【作者】 林雪迟；

【导师】 曹亚；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2008， 博士

【摘要】 信号转导和细胞周期调控理论认为:肿瘤的形成是细胞调节通路及其构成网络整体紊乱的结果。各种致瘤因素在不同的时空点异常激活细胞内信号转导通路,并引起调节失调,进而引发细胞周期重要检测点紊乱,从而造成肿瘤细胞恶性生长优势。EBV与鼻咽癌发病密切相关。在鼻咽癌细胞中,EBV潜伏感染时主要表达两种潜伏膜蛋白LMP1、LMP2,其中LMP1已被确证具有瘤蛋白功能,可以促进B淋巴细胞、上皮细胞转化,使永生化的细胞具有致瘤性。我们已经证实,EB病毒瘤蛋白LMP1可以异常激活AP-1、NFκB、STAT三条信号转导通路和磷酸化这三条信号通路中的EGFR,JNK,JAK,STAT,IκB,c-Jun等蛋白质分子,影响细胞周期进程,促进细胞的生长发育与分化增殖。EB病毒瘤蛋白LMP1调节的下游磷酸化蛋白质分子,重要的信号分子或下游功能效应蛋白质分子,远远没有得到全面地阐明。我们前期利用磷酸基团金属离子亲和层析(PMAC)技术富集磷酸化蛋白质策略,结合蛋白质组学高通量技术,在LMP1介导AP-1、NFκB和STAT三条信号转导通路的基础上,依据生物信息学预测,建立了LMP1介导的信号转导通路虚拟网络,获得了25个新的受LMP1调控的差异磷酸化蛋白质分子,其中包括Op18/stathmin。stathmin是一种遗传上高度保守的小分子磷蛋白,在淋巴瘤与乳腺癌中高表达,又被称为癌蛋白18(Oncoprotein 18,Op18)。Op18/stathmin集成细胞内外各种信号,以微管蛋白为作用底物,直接调控微管蛋白的聚合与解聚。微管是细胞骨架的主要成分,对维持细胞的形状,细胞内的运输,细胞黏附与移行,细胞增殖,分化和死亡至关重要。Op18/stathmin集成细胞内外不同的信号调节微管动力学,在细胞生物学性状的维持方面发挥重要作用。Op18/stathmin磷酸化与去磷酸化与细胞周期的进程密切相关,受细胞周期及与细胞周期相关的多种激酶调控。在LMP1调控的虚拟信号网络中,我们发现LMP1有可能通过MAPK与cdc2介导Op18/stathmin的信号通路的调控。MAPK是一类Ser/Thr蛋白激酶,是细胞内信号传导链的重要整合点,诱导细胞增殖、分化与生存。MAPK与野生型与突变型Op18均能结合。Op18的两个磷酸化位点Ser25/Ser38后都带有一个脯氨酸残基,成为合适的MAPK底物,MAPK家族中3个亚家族ERK、JNK、p38活化后,均能磷酸化Op18 Ser25/Ser38位点。Cdc2是一个典型的推动细胞周期演进的正性调控因子,是M期事件启动和进行的必要条件。cdc2激酶活性与cyclinB1周期性生产,累积和降解机制有关,有明显的细胞周期依赖性。cdc2激酶可以直接磷酸化Op18/stathmin的Ser25/Ser38位点,当Op18-25A/38A靶点突变,会导致缺陷细胞快速向G2期聚集,引起有丝分裂M期染色体分离缺陷。确立LMP1通过MAPK与cdc2介导的对Op18/stathmin信号通路,将进一步完善了LMP1信号通路的调控网络,确立Op18/stathmin为LMP1的一个新的效应分子提供分子证据,对阐明LMP1在癌变中的分子机制有重要的意义。1.LMP1对Op18/stathmin表达与磷酸化调节CNE1是LMP1阴性的鼻咽癌细胞,CNE1-LMP1是稳定表达LMP1的鼻咽癌低分化鳞状癌细胞;LMP1受DOX诱导表达的Tet-on-LMP1-HNE2鼻咽癌细胞;来源于SV40T转化的永生化鼻咽部上皮细胞NP69、NP69-PLNSX、NP69-LMP1的NP69细胞系列。通过不同细胞株的研究表明,鼻咽癌细胞与人永生化鼻咽部细胞,LMP1对Op18/stathmin表达没有影响,也没有时间与剂量依赖性;LMP1显著诱导Op18/stathmin磷酸化水平上调;但在鼻咽部黏膜上皮细胞转化的永生化细胞NP69系列,LMP1诱导Op18/stathmin磷酸化改变不明显。LMP1调节Op18/stathmin主要通过影响Op18/stathmin磷酸化实现的,其对肿瘤细胞与永生化的细胞作用机制可能存在一定的差异。2.LMP1通过MAPK对Op18/stathmin信号通路的调控在鼻咽癌细胞中,LMP1能否通过MAPK调控Op18/stathmin信号通路。LMP1阴性的CNE1与稳定表达LMP1的CNE1-LMP1鼻咽癌低分化鳞状癌细胞系,通过细胞同步化,特异阻断MAPK信号通路,免疫共沉淀,可溶性与聚合性微管蛋白的萃取,Western-blot分析等方法,研究LMP1对MAPK激酶活性影响,MAPK与Op18/stathmin相互作用的改变,微管动力学变化,及LMP1调控MAPK活性改变与细胞周期的相关性。研究发现,当细胞绝大部分处于G1/S期,LMP1上调p38、ERK、JNK/MAPK磷酸化,其中ERK磷酸化水平显著升高;当绝大部分细胞处于G2/M期时,与G1/S期刚好相反,LMP1负性调控p38、ERK和JNK/MAPK激酶磷酸化,ERK磷酸化水平下降最为显著。为了证实Op18/stathmin失稳定微管的活性,我们设计了靶向Op18/stathmin的siRNA重组载体,观察在Op18/stathmin基因表达被抑制情况下,微管动力学的改变。研究发现,RNAi有效抑制Op18/stathmin转录及Op18/stathmin蛋白表达;转染pGCsi.U6/neo/GFP空白质粒与pGCsi.U6/neo/GFP-NON无意义质粒的对照组中,可溶性微管蛋白水平无明显变化,但转染pGCsi.U6/neo/GFP-RNAi质粒的处理组,可溶性微管蛋白水平明显下降。证实,在鼻咽癌细胞中,活性Op18/stathmin促进微管解聚,抑制Op18/stathmin活性表达,将促进微管的稳定。采用免疫共沉淀方法,确定DZ1阻断LMP1表达的情况下,MAPK与Op18/stathmin相互作用及与LMP1调控关系。结果可见,随DZ1抑制浓度加大,与Op18/stathmin相互作用的MAPK磷酸化水平下降;DZ1阻断可导致可溶性微管蛋白比例增加,聚合性微管蛋白比例减少,与LMP1正性调控MAPK激酶活性,导致Op18/stathmin磷酸化水平增加,失稳定微管活性下降,促进微管聚合作用相一致。由于未经秋水仙胺处理细胞绝大多数处于G1/S期(83.9%),说明在G1/S期,LMP1通过诱导MAPK激酶表达与磷酸化介导Op18/stathmin磷酸化失活,促进微管聚合,保证间期微管的相对稳定。PD98059选择性阻断MAPK家族中的主要传导通路ERK/MAPK,随PD98059浓度抑制剂浓度增加,p-Op18水平有所下降,微管解聚,聚合性微管蛋白比例下降。