔加入150μlDMSO溶解结晶,选择波长490nm,在酶联免疫(ELISA)检测仪上测定各孑L吸光度。实验重复3次。绘制细胞生长曲线。
10、核浆蛋白分离
按照Thermo公司说明操作,取一定体积(100mg)的组织或者10×10~6。的细胞,依次加入体积比为200:11:100的试剂CERⅠ,CERⅡ和NER,分别提取细胞浆蛋白和细胞核蛋白。检测a-tubulin和Lamin B 1在两种细胞组分中的表达以验证核浆蛋白分离效果。
11、免疫共沉淀
提取的蛋白中加入4μg的诱饵蛋白抗体,充分混匀后4℃孵育2小时后加入25μl Protein G Microbeads,混匀后继续4℃孵育1小时。按照操作说明步骤,混合液过μ柱,SDS上样缓冲液冲洗μ柱,收集免疫共沉淀产物。
12、细胞周期同步化
GO期同步化(血清饥饿法):指数生长期的肺癌细胞,培养至汇合密度为30%时收集细胞,1000rpm离心5分钟,PBS洗2次后铺板接种,换成无血清的RPMI-1640/DMEM培养液,继续培养36小时后用于检测。
S期同步化(胸苷双阻断法):指数生长期的肺癌细胞,培养液中加入胸苷至终浓度2mmol/L,继续培养18小时。以1000rpm离心5小时,弃上清,用PBS清洗3次,去除胸苷阻滞。将细胞重新悬浮成细胞悬液,接种到培养瓶中继续培养16小时。培养液中再加入胸苷,使之终浓度为2mmol/L,继续培养12小时。用0.25%胰蛋白酶溶液消化后,换新鲜培养液,1000rpm离心,预热Hanks液清洗2次后接种入另一培养瓶内,继续培养2小时后用于检测。
13、流式细胞仪检测细胞周期
收集对数生长期细胞制成1×10~7个/ml细胞悬液。取1ml细胞悬液75%冷乙醇于4℃固定、离心后制成500μl的细胞悬液,加碘化丙啶染液于4℃孵育45分钟后上机检测,ModFitLT3.O软件分析细胞周期。
14、统计分析
各组资料利用SPSS for Window 13.0进行统计分析。p<0.05有统计学意义。
结果
1、Kaiso主要表达于肺癌组织细胞浆内并与肺癌患者不良预后相关
Kaiso在癌旁肺支气管上皮细胞中存在微量的浆表达,但按照我们制定的阳性标准判定为阴性表达。294例肺癌组织标本中Kaiso蛋白浆阳性表达187例,占63.61%,明显高于癌旁支气管上皮Kaiso的表达水平(p<0.005)。Western blot检测结果证实,肺癌组织中Kaiso蛋白的表达量要显著高于原发灶中Kaiso蛋白的表达量(t=10.610,n=20,p=0.000)。Kaiso浆阳性表达与TNM分期和淋巴结转移密切相关(均p<0.05),与肺癌患者的性别、年龄、分化程度、组织学类型均无明显相关性。III+IV期Kaiso浆阳性表达率(70.42%)显著高于I+II期(57.24%)(ρ=0.019),有淋巴结转移的肺癌病例Kaiso浆阳性表达率(71.2%,116/163)显著高于没有淋巴结转移的肺癌病例(28.8%,47/131)(ρ=0.003);原发灶和淋巴结转移癌的配对标本中,淋巴结转移癌中Kaiso的浆阳性率(90.9%)显著高于原发灶的Kaiso浆阳性率(72.7%)(ρ=0.001)。单因素分析结果显示,Kaiso蛋白浆表达与肺癌患者的不良预后密切相关(ρ=0.002)。Cox模型多因素分析结果显示Kaiso浆表达可能是影响肺癌患者预后的独立危险因素(ρ=0.054)。
2、Kaiso浆表达与p120ctn的浆表达具有较好的协同性,两种蛋白在肺癌组织处于结合状态
统计学分析表明,196例肺癌组织的原发灶中,Kaiso浆表达与p120ctn的浆表达存在显著的相关性(Kappa=0.269,ρ=0.000)。55例肺癌配对标本的原发灶和转移灶中,Kaiso与p120ctn的浆表达也存在显著相关性(Kappa=0.267,ρ=0.037)(Kappa=0.393,ρ=0.001)。免疫共沉淀检测了13例肺癌原发灶和淋巴结转移癌中Kaiso和p120ctn的结合情况(免疫组化检测表明,这13例组织的原发灶和转移灶中Kaiso和p120ctn均阳性表达),结果表明,无论原发灶还是转移灶,均能够检测到Kaiso-p120ctn复合物。
3、干扰肺癌细胞Kaiso核表达后,肺癌细胞的增殖和侵袭能力显著增强,核内基因matrilysin转录水平显著上调
转染shRNA-Kaiso质粒下调Kaiso的核表达或加抗体抑制Kaiso的核内功能后,受Kaiso抑制的基因matrilysin转录水平上调,证实Kaiso核内功能被取消,丧失了对下游靶基因的抑制作用。MTT法和基质胶侵袭实验结果显示,丧失Kaiso核内功能后,肺癌细胞的增殖能力和侵袭能力均显著增强。
4、Kaiso的蛋白表达量和核浆分布受p120ctn的调控,p120ctn 3A直接与Kaiso结合,而p120ctn 1A并不与Kaiso直接结合
将p120ctn高表达的肺癌细胞系BE1和p120ctn中等量表达的SPC-A-1细胞中的p120ctn稳定干扰后发现,虽然Kaiso的mRNA表达水平没有发生显著变化,但是Kaiso蛋白表达量显著下调。尤其引人关注的是,Kaiso的核浆分布发生了显著变化:稳定干扰p120ctn表达后,分布于细胞核的Kaiso明显减少,甚至消失,分布于细胞浆(尤其是核膜周区域)中的Kaiso增加。与之相反,SPC-A-1细胞在稳定干扰了p120ctn的表达后,细胞核内的Kaiso增加,细胞浆中的Kaiso明显减少
以p120ctn低表达的肺癌细胞系A549为基础,分别建立稳定过表达p120ctn亚型1A和3A的细胞系。RT-PCR检测结果表明,稳定过表达p120ctn亚型1A和3A均不影响Kaiso的mRNA表达,但都明显上调了Kaiso蛋白的表达量。p120ctn亚型1A和3A过表达均能影响Kaiso的核浆分布,但又存在差别:虽然p120ctn亚型1A和3A过表达均能够增加Kaiso在细胞浆和细胞核中的表达,但p120ctn亚型3A促进Kaiso核定位的能力要强于p120ctn亚型1A。免疫共沉淀结果显示,Kaiso只能与p120ctn亚型3直接结合,而并不与p120ctn亚型1直接结合。
5、Kaiso的核浆分布可能还与细胞周期有关
在体外培养的条件下,不同培养时间点Kaiso的核浆分布存在差异。应用细胞周期同步化的方法,把BE1和siRNA-BE1细胞同步化至GO期和S期后发现,Kaiso在GO期更多见于细胞浆,而在S期则更多见于细胞核。
结论
1、Kaiso在细胞浆和细胞核中的生物学作用不同。
肺癌组织中Kaiso的表达量明显高于癌旁肺组织,Kaiso的浆阳性表达与肺癌的分期、淋巴结转移、不良预后密切相关,可能是影响肺癌不良预后的独立危险因素。Kaiso在细胞核内抑制着matrilysin基因的转录,下调Kaiso的核表达使肺癌的增殖和侵袭能力显著上调。
2、p120ctn和Kaiso在肺癌中协同表达,p120ctn调控着Kaiso的蛋白表达量和核浆分布。
p120ctn与Kaiso在肺癌组织中协同浆表达,在肺癌原发灶及淋巴结转移癌中均能检测到Kaiso-p120ctn复合物。p120ctn亚型1和亚型3都可以影响Kaiso的蛋白表达量,但不影响Kaiso的转录水平。p120ctn亚型1和亚型3都能增加Kaiso的核、浆表达,但p120ctn亚型3对Kaiso核浆分布的调控能力要强于p120ctn亚型1。p120ctn亚型3很可能通过与Kaiso直接结合的方式调控Kaiso的表达和核浆分布,而p120ctn亚型1并不是通过直接结合的方式。
3、Kaiso的核浆分布可能还受到细胞周期的影响,但具体机制有待深入探讨。
The catenin p120(hereafter pl20ctn) is an Armadillo protein first identified as a prominent tyrosine kinase substrate implicated in cell transformation by Src,and in ligand-induced receptor signaling through various tyrosine kinase receptors.pl20ctn binds to the juxtamembrane domain of classical cadherins,where it modulates cell-cell adhesion by regulating cadherin turnover and stability at the cell surface.The recent identification of the transcription factor Kaiso as a binding partner for pl20ctn,suggest that pl20ctn may contribute to tumorigenesis and adhesion defects by regulating Kaiso.
Kaiso is a nuclear protein that plays a role in transcription repression.It contains an amino-terminal BTB/POZ domain(Broad Complex,Tramtrak,Brie a' brac/Pox virus and Zinc finger) and a carboxyl-terminal region with three zinc finger motifs of the C2H2 type.Whether Kaiso inhibits or promotes oncogenesis cannot be stated conclusively.It was considered as a negative regulator of Wnt signaling,since several target genes of the canonical Wnt signaling pathway(such as MTA2 and matrilysiri) and the non-canonical Wnt signaling pathway(such as Wnt 11)are inhibited by Kaiso and may function as a tumor suppressor.However,a recent report showed that Kaiso-null mice were healthy and did not develop tumors.Interestingly,when Kaiso-null mice were crossed with the well-characterized Apcmin/+ mice that develop intestinal polyps, polyp formation was delayed in the progeny.Furthermore,a newly performed study suggested that Kaiso was a methylation-dependent "opportunistic" oncogene that silences tumor suppressor genes in colon cancer cell lines.Despite these advances,the involvement of Kaiso in tumor,especially in lung cancer,is largely unknown.There has been only one report examining the Kaiso expression in lung cancer,and the
authors reported no Kaiso expression in 2 cases of lung squamous cell carcinomas. These situations promoted us to clarify the expression profile of Kaiso proteins as well as its relationship with p120ctn in lung cancer with a large sample size.
1.Patients and specimens
The primary tumors specimens were from patients with lung squamous cell carcinomas and adenocarcinoma who underwent complete resection in the First Affiliated Hospital of China Medical University None of the patients had received radiotherapy or chemotherapy before surgical resection,and all were treated with routine chemotherapy after the operation.Among these samples,some fresh specimens and corresponding normal tissue samples were stored at -70℃immediately after resection until the extraction of protein and RNA.
2.Immunohistochemical study and result assessment
Staining scores were determined by the percentage of positive cells per slide for membranous and cytoplasmic staining separately.As proposed previously,normal expression was defined when over 90%of the tumor cells showed cell membrane staining of p120ctn.When less than 90%of the tumor cells were stained for p120ctn at the cell membrane,the sample was labeled with "reduced membranous expression". When more than 10%of the tumor cells stained for cytoplasmic p120ctn expression, the sample was labeled with "ectopic cytoplasmic expression".A designation of either "reduced membranous expression" or "ectopic cytoplasmic expression" was defined as abnormal expression of p120ctn.For evaluation of the staining of Kaiso in tissue sections,we use the criteria as follows:0:less than 25%;1:26%-50%;2:51%-75%; and 3:more than 75%.The staining intensity was categorized by relative intensity as follows:l(weak);2(intermediate) and 3(strong).The proportion and intensity scores were then multiplied to obtain a total score.Score less than 1 was considered as negative,while scores of 2 or more were considered as positive.Cases were scored nuclear positive when≥5%of the cells reacted with the anti-Kaiso antibody.
3.Immunofluorescent staining
Tissues and cells grown on glass coverslips were fixed with ice-cold 100% methanol for 15 minutes at -20℃,followed by permeabilization with 0.2%Triton X-100.Kaiso was detected using two mouse monoclonal antibodies,and a polyclonal antibodyand pl20ctn was detected using mouse monoclonal antibodies.Primary antibodies were applied overnight at 4℃followed by incubation with secondary antibody conjugated to rhodamine/fluorescein isothiocyanate(FITC)-labeled.The nuclei were counterstained with propidium iodide/ 4,6 diamidino-2-phenylindole.The cells were examined with an Olympus IX51 fluorescent microscope(Olympus,Tokyo, Japan),and images were recorded with a CoolPIX 5400 camera(Nikon,Japan).
4.Western Blot
50μg proteins were separated by SDS-PAGE.After transferring to polyvinylidene fluoride membrane,the membrane was incubated overnight at 4℃with either the mouse monoclonal antibody against p120ctn and Kaiso.After incubation with peroxidase-coupled anti-mouse IgG at 37℃for 2 hours,the proteins were visualized using DAB/ECL.
5.RT-PCR
RT-PCR was performed with the RNA PCR Kit(AMV) Version 3.0,according to the manufacturer's instructions.
6.Cell culture
BE1,SPC,A549 cells were cultured in RPMI 1640 medium,containing 10%fetal calf serum,1OOIU/ml penicillin,and 100μg/ml streptomycin.Cells were grown on sterilized culture dishes glass and were passaged every 2 days with 0.25%trypsin.
7.Plasmid construction and transfection
The shRNA-Kaiso plasmids transfections were carried out using the Arrest-In~(TM) Transfection Reagent,according to the manufacturer's instructions.
The cells were stably transfected with the pl20ctn-siRNA plasmids or p120ctn plasmids using Lipofectamine 2000,following the manufacturer's instructions.The empty plasmid was used as a negative control.Selection was accomplished with G418. Transfected cells were cultured in RPMI 1640 medium containing 10%fetal calf serum andG418.
8.Matrigel invasion assay Following the manufacturer's instructions,in the upper chambers,5x10~5 BE1 cells were grown in serum-free medium on 8μm porous polycarbonate membranes, which were coated with Matrigel basement membrane matrix.The lower chambers were filled with RPMI 1640 medium containing 10%fetal calf serum.After incubation for 6,16,or 24 hours at 37℃in a humid atmosphere of 5%CO2 and 95%air,the cells that had migrated through the pores were fixed with methanol for 30 minutes and stained with hematoxylin.Then the number of cells counted visually using Nikon E200 microscope in five different fields under 200x magnifications per filter.
9.3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) Assay Cell proliferation was evaluated each day for four days after the MTT treatment. The absorbance,which is directly proportional to the number of living cells in the culture,was measured at 570 nm using a microplate reader(Model 550,Bio-Rad, Hercules,CA,USA).A blank with dimethyl sulfoxide(DMSO) alone was taken and subtracted from all values.
10.Cellular Fractionation
Cellular fractionation was performed using the NE-PER Nuclear and Cytoplasmic Extraction Reagents(Thermo Fisher Scientific Inc.Meridian Rd.,Rockford,USA) according to the manufacturer's instructions.Antibodies against a-tubulin and Lamin B 1 were used in control experiments.
11.Immunoprecipitation
Cells were washed twice with 5 ml of PBS followed by incubation on ice with lysis buffer containing 0.5%NP-40,50 mM Tris,150 Mm NaCl,1 mM phenylmethylsulfonyl fluoride,5 mg/ml leupeptin,2mg/ml aprotinin,1 mM sodium orthovanadate,and 1 mM EDTA for 5 minutes.Cells were harvested from the plates, and transferred to a 1.5 ml tube.The lysate was centrifuged at 16,000 g for 5 minutes at 4℃and the supernatant transferred to a new tube.Lysates were quantified by Bradford assay and equal amounts of total protein were used for immunoprecipitation with the anti-pl20ctn or anti-Kaiso pAb.The immunocomplexes were then subjected to SDS-PAGE.
12.Cell Synchronization
(1)GO synchronization(seum starvation method) Cells in the exponential growth phase were incubated at 37℃in 5%CO~2/95%air until they reached 30% confluence.The cells were harvested by centrifugation at 1000xg for 5min,washed with preheated PBS twice then seeded in the serum-free RPMI-1640 medium for further culturing for 36h.The cells that were cultured in serum-containing medium for 36 h at 37℃in 5%CO~2/95%air,harvested for testing after 24h,48h,and 72h.
(2)S phase Synchronization(Double Thymidine Block) Cells in the exponential growth phase had thymidine added at a final concentration of 2 mmol/L and were cultured at 37℃in 5%CO~2/95%air for 18 h.The cultures were centrifuged at 1000xg for 5min and the supernatant was discarded.The cells were washed 3 times with pre-heated PBS to remove the thymidine.Following resuspension,the cells were seeded in a culture flask and incubated for 16 h.Thymidine was added again at a final concentration of 2 mmol/L,and the cells were cultured for 12 h.After the cells were digested with 0.25%trypsin to remove them from the flask and fresh medium was added,followed by centrifugation at 1000xg,the cells were washed with pre-heated PBS twice and then seeded in another flask for an additional 2 h of culture.
After 48 hours of culture,cells from each experimental group were collected and digested with trypsin and fixed with 75%ice-cold ethanol at 4℃overnight.Cells (l×l0~6) were centrifuged at 1500 rpm 5 minutes,and were resuspended with 50μg/ml propidium iodide for 45 minutes in the dark before analysis.The percentages of cells in the different cell cycle phases were determined using a FACSCalibur Flow Cytometer with CellQuest 3.0 software.
14.Statistical analysis All statistical calculations were performed by SPSS 13.0 for Windows software,p values less than 0.05 were considered statistically significant. Results
1.Kaiso was expressed in the cytoplasm of lung cancer cells and is associated with the malignancy of NSCLC
Kaiso was primarily expressed in the cytoplasm of lung cancer tissues.Overall positive cytoplasmic expression rate was 63.61%(187/294).The positive cytoplasmic expression of Kaiso was higher in III+IV TNM stages of NSCLC,compared to I+II stages(ρ=0.019).The western blot analysis revealed that Kaiso expression was significant higher in tumorous tissues(t=10.610,n=20,ρ=0.000).A correlation between cytoplasmic Kaiso expression and lymph node metastasis was found(ρ=0.003).In paired cases,cytoplasmic expression of Kaiso was 72.7%in primary sites and 90.9%in lymph node metastases(ρ=0.001).The lung cancer-related 5-year survival rate was significantly lower in patients who were cytoplasmic Kaiso-positive (22.22%),compared to those with cytoplasmic Kaiso-negative tumors(64.00%)(ρ=0.005).Cytoplasmic Kaiso status may be an independent prognostic factor for the p-value(ρ=0.054).
2.Cytoplasmic Kaiso and p120ctn expression in lung cancer was correlated;the Kaiso-pl20ctn complex can be detected in lung cancer tissues
Cytoplasmic Kaiso and pl20ctn expression in lung cancer was related.In the 196 cases of primary lung cancer tissues,cytoplasmic expression of Kaiso correlated with that of cytoplasmic pl20ctn(Kappa=0.269,ρ=0.000).The same was true for the primary cancer and lymph node metastasis samples of the 55 paired(Kappa=0.267,ρ=0.037)(Kappa=0.393,ρ=0.00l).To verify that Kaiso was a bona fide pl20ctn binding partner in lung cancer tissues,we performed coprecipitation studies to determine whether pl20ctn and Kaiso interacted in vivo.Immunohistochemical study already showed that Kaiso and pl20ctn were synergistically expressed in 13 of 20 cases of lung cancer tissues,both in primary and lymph node metastases samples. Furthermore,co-immunoprecipitation results confirmed that Kaiso coprecipitated efficiently with pl20ctn-specific mAb from all these 13 paired lung cancer tissues.