在G1/S期,LMP1通过MAPK介导正性调控Op18/stathmin信号;在G2/M期,LMP1通过MAPK介导负性调控Op18/stathmin的信号;在MAPK家族,LMP1主要通过ERK/MAPK介导影响Op18/stathmin信号,是主要的调控途径。G2/M期,LMP1负性调节MAPK活性机制有多种可能性,如cdc2、p53等的参与。3.LMP1通过edc2调控Op18/stathmin信号通路由于Op18/stathmin信号传导与细胞周期密切相关,而cdc2是一种有丝分裂期依赖激酶,细胞周期的正向调控子,我们进一步检测cdc2活性变化与LMP1相关性,探索LMP1通过cdc2介导Op18/stathmin信号通路的调控。采用M期细胞同步化处理,cdc2激酶活性分析,免疫共沉淀分析cdc2与Op18/stathmin相互作用,微管动力学分析与免疫荧光分析等发现,LMP1不影响cdc2表达;M期,LMP1显著上调cdc2的Thr161磷酸化与cdc2激酶活性,Op18/stathmin磷酸化水平同步升高。CO-IP-Western-blot分析发现,LMP1促进cdc2与Op18/stathmin相互作用,这种相互作用在M期被显著增强;免疫荧光与Western-blot分析发现,有丝分裂期,LMP1诱导纺锤体微管形成,与Op18/stathmin磷酸化失活,促进微管聚合作用相一致。特异性靶向LMP1的脱氧核酶抑制剂DZ1抑制LMP1表达后,cdc2激酶活性下调,cdc2与Op18/stathmin相互作用减弱,聚合性微管蛋白比例下降;CDK1阻断剂Purvalanol A能有效抑制cdc2激酶活性,抑制Op18/stathmin磷酸化,促进微管解聚,进一步说明LMP1可以通过cdc2介导调节Op18/stathmin信号通路。小结我们采用鼻咽癌细胞为模型,采用细胞同步化,可溶性与聚合性微管蛋白萃取,免疫荧光,Western-blot分析等技术,结合信号传导中信号阻断策略,研究LMP1对Op18/stathmin信号通路的调控,取得了如下发现。1)LMP1不影响Op18/stathmin表达,但上调Op18/stathmin磷酸化。对鼻咽部上皮细胞转化的永生化细胞NP69系列影响不明显,提示在鼻咽癌细胞与鼻咽部黏膜上皮细胞,LMP1调节Op18/stathmin信号通路机制可能存在差异。2)在鼻咽癌细胞,首次证明LMP1对MAPK活性调控与细胞周期相关联。在G1/S期,LMP1正性调节MAPKs系列激酶;在G2/M期,负性调节MAPKs激酶活性,其中ERK/MAPK是其主要的调控途径;靶向Op18/stathmin的RNAi实验证实,Op18/stathmin在NPC细胞是一种微管失稳定活性分子,抑制Op18/stathmin的表达,会促进微管聚合。在G1/S期,LMP1促进MAPK与Op18/stathmin相互作用。LMP1通过MAPK介导的Op18/stathmin信号通路的调控,主要发生在G1/S期,与间期的微管稳定性有关。3)LMP1对细胞周期正向调控因子cdc2的表达无影响,但LMP1能上调cdc2的Thr161的磷酸化水平,促进M期cdd2激酶活性显著上升;在M期,LMP1诱导Op18/stathmin磷酸化水平显著上升,与cdc2激酶活性改变一致;LMP1促进cdc2与Op18/stathmin相互作用,促进有丝分裂期纺锤体微管的聚合。LMP1通过cdc2介导的Op18/stathmin信号通路的调控,主要发生在M期,与有丝分裂期纺锤体形成有关。以上结果证明:LMP1能通过MAPKs与cdc2激酶调控Op18/stathmin信号通路,影响微管聚合与解聚的动态平衡,这种作用与细胞周期密切相关,具有细胞周期特异性。两条信号通路作用机制不一样,其调控的生物学效应也有差异,在细胞间期,LMP1主要通过MAPK正性调控Op18/stathmin信号通路,维持间期微管的稳定:在M期,LMP1通过上调cdc2激酶活性调控Op18/stathmin信号,促进有丝分裂期纺锤体形成。这两条新的信号通路进一步完善了LMP1的调控网络,对阐明LMP1在癌变的分子机制提供了更多的依据。更多还原

【Abstract】 The theory on the regulation of signaling transduction and cell cycle considers that the aberrant activation of signaling pathway of regulated network can induce tumorigenesis,and the activation of various oncogene and oncoprotein signaling pathways which leads to the disruption of cell cycle check points and the preferred growth of cancer cells.Epstein-Barr virus(EBV)is closely associated with nasopharyngeal carcinoma(NPC),EBV infection is mainly characterized by the expression of typeⅡlatent proteins including latent membrane proteins (LMP)1,2.Of which LMP1 is an EBV encoded oncogenic protein that is able to induce tumorigenesis such as promoting cellular transformation of B lymphocytes and epithelial cells,and immortalized cells to neoplastic cells.Previous studies on LMP1 regulation demonstrated that EBV-encoded LMP1 abnormally activated nuclear factor-kappa B (NF-κB),activator protein 1(AP-1),and signal transducer and activator of transcription(STAT)signaling pathways by phosphorylating epidermal growth factor receptor(EGFR),c-Jun N-terminal