3.Down-regulating nuclear Kaiso increases matrilysin transcription and enhances the proliferative and invasive abilities of lung cancer cells
Since Kaiso primarily localized to the nucleus in vitro,it was conceivable to explore the biological role of nuclear Kaiso in lung cancers using in vitro cultured cells. The MTT assay results demonstrated that after down-regulating nuclear Kaiso by transfecting shRNA-Kaiso,the levels of proliferation were significantly higher in the shRNA-Kaiso group cells,compared to the control cells[ρ>0.05(day 1);ρ<0.05(day 2-3),n=3].For the Kaiso antibody addition groups,the growth rates were markedly different from the control cells at all three days.Meanwhile,the shRNA-Kaiso cells and the Kaiso antibody-treated cells showed increased invasion onto the lower surfaces of the Transwell filters,compared to control cells(ρ<0.0l).
To further confirm whether the enhancement of proliferative and invasive abilities contributes to Kaiso down-regulation in the nucleus,matrilysin mRNA levels were detected by RT-PCR.The results demonstrated that matrilysin mRNA increased significantly in both shRNA-Kaiso and Kaiso antibody-treated cells,compared with controls(ρ<0.01).
4.Expression and localization of Kaiso protein is affected by its binding partner,pl20ctn.Kaiso only directly interacts with pl20ctn isoform 3,but not pl20ctn isoform 1
We knocked down pl20ctn using siRNA in BE1 and SPC cells.Ablation of pl20ctn reduced the levels of Kaiso proteins,but not true for the mRNA of Kaiso. Furthermore,pl20ctn ablation altered the subcellular localization of Kaiso.The nuclear localizaiton of Kaiso was significantly reduced in siRNA-BEl cells,while that was significantly increased in siRNA-SPC cells.RhoA,but increased the activity of Cdc42 and Racl,and promoted proliferation and the invasive ability of lung cancer cells both in vitro and in vivo.
We overexpressed pl20ctn lA and 3A in A549 cells.Overexpression of pl20ctn 1A and 3A significantly increased the levels of Kaiso proteins,but not true for the mRNA of Kaiso.After pl20ctn 1A or 3A stable overexpression,more Kaiso can be observed in both cytoplasm and nucleus.But difference existed between pl20ctn-lA and pl20ctn-3A cells.In pl20ctn-lA cells,although more Kaiso can be coprecipitated from both cytoplasmic and nuclear extract,significant difference only found in cytoplasmic extract.As for pl20ctn 3A overexpression A549 cells,significant difference existed in both cytoplasmic and nuclear extract.Besides,Kaiso only directly interacts with pl20ctn isoform 3,but not pl20ctn isoform 1.
5.Subcellular loacalization of Kaiso may affected by cell cycle progress. We noticed that Kaiso subcelluar localization altered in different in vitro culture time points.More Kaiso was found in the cytoplasm of the BE1 and siRNA-BEl cells at stage Go,while more Kaiso was found in the nucleus at stageS.
1.Different subcellular localizations of Kaiso may play differential biological roles in NSCLC.
2.Expression and localization of Kaiso protein is affected by its binding partner, pl20ctn.Kaiso only directly interacts with pl20ctn isoform 3,but not pl20ctn isoform 1.
3.Subcellular loacalization of Kaiso may affected by cell cycle progress.
Kaiso是BTB/POZ蛋白家族的重要成员,其氨基端有一个POZ/BTB结构域,羧基端有三个C-2H-2锌指结构。氨基端POZ/BTB结构域的功能是:(一)Kaiso分子之间形成同二聚体;(二)与其他蛋白分子(如CTCF、HDAC等)形成异二聚体。羧基端锌指结构的功能是:(一)与一些肿瘤相关基因的启动子区域(如matrilsin、CDKN2A等)结合,抑制这些基因的表达;(二)与p120ctn结合。p120ctn是Kaiso最重要的结合蛋白,在特定条件下,它能够阻断Kaiso对下游基因的抑制,从而调控这些基因的表达。
Kaiso在肿瘤中发挥的作用还很不清楚。不同研究小组得出的结论甚至严重冲突,有研究认为Kaiso可能是一个抑癌因子,还有研究则得出了完全相反的结论。故Kaiso是促癌因子还是抑癌因子目前还没有定论,有待进一步阐明。
与p120ctn类似,转录因子Kaiso也能够在核/浆之间穿梭,但它在细胞核、细胞浆中是否都发挥着同样的功能还有待研究。此外,Kaiso核浆穿梭的调控机制还不是十分清楚。有研究表明肿瘤微环境影响了Kaiso的核浆分布,而p120ctn并不参与Kaiso的核浆穿梭过程;而另有研究却给出了相反的实验证据,即p120ctn在细胞浆中过表达后,Kaiso也与p120ctn共定位于细胞浆。因此,p120ctn究竟能否调控Kaiso的核浆穿梭还有待进一步研究。
曾有学者利用免疫组织化学的方法检测了多种人类良恶性组织(均为小样本)中Kaiso的表达情况,结果发现:Kaiso在多种实体恶性肿瘤中以细胞浆为主,而在体外培养的多种细胞系(如MDCK、NIH3T3、HT29、SW48细胞等),Kaiso以细胞核表达为主。由于即使是处于进展期的肿瘤组织,也只是少数细胞处于增殖阶段(S期+G2期),肿瘤体积的增大是由于肿瘤细胞凋亡减少造成的,而体外培养的细胞在一段时间内会有大量的细胞处于增殖期。因此,我们推测Kaiso在核或浆的分布,很有可能还与细胞周期有关。当细胞处于增殖阶段时,Kaiso入核。
本研究目的旨在探讨肺癌组织中p120ctn和Kaiso的表达情况及与临床病理学参数意义、肺癌患者预后的关系。在细胞水平上,应用shRNA干扰技术研究Kaiso的核内功能,通过稳定干扰和过表达p120ctn的表达,分析p120ctn亚型蛋白1和3对Kaiso表达和定位的影响。运用细胞周期同步化技术探讨细胞周期对Kaiso核浆分布的影响。
方法
1、肺癌组织
原发性肺癌标本及配对的淋巴结转移癌均来自中国医科大学第一附属医院外科1998-2005年手术切除的标本。所有患者术前均未接受放、化疗。其中部分患者保存有完整、详实的预后资料。标本经4%中性甲醛固定,石蜡包埋,HE常规染色后明确诊断。其中部分癌旁肺组织(远离肿瘤至少5厘米)和淋巴结转移癌离体后立即放入液氮后-70℃冰箱保存备用。
2、免疫组织化学染色及结果判定
标本用中性福尔马林溶液固定,石蜡包埋,制成4μm切片,经脱蜡,脱苯,水化后,采用链菌素抗生物素蛋白-过氧化物酶免疫组化法(S-P法)检测Kaiso和p120ctn蛋白表达。单克隆抗体Kaiso和p120ctn 4℃孵育过夜。以癌旁肺支气管粘膜上皮细胞着色强度和定位为内对照,PBS缓冲液代替一抗为阴性对照。结果判定:高倍视野(×400)下选阳性信号最强区域计数200个肿瘤细胞中阳性细胞数,按Kaiso表达百分率分为以下四个等级:<25%为0分,26%-50%为1分,51%-75%为2分,>75%为3分。根据免疫组化染色强度分为三个等级:浅黄色计为1分,棕黄色计为2分,黄褐色计为3分。以阳性细胞率和染色强度的分值乘积作为每一例的积分,积分为0和1分者判定为阴性,积分≥2为阳性。当Kaiso核阳性信号≥5%时定为核阳性表达。p120ctn正常表达的阳性信号定位于细胞膜,表现为膜着色清楚、连续且阳性细胞数≥90%;膜表达下降表现为膜着色淡且不连续,阳性细胞数<90%;而超过10%的肿瘤细胞胞浆出现阳性信号定为异位表达。p120ctn的异位表达和膜表达下降统归为p120ctn异常表达。
3、间接免疫荧光检测
固定后的冰冻组织切片和细胞爬片用0.2%Triton X-100处理15分钟,非免疫动物血清封闭,一抗为单克隆抗体小鼠抗人Kaiso和小鼠抗人p120ctn 4℃孵育过夜;二抗为异硫氰酸荧光素(FITC)标记或罗丹明标记(TRITC)标记的山羊抗小鼠IgG,碘化丙啶或DAPI进行核复染。50%缓冲甘油封片,荧光显微镜Olympus IX51于波长为527nm处观察罗丹明荧光强度,于372nm波长处观察DAPI的荧光染色观察并照相。阴性对照实验:用等量的0.01mol/LPBS代替一抗。
4、Western BlOt
将含80μg总蛋白的裂解产物进行SDS-PAGE电泳后,转印至PVDF膜,单克隆抗体Kaiso或p120ctn4~C孵育过夜,辣根过氧化物酶标记二抗37℃孵育2小时后DAB或ECL显色。
5、RT-PCR
采用Trizol~TM试剂提取肺癌组织或培养细胞的总RNA,利用RNA PCR Kit(AMV)Ver.3.0试剂盒进行反转录。分别扩增Kaiso、p120ctn和matrilysin,以β-actin为内参。扩增产物经1.5%琼脂糖凝胶电泳后,成像分析。
6、细胞培养
在37℃,含5%CO_2的培养箱内培养人类肺癌细胞系BE1、SPC-A-1、LTEP-A-2和A549细胞系,培养基分别为含10%灭活胎牛血清的RPMI 1640或高糖DMEM,贴壁生长,每2-3天胰酶消化传代一次。
7、质粒构建与转染
shRNA干扰质粒导入感受态宿主细菌E.coli中扩增、纯化,使用Arrest-In~(TM)Transfection Reagent转染试剂将质粒分别转染至肺癌细胞系LTEP-A-2、SPC-A-1和BE1中,通过Western Blot和RT-PCR鉴定转染效果。
将含有目的基因的载体导入感受态宿主细菌DH5a中扩增、纯化,测序验证后,使用Lipofect 2000转染试剂将质粒分别转染至肺癌细胞系A549中,并以G418筛选出阳性细胞克隆,通过Western Blot和RT-PCR鉴定转染效果后,挑取转染成功的单克隆细胞在含有G418的RPMI 1640或高糖DMEM培养液中继续培养。
8、体外基质胶侵袭实验检测细胞侵袭能力
按照BD公司说明操作,取100μl细胞悬液(5×10~6个/m1)滴入上室内,下室加入含有10%胎牛血清的RPMI 1640培养液,上下室之间用孔径为8μm的聚碳酸酯微孔滤膜分开,将小室置37℃,5%CO_2条件下放置后,弃去上室内液体,擦尽膜上Matrigel胶,100%甲醇固定30分钟,常规苏木素染色,光镜下计数细胞数。
9、四甲基偶氮唑盐(tetrazolium,MTT)法检测细胞增殖能力
取对数生长期的肺癌细胞悬液(细胞密度3×10~5个/ml)分别接种于4个96孔板培养板,每板分6组:空白组(A)(B),干扰对照组(C)、干扰组(D)、抗体抑制对照组(E)和抗体移植组(F)。培养第24、48、72小时分别加入MTT(5mg/m1)20μl,继续孵育4小时后,每孔加入150μlDMSO溶解结晶,选择波长490nm,在酶联免疫(ELISA)检测仪上测定各孑L吸光度。实验重复3次。绘制细胞生长曲线。
10、核浆蛋白分离
按照Thermo公司说明操作,取一定体积(100mg)的组织或者10×10~6。的细胞,依次加入体积比为200:11:100的试剂CERⅠ,CERⅡ和NER,分别提取细胞浆蛋白和细胞核蛋白。检测a-tubulin和Lamin B 1在两种细胞组分中的表达以验证核浆蛋白分离效果。
11、免疫共沉淀
提取的蛋白中加入4μg的诱饵蛋白抗体,充分混匀后4℃孵育2小时后加入25μl Protein G Microbeads,混匀后继续4℃孵育1小时。按照操作说明步骤,混合液过μ柱,SDS上样缓冲液冲洗μ柱,收集免疫共沉淀产物。
12、细胞周期同步化
GO期同步化(血清饥饿法):指数生长期的肺癌细胞,培养至汇合密度为30%时收集细胞,1000rpm离心5分钟,PBS洗2次后铺板接种,换成无血清的RPMI-1640/DMEM培养液,继续培养36小时后用于检测。
S期同步化(胸苷双阻断法):指数生长期的肺癌细胞,培养液中加入胸苷至终浓度2mmol/L,继续培养18小时。以1000rpm离心5小时,弃上清,用PBS清洗3次,去除胸苷阻滞。将细胞重新悬浮成细胞悬液,接种到培养瓶中继续培养16小时。培养液中再加入胸苷,使之终浓度为2mmol/L,继续培养12小时。用0.25%胰蛋白酶溶液消化后,换新鲜培养液,1000rpm离心,预热Hanks液清洗2次后接种入另一培养瓶内,继续培养2小时后用于检测。
13、流式细胞仪检测细胞周期
收集对数生长期细胞制成1×10~7个/ml细胞悬液。取1ml细胞悬液75%冷乙醇于4℃固定、离心后制成500μl的细胞悬液,加碘化丙啶染液于4℃孵育45分钟后上机检测,ModFitLT3.O软件分析细胞周期。
14、统计分析
各组资料利用SPSS for Window 13.0进行统计分析。p<0.05有统计学意义。
结果
1、Kaiso主要表达于肺癌组织细胞浆内并与肺癌患者不良预后相关
Kaiso在癌旁肺支气管上皮细胞中存在微量的浆表达,但按照我们制定的阳性标准判定为阴性表达。294例肺癌组织标本中Kaiso蛋白浆阳性表达187例,占63.61%,明显高于癌旁支气管上皮Kaiso的表达水平(p<0.005)。Western blot检测结果证实,肺癌组织中Kaiso蛋白的表达量要显著高于原发灶中Kaiso蛋白的表达量(t=10.610,n=20,p=0.000)。Kaiso浆阳性表达与TNM分期和淋巴结转移密切相关(均p<0.05),与肺癌患者的性别、年龄、分化程度、组织学类型均无明显相关性。III+IV期Kaiso浆阳性表达率(70.42%)显著高于I+II期(57.24%)(ρ=0.019),有淋巴结转移的肺癌病例Kaiso浆阳性表达率(71.2%,116/163)显著高于没有淋巴结转移的肺癌病例(28.8%,47/131)(ρ=0.003);原发灶和淋巴结转移癌的配对标本中,淋巴结转移癌中Kaiso的浆阳性率(90.9%)显著高于原发灶的Kaiso浆阳性率(72.7%)(ρ=0.001)。单因素分析结果显示,Kaiso蛋白浆表达与肺癌患者的不良预后密切相关(ρ=0.002)。Cox模型多因素分析结果显示Kaiso浆表达可能是影响肺癌患者预后的独立危险因素(ρ=0.054)。
2、Kaiso浆表达与p120ctn的浆表达具有较好的协同性,两种蛋白在肺癌组织处于结合状态
统计学分析表明,196例肺癌组织的原发灶中,Kaiso浆表达与p120ctn的浆表达存在显著的相关性(Kappa=0.269,ρ=0.000)。55例肺癌配对标本的原发灶和转移灶中,Kaiso与p120ctn的浆表达也存在显著相关性(Kappa=0.267,ρ=0.037)(Kappa=0.393,ρ=0.001)。免疫共沉淀检测了13例肺癌原发灶和淋巴结转移癌中Kaiso和p120ctn的结合情况(免疫组化检测表明,这13例组织的原发灶和转移灶中Kaiso和p120ctn均阳性表达),结果表明,无论原发灶还是转移灶,均能够检测到Kaiso-p120ctn复合物。
3、干扰肺癌细胞Kaiso核表达后,肺癌细胞的增殖和侵袭能力显著增强,核内基因matrilysin转录水平显著上调
转染shRNA-Kaiso质粒下调Kaiso的核表达或加抗体抑制Kaiso的核内功能后,受Kaiso抑制的基因matrilysin转录水平上调,证实Kaiso核内功能被取消,丧失了对下游靶基因的抑制作用。MTT法和基质胶侵袭实验结果显示,丧失Kaiso核内功能后,肺癌细胞的增殖能力和侵袭能力均显著增强。
4、Kaiso的蛋白表达量和核浆分布受p120ctn的调控,p120ctn 3A直接与Kaiso结合,而p120ctn 1A并不与Kaiso直接结合
将p120ctn高表达的肺癌细胞系BE1和p120ctn中等量表达的SPC-A-1细胞中的p120ctn稳定干扰后发现,虽然Kaiso的mRNA表达水平没有发生显著变化,但是Kaiso蛋白表达量显著下调。尤其引人关注的是,Kaiso的核浆分布发生了显著变化:稳定干扰p120ctn表达后,分布于细胞核的Kaiso明显减少,甚至消失,分布于细胞浆(尤其是核膜周区域)中的Kaiso增加。与之相反,SPC-A-1细胞在稳定干扰了p120ctn的表达后,细胞核内的Kaiso增加,细胞浆中的Kaiso明显减少
以p120ctn低表达的肺癌细胞系A549为基础,分别建立稳定过表达p120ctn亚型1A和3A的细胞系。RT-PCR检测结果表明,稳定过表达p120ctn亚型1A和3A均不影响Kaiso的mRNA表达,但都明显上调了Kaiso蛋白的表达量。p120ctn亚型1A和3A过表达均能影响Kaiso的核浆分布,但又存在差别:虽然p120ctn亚型1A和3A过表达均能够增加Kaiso在细胞浆和细胞核中的表达,但p120ctn亚型3A促进Kaiso核定位的能力要强于p120ctn亚型1A。免疫共沉淀结果显示,Kaiso只能与p120ctn亚型3直接结合,而并不与p120ctn亚型1直接结合。
5、Kaiso的核浆分布可能还与细胞周期有关
在体外培养的条件下,不同培养时间点Kaiso的核浆分布存在差异。应用细胞周期同步化的方法,把BE1和siRNA-BE1细胞同步化至GO期和S期后发现,Kaiso在GO期更多见于细胞浆,而在S期则更多见于细胞核。
结论
1、Kaiso在细胞浆和细胞核中的生物学作用不同。
肺癌组织中Kaiso的表达量明显高于癌旁肺组织,Kaiso的浆阳性表达与肺癌的分期、淋巴结转移、不良预后密切相关,可能是影响肺癌不良预后的独立危险因素。Kaiso在细胞核内抑制着matrilysin基因的转录,下调Kaiso的核表达使肺癌的增殖和侵袭能力显著上调。
2、p120ctn和Kaiso在肺癌中协同表达,p120ctn调控着Kaiso的蛋白表达量和核浆分布。
p120ctn与Kaiso在肺癌组织中协同浆表达,在肺癌原发灶及淋巴结转移癌中均能检测到Kaiso-p120ctn复合物。p120ctn亚型1和亚型3都可以影响Kaiso的蛋白表达量,但不影响Kaiso的转录水平。p120ctn亚型1和亚型3都能增加Kaiso的核、浆表达,但p120ctn亚型3对Kaiso核浆分布的调控能力要强于p120ctn亚型1。p120ctn亚型3很可能通过与Kaiso直接结合的方式调控Kaiso的表达和核浆分布,而p120ctn亚型1并不是通过直接结合的方式。
3、Kaiso的核浆分布可能还受到细胞周期的影响,但具体机制有待深入探讨。
The catenin p120(hereafter pl20ctn) is an Armadillo protein first identified as a prominent tyrosine kinase substrate implicated in cell transformation by Src,and in ligand-induced receptor signaling through various tyrosine kinase receptors.pl20ctn binds to the juxtamembrane domain of classical cadherins,where it modulates cell-cell adhesion by regulating cadherin turnover and stability at the cell surface.The recent identification of the transcription factor Kaiso as a binding partner for pl20ctn,suggest that pl20ctn may contribute to tumorigenesis and adhesion defects by regulating Kaiso.