kinase (JNK),Janus kinase(JAK),and other moleculars.Activation of these signaling pathways by LMP1 has been linked to cell cycle progression, to cell growth and proliferation,but the downstream target proteins regulated by LMP1 have not been thoroughly identified.Using phosphate metal affinity chromatography(PMAC)to enrich phosphoproteins,combined with matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry(MALDI-TOF-MS)analysis, this study showed that phosphorylation levels of 25 novel proteins for LMP1 expression were changed obviously,including Op18/stathmin, which are possible downstream target molecules of LMP1.On the basis of these results,a suppositional signaling network for LMP1 regulation was established through prediction via bioinformatics.This study focused on Op18/stathmin,a small-molecule phosphoprotein in the complicated suppositional signaling network regulated by LMP1.Op18/stathmin is genetically a highly conserved, small cytosolic phosphoprotein,which is expressed at high levels in tumors such as leukemia and breast carcinomas,so also named oncoprotein 18.Op18/stathmin has been hypothesized to relay the integration of diverse cell signals to regulate microtubule dynamics. Microtubule dynamic equilibrium is crucial to cell morphological stabilization,substance transportation,cell division and proliferation, tumor migration and invasion,and so on.Similarly,Op18/stathmin, as a main regulator of microtubule dynamics,plays a significant role in maintaining cell biological characteristics,the phosphorylation/ dephosphorylation of Op18/stathmin is tightly linked to cell cycle progression.The suppositional signaling network on LMP1 regulation suggests that LMP1 maybe regulate Op18/stathmin signaling pathway by MAPK or cdc2 mediation.MAPK is a type of Ser/Thr kinases which can combine with wild type or mutant Op18/stathmin.MAPK family mainly consists of three subfamilies,extracelluar signal regulated protein kinase(Erk),Jun N-terminus kinase(JNK)and p38.Studies had testified that Op18/stathmin is a good substrate of MAPK for both Set25 and Set38 of Op18/stathmin’s phosphorylation sites with a proline residue.MAPK prefers to phosphorylate Ser25 and Ser38 of Op18/stathmin.Activated p38,Erk,JNK all can phosphorylate Op18/stathmin at Ser25/Ser38.Cdc2 is a crucial kinase to start M phase event during cell cycle progression,and a positive regulator promoting cell cycle processes. Cdc2 activation depends on cyclin B1 production,accumulation, degradation and cell cycle progression.It is confirmed that cdc2 can directly phosphorylate Ser25 and Ser38 of Op18/stathmin,the expression of cdc2 kinase target site-deficient mutant of Op18/stathmin (Op18-Ser25A,38A)resulted in rapid accumulation of cells at G2 phase of cell cycle,and a serious defect of mitotic chromosome segregation.This study focused on these two new pathways of