Kaiso is a nuclear protein that plays a role in transcription repression.It contains an amino-terminal BTB/POZ domain(Broad Complex,Tramtrak,Brie a' brac/Pox virus and Zinc finger) and a carboxyl-terminal region with three zinc finger motifs of the C2H2 type.Whether Kaiso inhibits or promotes oncogenesis cannot be stated conclusively.It was considered as a negative regulator of Wnt signaling,since several target genes of the canonical Wnt signaling pathway(such as MTA2 and matrilysiri) and the non-canonical Wnt signaling pathway(such as Wnt 11)are inhibited by Kaiso and may function as a tumor suppressor.However,a recent report showed that Kaiso-null mice were healthy and did not develop tumors.Interestingly,when Kaiso-null mice were crossed with the well-characterized Apcmin/+ mice that develop intestinal polyps, polyp formation was delayed in the progeny.Furthermore,a newly performed study suggested that Kaiso was a methylation-dependent "opportunistic" oncogene that silences tumor suppressor genes in colon cancer cell lines.Despite these advances,the involvement of Kaiso in tumor,especially in lung cancer,is largely unknown.There has been only one report examining the Kaiso expression in lung cancer,and the
authors reported no Kaiso expression in 2 cases of lung squamous cell carcinomas. These situations promoted us to clarify the expression profile of Kaiso proteins as well as its relationship with p120ctn in lung cancer with a large sample size.
1.Patients and specimens
The primary tumors specimens were from patients with lung squamous cell carcinomas and adenocarcinoma who underwent complete resection in the First Affiliated Hospital of China Medical University None of the patients had received radiotherapy or chemotherapy before surgical resection,and all were treated with routine chemotherapy after the operation.Among these samples,some fresh specimens and corresponding normal tissue samples were stored at -70℃immediately after resection until the extraction of protein and RNA.
2.Immunohistochemical study and result assessment
Staining scores were determined by the percentage of positive cells per slide for membranous and cytoplasmic staining separately.As proposed previously,normal expression was defined when over 90%of the tumor cells showed cell membrane staining of p120ctn.When less than 90%of the tumor cells were stained for p120ctn at the cell membrane,the sample was labeled with "reduced membranous expression". When more than 10%of the tumor cells stained for cytoplasmic p120ctn expression, the sample was labeled with "ectopic cytoplasmic expression".A designation of either "reduced membranous expression" or "ectopic cytoplasmic expression" was defined as abnormal expression of p120ctn.For evaluation of the staining of Kaiso in tissue sections,we use the criteria as follows:0:less than 25%;1:26%-50%;2:51%-75%; and 3:more than 75%.The staining intensity was categorized by relative intensity as follows:l(weak);2(intermediate) and 3(strong).The proportion and intensity scores were then multiplied to obtain a total score.Score less than 1 was considered as negative,while scores of 2 or more were considered as positive.Cases were scored nuclear positive when≥5%of the cells reacted with the anti-Kaiso antibody.
3.Immunofluorescent staining
Tissues and cells grown on glass coverslips were fixed with ice-cold 100% methanol for 15 minutes at -20℃,followed by permeabilization with 0.2%Triton X-100.Kaiso was detected using two mouse monoclonal antibodies,and a polyclonal antibodyand pl20ctn was detected using mouse monoclonal antibodies.Primary antibodies were applied overnight at 4℃followed by incubation with secondary antibody conjugated to rhodamine/fluorescein isothiocyanate(FITC)-labeled.The nuclei were counterstained with propidium iodide/ 4,6 diamidino-2-phenylindole.The cells were examined with an Olympus IX51 fluorescent microscope(Olympus,Tokyo, Japan),and images were recorded with a CoolPIX 5400 camera(Nikon,Japan).
4.Western Blot
50μg proteins were separated by SDS-PAGE.After transferring to polyvinylidene fluoride membrane,the membrane was incubated overnight at 4℃with either the mouse monoclonal antibody against p120ctn and Kaiso.After incubation with peroxidase-coupled anti-mouse IgG at 37℃for 2 hours,the proteins were visualized using DAB/ECL.
5.RT-PCR
RT-PCR was performed with the RNA PCR Kit(AMV) Version 3.0,according to the manufacturer's instructions.
6.Cell culture
BE1,SPC,A549 cells were cultured in RPMI 1640 medium,containing 10%fetal calf serum,1OOIU/ml penicillin,and 100μg/ml streptomycin.Cells were grown on sterilized culture dishes glass and were passaged every 2 days with 0.25%trypsin.
7.Plasmid construction and transfection
The shRNA-Kaiso plasmids transfections were carried out using the Arrest-In~(TM) Transfection Reagent,according to the manufacturer's instructions.
The cells were stably transfected with the pl20ctn-siRNA plasmids or p120ctn plasmids using Lipofectamine 2000,following the manufacturer's instructions.The empty plasmid was used as a negative control.Selection was accomplished with G418. Transfected cells were cultured in RPMI 1640 medium containing 10%fetal calf serum andG418.
8.Matrigel invasion assay Following the manufacturer's instructions,in the upper chambers,5x10~5 BE1 cells were grown in serum-free medium on 8μm porous polycarbonate membranes, which were coated with Matrigel basement membrane matrix.The lower chambers were filled with RPMI 1640 medium containing 10%fetal calf serum.After incubation for 6,16,or 24 hours at 37℃in a humid atmosphere of 5%CO2 and 95%air,the cells that had migrated through the pores were fixed with methanol for 30 minutes and stained with hematoxylin.Then the number of cells counted visually using Nikon E200 microscope in five different fields under 200x magnifications per filter.
9.3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) Assay Cell proliferation was evaluated each day for four days after the MTT treatment. The absorbance,which is directly proportional to the number of living cells in the culture,was measured at 570 nm using a microplate reader(Model 550,Bio-Rad, Hercules,CA,USA).A blank with dimethyl sulfoxide(DMSO) alone was taken and subtracted from all values.
10.Cellular Fractionation
Cellular fractionation was performed using the NE-PER Nuclear and Cytoplasmic Extraction Reagents(Thermo Fisher Scientific Inc.Meridian Rd.,Rockford,USA) according to the manufacturer's instructions.Antibodies against a-tubulin and Lamin B 1 were used in control experiments.
11.Immunoprecipitation
Cells were washed twice with 5 ml of PBS followed by incubation on ice with lysis buffer containing 0.5%NP-40,50 mM Tris,150 Mm NaCl,1 mM phenylmethylsulfonyl fluoride,5 mg/ml leupeptin,2mg/ml aprotinin,1 mM sodium orthovanadate,and 1 mM EDTA for 5 minutes.Cells were harvested from the plates, and transferred to a 1.5 ml tube.The lysate was centrifuged at 16,000 g for 5 minutes at 4℃and the supernatant transferred to a new tube.Lysates were quantified by Bradford assay and equal amounts of total protein were used for immunoprecipitation with the anti-pl20ctn or anti-Kaiso pAb.The immunocomplexes were then subjected to SDS-PAGE.
12.Cell Synchronization
(1)GO synchronization(seum starvation method) Cells in the exponential growth phase were incubated at 37℃in 5%CO~2/95%air until they reached 30% confluence.The cells were harvested by centrifugation at 1000xg for 5min,washed with preheated PBS twice then seeded in the serum-free RPMI-1640 medium for further culturing for 36h.The cells that were cultured in serum-containing medium for 36 h at 37℃in 5%CO~2/95%air,harvested for testing after 24h,48h,and 72h.
(2)S phase Synchronization(Double Thymidine Block) Cells in the exponential growth phase had thymidine added at a final concentration of 2 mmol/L and were cultured at 37℃in 5%CO~2/95%air for 18 h.The cultures were centrifuged at 1000xg for 5min and the supernatant was discarded.The cells were washed 3 times with pre-heated PBS to remove the thymidine.Following resuspension,the cells were seeded in a culture flask and incubated for 16 h.Thymidine was added again at a final concentration of 2 mmol/L,and the cells were cultured for 12 h.After the cells were digested with 0.25%trypsin to remove them from the flask and fresh medium was added,followed by centrifugation at 1000xg,the cells were washed with pre-heated PBS twice and then seeded in another flask for an additional 2 h of culture.
After 48 hours of culture,cells from each experimental group were collected and digested with trypsin and fixed with 75%ice-cold ethanol at 4℃overnight.Cells (l×l0~6) were centrifuged at 1500 rpm 5 minutes,and were resuspended with 50μg/ml propidium iodide for 45 minutes in the dark before analysis.The percentages of cells in the different cell cycle phases were determined using a FACSCalibur Flow Cytometer with CellQuest 3.0 software.
14.Statistical analysis All statistical calculations were performed by SPSS 13.0 for Windows software,p values less than 0.05 were considered statistically significant. Results
1.Kaiso was expressed in the cytoplasm of lung cancer cells and is associated with the malignancy of NSCLC
Kaiso was primarily expressed in the cytoplasm of lung cancer tissues.Overall positive cytoplasmic expression rate was 63.61%(187/294).The positive cytoplasmic expression of Kaiso was higher in III+IV TNM stages of NSCLC,compared to I+II stages(ρ=0.019).The western blot analysis revealed that Kaiso expression was significant higher in tumorous tissues(t=10.610,n=20,ρ=0.000).A correlation between cytoplasmic Kaiso expression and lymph node metastasis was found(ρ=0.003).In paired cases,cytoplasmic expression of Kaiso was 72.7%in primary sites and 90.9%in lymph node metastases(ρ=0.001).The lung cancer-related 5-year survival rate was significantly lower in patients who were cytoplasmic Kaiso-positive (22.22%),compared to those with cytoplasmic Kaiso-negative tumors(64.00%)(ρ=0.005).Cytoplasmic Kaiso status may be an independent prognostic factor for the p-value(ρ=0.054).
2.Cytoplasmic Kaiso and p120ctn expression in lung cancer was correlated;the Kaiso-pl20ctn complex can be detected in lung cancer tissues
Cytoplasmic Kaiso and pl20ctn expression in lung cancer was related.In the 196 cases of primary lung cancer tissues,cytoplasmic expression of Kaiso correlated with that of cytoplasmic pl20ctn(Kappa=0.269,ρ=0.000).The same was true for the primary cancer and lymph node metastasis samples of the 55 paired(Kappa=0.267,ρ=0.037)(Kappa=0.393,ρ=0.00l).To verify that Kaiso was a bona fide pl20ctn binding partner in lung cancer tissues,we performed coprecipitation studies to determine whether pl20ctn and Kaiso interacted in vivo.Immunohistochemical study already showed that Kaiso and pl20ctn were synergistically expressed in 13 of 20 cases of lung cancer tissues,both in primary and lymph node metastases samples. Furthermore,co-immunoprecipitation results confirmed that Kaiso coprecipitated efficiently with pl20ctn-specific mAb from all these 13 paired lung cancer tissues.
3.Down-regulating nuclear Kaiso increases matrilysin transcription and enhances the proliferative and invasive abilities of lung cancer cells
Since Kaiso primarily localized to the nucleus in vitro,it was conceivable to explore the biological role of nuclear Kaiso in lung cancers using in vitro cultured cells. The MTT assay results demonstrated that after down-regulating nuclear Kaiso by transfecting shRNA-Kaiso,the levels of proliferation were significantly higher in the shRNA-Kaiso group cells,compared to the control cells[ρ>0.05(day 1);ρ<0.05(day 2-3),n=3].For the Kaiso antibody addition groups,the growth rates were markedly different from the control cells at all three days.Meanwhile,the shRNA-Kaiso cells and the Kaiso antibody-treated cells showed increased invasion onto the lower surfaces of the Transwell filters,compared to control cells(ρ<0.0l).
To further confirm whether the enhancement of proliferative and invasive abilities contributes to Kaiso down-regulation in the nucleus,matrilysin mRNA levels were detected by RT-PCR.The results demonstrated that matrilysin mRNA increased significantly in both shRNA-Kaiso and Kaiso antibody-treated cells,compared with controls(ρ<0.01).
4.Expression and localization of Kaiso protein is affected by its binding partner,pl20ctn.Kaiso only directly interacts with pl20ctn isoform 3,but not pl20ctn isoform 1
We knocked down pl20ctn using siRNA in BE1 and SPC cells.Ablation of pl20ctn reduced the levels of Kaiso proteins,but not true for the mRNA of Kaiso. Furthermore,pl20ctn ablation altered the subcellular localization of Kaiso.The nuclear localizaiton of Kaiso was significantly reduced in siRNA-BEl cells,while that was significantly increased in siRNA-SPC cells.RhoA,but increased the activity of Cdc42 and Racl,and promoted proliferation and the invasive ability of lung cancer cells both in vitro and in vivo.
We overexpressed pl20ctn lA and 3A in A549 cells.Overexpression of pl20ctn 1A and 3A significantly increased the levels of Kaiso proteins,but not true for the mRNA of Kaiso.After pl20ctn 1A or 3A stable overexpression,more Kaiso can be observed in both cytoplasm and nucleus.But difference existed between pl20ctn-lA and pl20ctn-3A cells.In pl20ctn-lA cells,although more Kaiso can be coprecipitated from both cytoplasmic and nuclear extract,significant difference only found in cytoplasmic extract.As for pl20ctn 3A overexpression A549 cells,significant difference existed in both cytoplasmic and nuclear extract.Besides,Kaiso only directly interacts with pl20ctn isoform 3,but not pl20ctn isoform 1.
5.Subcellular loacalization of Kaiso may affected by cell cycle progress. We noticed that Kaiso subcelluar localization altered in different in vitro culture time points.More Kaiso was found in the cytoplasm of the BE1 and siRNA-BEl cells at stage Go,while more Kaiso was found in the nucleus at stageS.
1.Different subcellular localizations of Kaiso may play differential biological roles in NSCLC.
2.Expression and localization of Kaiso protein is affected by its binding partner, pl20ctn.Kaiso only directly interacts with pl20ctn isoform 3,but not pl20ctn isoform 1.
3.Subcellular loacalization of Kaiso may affected by cell cycle progress.
引文
1 Keirsebilck A,Bonne S,Staes K,et al:Molecular cloning of the human pl20ctn catenin gene(CTNND1):expression of multiple alternatively spliced isoforms.Genomics 1998,50:129-146.
2 Anastasiadis PZ,Reynolds AB:The p120 catenin family:complex roles in adhesion,signaling and cancer.J Cell Sci 2000,113(Pt 8):1319-1334.
3 Golenhofen N,Drenckhahn D:The catenin,p120ctn,is a common membrane-associated protein in various epithelial and non-epithelial cells and tissues.Histochem Cell Biol 2000,114:147-155.
4 Anastasiadis PZ,Moon SY,Thoreson MA,et al:Inhibition of RhoA by p120 catenin.Nat Cell Biol 2000,2:637-644.
5 Noren NK,Liu BP,Burridge K,et al:p120 catenin regulates the actin cytoskeleton via Rho family GTPases.J Cell Biol 2000,150:567-580.
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7 Collins T,Stone JR,Williams AJ:All in the family:the BTB/POZ,KRAB,and SCAN domains.Mol Cell Biol 2001,21:3609-3615.
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1 Daniel JM,Reynolds AB.The catenin p120(ctn) interacts with Kaiso,a novel BTB/POZ domain zinc finger transcription factor.Mol Cell Biol 1999;19:3614-3623.
2 Maeda T,Hobbs RM,Merghoub T,et al.Role of the proto-oncogene Pokemon in cellular transformation and ARF repression.Nature 2005;433:278-285.
3 Chen WY,Wang DH,Yen RC,et al.Tumor suppressor HIC1 directly regulates SIRT1 to modulate p53-dependent DNA-damage responses.Cell 2005;123:437-448.
4 van Roy FM,McCrea PD.A role for Kaiso-p120ctn complexes in cancer?Nat Rev Cancer 2005;5:956-964.
5 Prokhortchouk A,Sansom O,Selfridge J,et al.Kaiso-deficient mice show resistance to intestinal cancer.Mol Cell Biol 2006;26:199-208.
6 Lopes EC,Valls E,Figueroa ME,et al.Kaiso contributes to DNA methylation-dependent silencing of tumor suppressor genes in colon cancer cell lines.Cancer Res 2008;68:7258-7263.
7 Soubry A,van Hengel J,Parthoens E,et al.Expression and nuclear location of the transcriptional repressor Kaiso is regulated by the tumor microenvironment.Cancer Res 2005;65:2224-2233.
8 Travis WD BE,Muller-Hermelink HK,Harris CC.World Health Organization Classification of Tumours:Pathology and Genetics of Tumours of the Lung,Pleura,Thymus and Heart.Lyon:IARC Press;.2004.
9 Sobin DH WC.International Union Against Cancer(UICC):TNM Classification of Malignant Tumours.6th ed.New York:Wiley-Liss;2002.
10 Kelly KF,Daniel JM.POZ for effect--POZ-ZF transcription factors in cancer and development.Trends Cell Biol 2006;16:578-587.
11 Spring CM,Kelly KF,O'Kelly I,et al.The catenin p120ctn inhibits Kaiso-mediated transcriptional repression of the beta-catenin/TCF target gene matrilysin.Exp Cell Res 2005;305:253-265.
12 Keirsebilck A,Bonne S,Staes K,et al.Molecular cloning of the human p120ctn catenin gene (CTNND1):expression of multiple alternatively spliced isoforms.Genomics 1998;50:129-146.
13 Anastasiadis PZ,Reynolds AB.The p120 catenin family:complex roles in adhesion,signaling and cancer.J Cell Sci 2000;113(Pt 8):1319-1334.
14 Golenhofen N,Drenckhahn D.The catenin,p120ctn,is a common membrane-associated protein in various epithelial and non-epithelial cells and tissues.Histochem Cell Biol 2000;114:147-155.
15 Anastasiadis PZ,Moon SY,Thoreson MA,et al.Inhibition of RhoA by p120 catenin.Nat Cell Biol 2000;2:637-644.
16 Noren NK,Liu BP,Burridge K,et al.p120 catenin regulates the actin cytoskeleton via Rho family GTPases.J Cell Biol 2000;150:567-580.
17 Park JI,Kim SW,Lyons JP,et al.Kaiso/p120-catenin and TCF/beta-catenin complexes coordinately regulate canonical Wnt gene targets.Dev Cell 2005;8:843-854.
18 Daniel JM.Dancing in and out of the nucleus:p120(ctn) and the transcription factor Kaiso.Biochim Biophys Acta 2007;1773:59-68.
19 Vinay Kumar NF,Abul Abbas.Biology of Tumor Growth:Benign and Malignant Neoplasms.In:inay Kumar,Nelso Fausto,Abul Abbas,editors.Robbins and Cotran Pathologic Basis of Disease,7th.2005;276-277.
20 Liu Y,Wang Y,Zhang Y,et al.Abnormal expression of p120-catenin,E-cadherin,and small GTPases is significantly associated with malignant phenotype of human lung cancer.Lung Cancer 2009;63:375-382.
21 Wang EH,Liu Y,Xu HT,et al.Abnormal expression and clinicopathologic significance of p120-catenin in lung cancer.Histol Histopathol 2006;21:841-847.
22 Liu Y,Xu HT,Dai SD,et al.Reduction of p120(ctn) isoforms 1 and 3 is significantly associated with metastatic progression of human lung cancer.Apmis 2007;115:848-856.
23 Kim SW,Park JI,Spring CM,et al.Non-canonical Wnt signals are modulated by the Kaiso transcriptional repressor and p120-catenin.Nat Cell Biol 2004;6:1212-1220.
24 Park JI,Ji H,Jun S,et al.Frodo links Dishevelled to the p120-catenin/Kaiso pathway:distinct catenin subfamilies promote Wnt signals.Dev Cell 2006;11:683-695.
25 Kelly KF,Otchere AA,Graham M,et al.Nuclear import of the BTB/POZ transcriptional regulator Kaiso.J Cell Sci 2004;117:6143-6152.
26 Mo YY,Reynolds AB.Identification of murine p120 isoforms and heterogeneous expression of pl20cas isoforms in human tumor cell lines.Cancer Res 1996;56:2633-2640.