cdc2 and MAPK mediation via which LMP1 regulates Op18/stathmin signaling.These established pathways not only perfect the LMP1 regulation network,but so provide new insight elucidating the molecular mechanism of LMP1 that leads to carcinogenesis.1.Effects of LMP1 regulation on the expression and the phosphorylation of Op18/stathminCNE1 is an LMP1-negative poorly differentiated NPC cell line,the CNE1-LMP1 cell line stably expressing LMP1 was established in our laboratory;Tet-on-LMP1 HNE2 is a NPC cell line in which the expression of LMP1 would be turned on by Doxycline;NP69 series cell lines including NP69,NP69-PLNSX and NP69-LMP1 cells derived from a newly established,SV40T-immortalized nasopharyngeal(NP) cells,of which NP69-LMP1 is a NP cell line expressing LMP1. Western-blot analysis showed that Op18/stathmin expression was not affected by LMP1 among CNE1,CNE1-LMP1 and NP69 series cell lines.Tet-on-LMP1 HNE2 cells,in which the expression of LMP1 was increased with Doxycline addition in a dose-dependent manner,western blot analysis performed in Tet-on-LMP1 HNE2 cells treated with Doxycline at different dose and time points showed that the expression of Op18/stathmin was not affected with LMP1 induction in time and dose-dependent manners.This study also demonstrated that LMP1 enhanced the phosphorylation of Op18/stathmin between CNE1 and CNE1-LMP1 cells.The level of phosphorylation of Op18/stathmin was also markedly up-regulated by LMP1 in the Tet-on-LMP1 cells treated with Doxycline.Analysis of the status of Op18/stathmin phosphorylation of NP69 series cells,which derived from immortalized nasopharyngeal (NP)cells,showed that the change of phosphorylation of Op18/stathmin was not obvious by LMP1 induction.All these results demonstrated that LMP1 could regulate Op18/stathmin signaling through promoting the phosphorylation of Op18/stathmin,but the mechanism by which LMP1 regulates Op18/stathmin signaling may be different between NPC cells and normal epithelial cells.2.Regulation of MAPK mediated Op18/stathmin signaling pathway triggered by EBV encoded LMP1 in nasopharyngeal carcinoma cellsTo testify whether LMP1 can regulate Op18/stathmin signaling by MAPK mediation,we still chose CNE1 and CNE1-LMP1 cell models, took multiple methods including cell synchronization with colcemid, flow cytometric analysis,CO-IP-Western-blot and Extraction of solubilized and polymerized microtubulin to examine the change of MAPK activity,the interaction of MAPK with Op18/stathmin,the status of microtubule dynamics and the correlativity of cell cycle progression for LMP1.FACS analysis showed that the majority of cells untreated with cocemid stayed at G1/S.LMP1 enhanced the levels of the phosphorylation of p38,ERK,JNK/MAPK,of which the change of phosphorylation of ERK/MAPK was the most obvious when these cells stayed at G1/S phase;On the contrary,LMP1 