27 Daniel JM,Spring CM,Crawford HC,et al.The p120(ctn)-binding partner Kaiso is a bi-modal DNA-binding protein that recognizes both a sequence-specific consensus and methylated CpG dinucleotides.Nucleic Acids Res 2002;30:2911-2919.
28 Ireton RC,Davis MA,van Hengel J,et al.A novel role for p120 catenin in E-cadherin function.J Cell Biol 2002;159:465-476.
29 Rodova M,Kelly KF,VanSaun M,et al.Regulation of the rapsyn promoter by kaiso and delta-catenin.Mol Cell Biol 2004;24:7188-7196.
30 Na J,Lykke-Andersen K,Torres Padilla ME,et al.Dishevelled proteins regulate cell adhesion in mouse blastocyst and serve to monitor changes in Wnt signaling.Dev Biol 2007;302:40-49.
31 Kelly KF,Spring CM,Otchere AA,et al.NLS-dependent nuclear localization of pl20ctn is necessary to relieve Kaiso-mediated transcriptional repression.J Cell Sci 2004;117:2675-2686.
32 Yanagisawa M,Kaverina IN,Wang A,et al.A novel interaction between kinesin and p120 modulates p120 localization and function.J Biol Chem 2004;279:9512-9521.
33 Kim SW,Fang X,Ji H,et al.Isolation and characterization of XKaiso,a transcriptional repressor that associates with the catenin Xp120(ctn) in Xenopus laevis.J Biol Chem 2002;277:8202-8208.
34 Zhang BZ,Gu LK,Deng DJ.[Methylation specific binding activity of zinc finger protein Kaiso].Zhonghua Yu Fang Yi Xue Za Zhi 2007;41 Suppl:43-46.
35 Feng SY,Ota K,Yamada Y,et al.A yeast one-hybrid system to detect methylation-dependent DNA-protein interactions.Biochem Biophys Res Commun 2004;313:922-925.
36 Kondapalli J,Flozak AS,Albuquerque ML.Laminar shear stress differentially modulates gene expression of p120 catenin,Kaiso transcription factor,and vascular endothelial cadherin in human coronary artery endothelial cells.J Biol Chem 2004;279:11417-11424.
37 Barker N,van Es JH,Kuipers J,et al.Identification of stem cells in small intestine and colon by marker gene Lgr5.Nature 2007;449:1003-1007.
38 Woll PS,Morris JK,Painschab MS,et al.Wnt signaling promotes hemato-endothelial cell development from human embryonic stem cells.Blood 2007;
39 Iioka H,Doerner SK,Tamai K.Kaiso is a bimodal modulator for Wnt/beta-catenin signaling.FEBS Lett 2009;583:627-632.
40 Defossez PA,Kelly KF,Filion GJ,et al.The human enhancer blocker CTC-binding factor interacts with the transcription factor Kaiso.J Biol Chem 2005;280:43017-43023.
41 Myster SH,Cavallo R,Anderson CT,et al.Drosophila p120catenin plays a supporting role in cell adhesion but is not an essential adherens junction component.J Cell Biol 2003;160:433-449.
42 Delia Ragione F,Tiunova A,Vacca M,et al.The X-linked methyl binding protein gene Kaiso is highly expressed in brain but is not mutated in Rett syndrome patients.Gene 2006;373:83-89.
43 Xu X,Li WE,Huang GY,et al.N-cadherin and Cx43alphal gap junctions modulates mouse neural crest cell motility via distinct pathways.Cell Commun Adhes 2001;8:321-324.
44 Geis K,Aberle H,Kuhl M,et al.Expression of the Armadillo family member p120cas1B in Xenopus embryos affects head differentiation but not axis formation.Dev Genes Evol 1998;207:471-481.
45 Hecht A,Litterst CM,Huber O,et al.Functional characterization of multiple transactivating elements in beta-catenin,some of which interact with the TATA-binding protein in vitro.J Biol Chem 1999;274:18017-18025.
46 Papkoff J.Regulation of complexed and free catenin pools by distinct mechanisms.Differential effects of Wnt-1 and v-Src.J Biol Chem 1997;272:4536-4543.
47 Xu X,Li WE,Huang GY,et al.Modulation of mouse neural crest cell motility by N-cadherin and connexin 43 gap junctions.J Cell Biol 2001;154:217-230.
48 Aberle H,Bauer A,Stappert J,et al.beta-catenin is a target for the ubiquitin-proteasome pathway.Embo J 1997;16:3797-3804.
49 Gumbiner BM.Coordinate gene regulation by two different catenins.Dev Cell 2005;8:795-796.
50 Perez-Moreno M,Fuchs E.Catenins:keeping cells from getting their signals crossed.Dev Cell 2006;11:601-612.
51 Amini Nik S,Ebrahim RP,Van Dam K,et al.TGF-beta modulates beta-Catenin stability and signaling in mesenchymal proliferations.Exp Cell Res 2007;313:2887-2895.
52 Wittier L,Shin EH,Grote P,et al.Expression of Msgn1 in the presomitic mesoderm is controlled by synergism of WNT signalling and Tbx6.EMBO Rep 2007;8:784-789.
53 Cohen ED,Wang Z,Lepore JJ,et al.Wnt/beta-catenin signaling promotes expansion of Isl-1-positive cardiac progenitor cells through regulation of FGF signaling.J Clin Invest 2007;117:1794-1804.
2 Anastasiadis PZ,Reynolds AB:The p120 catenin family:complex roles in adhesion,signaling and cancer.J Cell Sci 2000,113(Pt 8):1319-1334.
3 Golenhofen N,Drenckhahn D:The catenin,p120ctn,is a common membrane-associated protein in various epithelial and non-epithelial cells and tissues.Histochem Cell Biol 2000,114:147-155.
4 Anastasiadis PZ,Moon SY,Thoreson MA,et al:Inhibition of RhoA by p120 catenin.Nat Cell Biol 2000,2:637-644.
5 Noren NK,Liu BP,Burridge K,et al:p120 catenin regulates the actin cytoskeleton via Rho family GTPases.J Cell Biol 2000,150:567-580.
6 Daniel JM,Reynolds AB:The catenin p120(ctn) interacts with Kaiso,a novel BTB/POZ domain zinc finger transcription factor.Mol Cell Biol 1999,19:3614-3623.
7 Collins T,Stone JR,Williams AJ:All in the family:the BTB/POZ,KRAB,and SCAN domains.Mol Cell Biol 2001,21:3609-3615.
8 Kelly KF,Otchere AA,Graham M,et al:Nuclear import of the BTB/POZ transcriptional regulator Kaiso.J Cell Sci 2004,117:6143-6152.
9 Kelly KF,Daniel JM:POZ for effect--POZ-ZF transcription factors in cancer and development.Trends Cell Biol 2006,16:578-587.
10 Kelly KF,Spring CM,Otchere AA,et al:NLS-dependent nuclear localization of p120ctn is necessary to relieve Kaiso-mediated transcriptional repression.J Cell Sci 2004,117:2675-2686.
11 Kim SW,Park JI,Spring CM,et al:Non-canonical Wnt signals are modulated by the Kaiso transcriptional repressor and p120-catenin.Nat Cell Biol 2004,6:1212-1220.
12 Park JI,Kim SW,Lyons JP,et al:Kaiso/p120-catenin and TCF/beta-catenin complexes coordinately regulate canonical Wnt gene targets.Dev Cell 2005,8:843-854.
13 Soubry A,van Hengel J,Parthoens E,et al:Expression and nuclear location of the transcriptional repressor Kaiso is regulated by the tumor microenvironment.Cancer Res 2005,65:2224-2233.
14 Park JI,Ji H,Jun S,et al:Frodo links Dishevelled to the p120-catenin/Kaiso pathway:distinct catenin subfamilies promote Wnt signals.Dev Cell 2006,11:683-695.
15 Vinay Kumar NF,Abul Abbas.:Biology of Tumor Growth:Benign and Malignant Neoplasms.In:inay Kumar,Nelso Fausto,Abul Abbas,editors.Robbins and Cotran Pathologic Basis of Disease,7th.2005:276-277.
16 Travis WD BE,Muller-Hermelink HK,Harris CC:World Health Organization Classification of Tumours:Pathology and Genetics of Tumours of the Lung,Pleura,Thymus and Heart.Lyon:IARC Press;.2004.
17 Sobin DH WC:International Union Against Cancer(UICC):TNM Classification of Malignant Tumours.6th ed.New York:Wiley-Liss;.2002.
18 Liu Y,Wang Y,Zhang Y,et al:Abnormal expression of p120-catenin,E-cadherin,and small GTPases is significantly associated with malignant phenotype of human lung cancer.Lung Cancer 2009,63:375-382.
19 Wang EH,Liu Y,Xu HT,et al:Abnormal expression and clinicopathologic significance of pl20-catenin in lung cancer.Histol Histopathol 2006,21:841-847.
20 Liu Y,Xu HT,Dai SD,et al:Reduction of p120(ctn) isoforms 1 and 3 is significantly associated with metastatic progression of human lung cancer.Apmis 2007,115:848-856.
21 Daniel JM:Dancing in and out of the nucleus:pl20(ctn) and the transcription factor Kaiso.Biochim Biophys Acta 2007,1773:59-68.
22 Roczniak-Ferguson A,Reynolds AB:Regulation of p120-catenin nucleocytoplasmic shuttling activity.J Cell Sci 2003,116:4201-4212.
23 Spring CM,Kelly KF,O'Kelly I,et al:The catenin p120ctn inhibits Kaiso-mediated transcriptional repression of the beta-catenin/TCF target gene matrilysin.Exp Cell Res 2005,305:253-265.
1 Daniel JM,Reynolds AB.The catenin p120(ctn) interacts with Kaiso,a novel BTB/POZ domain zinc finger transcription factor.Mol Cell Biol 1999;19:3614-3623.
2 Maeda T,Hobbs RM,Merghoub T,et al.Role of the proto-oncogene Pokemon in cellular transformation and ARF repression.Nature 2005;433:278-285.
3 Chen WY,Wang DH,Yen RC,et al.Tumor suppressor HIC1 directly regulates SIRT1 to modulate p53-dependent DNA-damage responses.Cell 2005;123:437-448.
4 van Roy FM,McCrea PD.A role for Kaiso-p120ctn complexes in cancer?Nat Rev Cancer 2005;5:956-964.
5 Prokhortchouk A,Sansom O,Selfridge J,et al.Kaiso-deficient mice show resistance to intestinal cancer.Mol Cell Biol 2006;26:199-208.
6 Lopes EC,Valls E,Figueroa ME,et al.Kaiso contributes to DNA methylation-dependent silencing of tumor suppressor genes in colon cancer cell lines.Cancer Res 2008;68:7258-7263.
7 Soubry A,van Hengel J,Parthoens E,et al.Expression and nuclear location of the transcriptional repressor Kaiso is regulated by the tumor microenvironment.Cancer Res 2005;65:2224-2233.
8 Travis WD BE,Muller-Hermelink HK,Harris CC.World Health Organization Classification of Tumours:Pathology and Genetics of Tumours of the Lung,Pleura,Thymus and Heart.Lyon:IARC Press;.2004.
9 Sobin DH WC.International Union Against Cancer(UICC):TNM Classification of Malignant Tumours.6th ed.New York:Wiley-Liss;2002.
10 Kelly KF,Daniel JM.POZ for effect--POZ-ZF transcription factors in cancer and development.Trends Cell Biol 2006;16:578-587.
11 Spring CM,Kelly KF,O'Kelly I,et al.The catenin p120ctn inhibits Kaiso-mediated transcriptional repression of the beta-catenin/TCF target gene matrilysin.Exp Cell Res 2005;305:253-265.
12 Keirsebilck A,Bonne S,Staes K,et al.Molecular cloning of the human p120ctn catenin gene (CTNND1):expression of multiple alternatively spliced isoforms.Genomics 1998;50:129-146.
13 Anastasiadis PZ,Reynolds AB.The p120 catenin family:complex roles in adhesion,signaling and cancer.J Cell Sci 2000;113(Pt 8):1319-1334.
14 Golenhofen N,Drenckhahn D.The catenin,p120ctn,is a common membrane-associated protein in various epithelial and non-epithelial cells and tissues.Histochem Cell Biol 2000;114:147-155.
15 Anastasiadis PZ,Moon SY,Thoreson MA,et al.Inhibition of RhoA by p120 catenin.Nat Cell Biol 2000;2:637-644.
16 Noren NK,Liu BP,Burridge K,et al.p120 catenin regulates the actin cytoskeleton via Rho family GTPases.J Cell Biol 2000;150:567-580.
17 Park JI,Kim SW,Lyons JP,et al.Kaiso/p120-catenin and TCF/beta-catenin complexes coordinately regulate canonical Wnt gene targets.Dev Cell 2005;8:843-854.
18 Daniel JM.Dancing in and out of the nucleus:p120(ctn) and the transcription factor Kaiso.Biochim Biophys Acta 2007;1773:59-68.
19 Vinay Kumar NF,Abul Abbas.Biology of Tumor Growth:Benign and Malignant Neoplasms.In:inay Kumar,Nelso Fausto,Abul Abbas,editors.Robbins and Cotran Pathologic Basis of Disease,7th.2005;276-277.
20 Liu Y,Wang Y,Zhang Y,et al.Abnormal expression of p120-catenin,E-cadherin,and small GTPases is significantly associated with malignant phenotype of human lung cancer.Lung Cancer 2009;63:375-382.
21 Wang EH,Liu Y,Xu HT,et al.Abnormal expression and clinicopathologic significance of p120-catenin in lung cancer.Histol Histopathol 2006;21:841-847.
22 Liu Y,Xu HT,Dai SD,et al.Reduction of p120(ctn) isoforms 1 and 3 is significantly associated with metastatic progression of human lung cancer.Apmis 2007;115:848-856.
23 Kim SW,Park JI,Spring CM,et al.Non-canonical Wnt signals are modulated by the Kaiso transcriptional repressor and p120-catenin.Nat Cell Biol 2004;6:1212-1220.
24 Park JI,Ji H,Jun S,et al.Frodo links Dishevelled to the p120-catenin/Kaiso pathway:distinct catenin subfamilies promote Wnt signals.Dev Cell 2006;11:683-695.
25 Kelly KF,Otchere AA,Graham M,et al.Nuclear import of the BTB/POZ transcriptional regulator Kaiso.J Cell Sci 2004;117:6143-6152.
26 Mo YY,Reynolds AB.Identification of murine p120 isoforms and heterogeneous expression of pl20cas isoforms in human tumor cell lines.Cancer Res 1996;56:2633-2640.
27 Daniel JM,Spring CM,Crawford HC,et al.The p120(ctn)-binding partner Kaiso is a bi-modal DNA-binding protein that recognizes both a sequence-specific consensus and methylated CpG dinucleotides.Nucleic Acids Res 2002;30:2911-2919.
28 Ireton RC,Davis MA,van Hengel J,et al.A novel role for p120 catenin in E-cadherin function.J Cell Biol 2002;159:465-476.
29 Rodova M,Kelly KF,VanSaun M,et al.Regulation of the rapsyn promoter by kaiso and delta-catenin.Mol Cell Biol 2004;24:7188-7196.
30 Na J,Lykke-Andersen K,Torres Padilla ME,et al.Dishevelled proteins regulate cell adhesion in mouse blastocyst and serve to monitor changes in Wnt signaling.Dev Biol 2007;302:40-49.
31 Kelly KF,Spring CM,Otchere AA,et al.NLS-dependent nuclear localization of pl20ctn is necessary to relieve Kaiso-mediated transcriptional repression.J Cell Sci 2004;117:2675-2686.
32 Yanagisawa M,Kaverina IN,Wang A,et al.A novel interaction between kinesin and p120 modulates p120 localization and function.J Biol Chem 2004;279:9512-9521.
33 Kim SW,Fang X,Ji H,et al.Isolation and characterization of XKaiso,a transcriptional repressor that associates with the catenin Xp120(ctn) in Xenopus laevis.J Biol Chem 2002;277:8202-8208.
34 Zhang BZ,Gu LK,Deng DJ.[Methylation specific binding activity of zinc finger protein Kaiso].Zhonghua Yu Fang Yi Xue Za Zhi 2007;41 Suppl:43-46.
35 Feng SY,Ota K,Yamada Y,et al.A yeast one-hybrid system to detect methylation-dependent DNA-protein interactions.Biochem Biophys Res Commun 2004;313:922-925.
36 Kondapalli J,Flozak AS,Albuquerque ML.Laminar shear stress differentially modulates gene expression of p120 catenin,Kaiso transcription factor,and vascular endothelial cadherin in human coronary artery endothelial cells.J Biol Chem 2004;279:11417-11424.
37 Barker N,van Es JH,Kuipers J,et al.Identification of stem cells in small intestine and colon by marker gene Lgr5.Nature 2007;449:1003-1007.
38 Woll PS,Morris JK,Painschab MS,et al.Wnt signaling promotes hemato-endothelial cell development from human embryonic stem cells.Blood 2007;
39 Iioka H,Doerner SK,Tamai K.Kaiso is a bimodal modulator for Wnt/beta-catenin signaling.FEBS Lett 2009;583:627-632.
40 Defossez PA,Kelly KF,Filion GJ,et al.The human enhancer blocker CTC-binding factor interacts with the transcription factor Kaiso.J Biol Chem 2005;280:43017-43023.
41 Myster SH,Cavallo R,Anderson CT,et al.Drosophila p120catenin plays a supporting role in cell adhesion but is not an essential adherens junction component.J Cell Biol 2003;160:433-449.
42 Delia Ragione F,Tiunova A,Vacca M,et al.The X-linked methyl binding protein gene Kaiso is highly expressed in brain but is not mutated in Rett syndrome patients.Gene 2006;373:83-89.
43 Xu X,Li WE,Huang GY,et al.N-cadherin and Cx43alphal gap junctions modulates mouse neural crest cell motility via distinct pathways.Cell Commun Adhes 2001;8:321-324.
44 Geis K,Aberle H,Kuhl M,et al.Expression of the Armadillo family member p120cas1B in Xenopus embryos affects head differentiation but not axis formation.Dev Genes Evol 1998;207:471-481.
45 Hecht A,Litterst CM,Huber O,et al.Functional characterization of multiple transactivating elements in beta-catenin,some of which interact with the TATA-binding protein in vitro.J Biol Chem 1999;274:18017-18025.
46 Papkoff J.Regulation of complexed and free catenin pools by distinct mechanisms.Differential effects of Wnt-1 and v-Src.J Biol Chem 1997;272:4536-4543.
47 Xu X,Li WE,Huang GY,et al.Modulation of mouse neural crest cell motility by N-cadherin and connexin 43 gap junctions.J Cell Biol 2001;154:217-230.
48 Aberle H,Bauer A,Stappert J,et al.beta-catenin is a target for the ubiquitin-proteasome pathway.Embo J 1997;16:3797-3804.
49 Gumbiner BM.Coordinate gene regulation by two different catenins.Dev Cell 2005;8:795-796.
50 Perez-Moreno M,Fuchs E.Catenins:keeping cells from getting their signals crossed.Dev Cell 2006;11:601-612.
51 Amini Nik S,Ebrahim RP,Van Dam K,et al.TGF-beta modulates beta-Catenin stability and signaling in mesenchymal proliferations.Exp Cell Res 2007;313:2887-2895.
52 Wittier L,Shin EH,Grote P,et al.Expression of Msgn1 in the presomitic mesoderm is controlled by synergism of WNT signalling and Tbx6.EMBO Rep 2007;8:784-789.
53 Cohen ED,Wang Z,Lepore JJ,et al.Wnt/beta-catenin signaling promotes expansion of Isl-1-positive cardiac progenitor cells through regulation of FGF signaling.J Clin Invest 2007;117:1794-1804.