negatively regulated the phosphorylation of MAPK in these cells stayed at G2/M phase. Similarly,the down-regulation of the levels of ERK/MAPK phosphorylation was the most obvious in MAPK family.The activity of Op18/stathmin destabilizing microtubules was analyzed,the recombinant carrier targeted Op18/stathmin was constructed to study the status of microtubule dynamics when the experession of Op18/stathmin was inhibited.This study showed that RNAi effectively inhibited the transcription and expression of Op18/stathmin,resulted in the decrease of levels of solublized microtubulin.All evidences indicated that Op18/stathmin can regulate microtubule dynamics and promotes microtubule depolymerization in NPC.Inhibition of LMP1 expression by LMP1-specific DNAzyme DZ1 attenuated the phosphorylation of MAPK and the interaction of MAPK with Op18/stathmin,and the levels of the polymerized tubulin was markedly down-regulated,which was accordant with the regulation of Op18/stathmin dephosphorylation.When PD98059 selectively blocked the ERK/MAPK signaling in CNE1-LMP1 cells,the phosphorylation of Op18/stathmin was weakened with the ERK/MAPK signaling repression,and the solublized tubulin was increased.These results indicated that LMP1 positively regulates Op18/stathmin signaling by MAPK mediation at G1/S phase,but at G2/M phase,LMP1 negatively regulates Op18/stathmin signaling by MAPK mediation;LMP1 mainly depends on ERK/MAPK pathway to regulate Op18/stathmin signaling in MAPK family;There are multiple possibilities on the mechanism by which LMP1 negatively regulates Op18/stathmin signaling,such as cdc2,p53 involved in.3.LMP1 regulates Op18/stathmin signaling pathway by cdc2 mediation at G2/M phase in nasopharyngeal carcinoma cellsThe signaling of Op18/stathmin is closely associated with cell cycle progression,and cdc2 is a positive regulation factor of cell cycle.We inspected the activity change of cdc2 induced by LMP1 at M phase and the possibility that LMP1 regulates Op18/stathmin signaling by cdc2 mediation.By these ways such as extraction of solublized and polymerized microtubulin,cell synchronization,cdc2 kinase activity assay, co-immunoprecipitation analysis and immunofluorescence staining, studies showed that LMP1 didn’t affect the expression of cdc2 and Op18/stathmin whether at different phase;LMP1 upregulated the levels of phosphorylation of cdc2 on threonine 161 and cdc2 kinase activity, the levels of phosphorylated Op18/stathmin was greatly enhanced at M phase related to G1/S phase.Inhibition LMP1 expression by LMP1-specific DNAzyme DZ1 attenuated the interaction of cdc2 with Op18/stathmin and promoted microtubule depolymerization,cdc2 kinase activity was also weakened;the inhibitor of CDK1 Purvalanol A effectively inhibited cdc2 kinase activation and the phosphorylation of Op18/stathmin,and promoted microtubule depolymerization.All these results indicated that LMP1 regulates cdc2 signaling by cdc2 mediation at M phase. Summary:Using stratges including cell synchronization,solublized microtubulin and polymerized microtubulin extraction, immunofluorescence staining,CO-IP-Western-blot analysis and signaling blockage to explore the regulation of Op18/stathmin signaling induced by LMP1,we gained some innovative findings as follows:1)Studies firstly demonstrated that LMP1 doesn’t affect the expression of Op18/stathmin,but enhanced the phosphorylation of Op18/stathmin;the status of Op18/stathmin phosphorylation of NP69 series cell lines which derived from immortalized nasopharyngeal(NP) cells showed that the change of phosphorylation of Op18/stathmin was not obvious by LMP1 induction,which indicated that the mechanism by which LMP1 regulates Op18/stathmin signaling in NPC isn’t the same with normal tissues.2)It is first to find that the activity of MAPK induced by LMP1 is associated with cell cyle progression.At G1/S phase,LMP1 positively regulates the activation of MAPK,on the contrary,negatively regulates MAPK activity at G2/M phase;ERK/MAPK is the mainly pathways regulated by LMP1 in MAPK family.RNAi showed that Op18/stathmin regulated microtubule dynamics and promoted microtubules depolymerization in NPC.LMP1 promoted the interaction of MAPK with Op18/stathmin and microtubule polymerization at G1/S phase.LMP1 regulated Op18/stathmin signaling by MAPK mediation mainly acts to keep interphase microtubule stable.3)It is first to demonstrate that LMP1 regulates Op18/stathmin signaling by cdc2 mediation at mitotic phase.LMP1 up-regulated the phosphorylation of cdc2 on threonine161 and cdc2 kinase activity,but doesn’t change the expression of cdc2.LMP1 promotes the phosphorylation of op18/stathmin and spindle assembly,enhances the interaction cdc2 with Op18/stathmin,cdc2 mediated Op18/stathmin signaling regulated by LMP1 mainly functions at mitotic phase,which is associated with mitotic spindles assembly.All above provide experimental proof on LMP1 regulated Op18/stathmin signaling by cdc2 and MAPK mediation.The regulation influences the equilibria of microtubule dynamics which is associated with cell cycle,and is characterized by time-phase speciality.The mechanism by which LMP1 regulates Op18/stathmin signaling is different at different phase.At G1/S phase,LMP positively regulates Op18/stathmin signaling by MAPK mediation,which mainly keep interphase microtubule stable;At M phase,LMP1 up-regulates cdc2 kinase activity to regulate Op18/stathmin signaling,which mainly promotes spindle formation.These results reveal two new pathways via which LMP1 regulates Op18/stathmin signaling by cdc2 and MAPK mediation.These new pathways not only perfect the LMP1 regulation network,but also provide new insights elucidating the molecular mechanism of LMP1 that leads to tumorigenesis.更多还原