【作者】 翟惠虹；

【导师】 樊代明；

【作者基本信息】 第四军医大学 ， 内科学， 2007， 博士

【摘要】 CacyBP （CalCyclin（S100A6）-binding-protein,钙周期素结合蛋白）,是1998年发现的以钙依赖的方式结合CalCyclin的蛋白;2001在研究P53刺激下β-catenin泛素化降解新通路时发现,SIP（Siah-1 Interacting Protein）,通过与泛肽连接酶（Skp1-Cullin-F-box,SCF Ebi）结合,参与P53刺激下β-catenin的降解。进一步的研究发现:SIP即CacyBP,因此目前命名为CacyBP/SIP。CacyBP/SIP是S100家族的靶蛋白,S100家族是Ca²⁺结合蛋白家族中最大的亚类,以组织特异性的方式作为钙信号的传导器,与特异的靶蛋白相互作用,介导钙信号对细胞的调控。现发现CacyBP/SIP可与S100A1、A6、A12、B、P结合,它们均与肿瘤的进展/转移有关。近有研究表明CacyBP/SIP与细胞分化、发育有关。在研究小鼠受孕子宫发育时发现,随着时间的推移,CacyBP/SIP的表达逐渐升高,第7天时达高峰,其表达受雌、孕激素的调节,表明它与受孕子宫内膜细胞的发育有关;心肌肥大时CacyBP/SIP表达上调,随后的研究发现它可促进H9C2细胞及小鼠心肌的分化及DNA合成。已有两家研究机构报道,在神经细胞内CacyBP/SIP具有依赖于Ca²⁺浓度的核转位及磷酸化现象。Ca²⁺是细胞内最重要的第二信使,通过其浓度的变化来传递信息。在神经细胞内Ca²⁺浓度升高,CacyBP/SIP转位至细胞核,同时发生磷酸化;细胞内Ca²⁺浓度降低时,CacyBP/SIP转位至细胞浆,同时发生去磷酸化。蛋白转位至细胞核且发生磷酸化对于传递细胞外信号,调控下游基因表达具有重要意义。但这种现象究竟有何意义呢?本课题将对此进行探讨。【目的】1、CacyBP/SIP单克隆抗体的制备;2、CacyBP/SIP在正常组织和肿瘤组织中的表达分布;3、CacyBP/SIP在结肠癌组织中的表达及功能;4、CacyBP/SIP核转位影响结肠癌细胞功能的可能机制。【方法】1、应用淋巴细胞杂瘤技术制备小鼠源性抗人CacyBP/SIP MAb,采用Western Blot及ELISA等方法鉴定抗体的特异性和敏感性;2、应用正辛酸-饱和硫酸铵方法纯化抗体,通过SP免疫组织化学技术,观察CacyBP/SIP在正常组织和肿瘤组织中的分布;3、利用免疫组化、Western Blot观察CacyBP/SIP在结肠癌、癌旁组织、结肠癌细胞中的表达和细胞定位;4、通过基因重组方法构建CacyBP/SIP的siRNA载体、全长载体、C端截短体;5、间接免疫荧光细胞内染色、Western Blot检测CacyBP/SIP在胃泌素诱导下在结肠癌细胞中的定位;6、利用细胞MTT实验、平皿克隆形成试验、细胞周期检测等研究CacyBP/SIP入核对结肠癌细胞SW480和HT29增殖的影响;7、通过Western Blot、激酶实验、co-IP观察CacyBP/SIP入核后细胞周期蛋白及活性的变化;8、应用蛋白酶抑制剂通过抑制泛素-蛋白酶体通路探讨细胞周期蛋白改变的机制;9、利用激光共聚焦观察C端截短体在细胞内定位;10、利用Western Blot、co-IP观察CacyBP/SIP截短体转染细胞后细胞周期蛋白表达变化。【结果】1制备了CacyBP/SIP单克隆抗体获得3株抗CacyBP/SIP的单克隆抗体,其亚型均为IgG（κ）亚类,间接ELISA测定腹水效价达1×10^-7,Western Blot及ELISA表明三株MAb具有较高的特异性与敏感性,均能识别组织及细胞内天然及变性CacyBP/SIP蛋白。2 CacyBP/SIP在正常及肿瘤组织中的分布以获得单抗为工具,较系统研究CacyBP/SIP在正常组织及肿瘤组织中的表达。通过IHC染色发现:心脏、脑中CacyBP/SIP呈强染色,主要表达于心肌细胞、神经元及神经胶质细胞;在胃、结肠、肝等弱表达或表达缺失。在大多数腺上皮起源的肿瘤中,CacyBP/SIP均着色,包括胃癌、结肠癌、直肠癌等,其中在胰腺癌中显示出较强的着色,在鼻咽癌中着色最强。CacyBP/SIP亦可在鳞状上皮起源的肿瘤中着色,如肺鳞癌及食道鳞癌。我们还发现CacyBP/SIP在其它细胞来源的肿瘤中着色:如膀胱/输尿管移行细胞癌,神经胶质瘤,骨肉瘤。在肝癌、黑色素瘤及卵巢癌中着色罕见或缺失。CacyBP/SIP在多数正常组织不表达或弱表达,而在多数肿瘤组织中表达或表达增强,这一结果提示CacyBP/SIP可能在肿瘤的发生发展起作用。3 CacyBP/SIP在结肠癌中高表达我们利用免疫组化技术检测了CacyBP/SIP在10例正常结肠粘膜,50例结肠癌及癌旁组织中的表达,发现CacyBP/SIP主要定位于结肠癌细胞的胞浆和胞核,在正常结肠粘膜表达缺失,结肠癌表达阳性率（51%）,明显高于癌旁组织（26%,p<0.05）。Western Blot显示CacyBP/SIP在结肠癌中的表达明显高于相应癌旁组织（p<0.05）。对3种结肠癌细胞系中CacyBP/SIP表达显示,HT29及SW480细胞中均表达CacyBP/SIP。以上研究结果说明CacyBP/SIP在结肠癌中高表达,可能在结肠癌的发生发展中起作用。4胃泌素可诱导CacyBP/SIP转位至细胞核胃泌素作为内分泌激素,除具有促进胃酸分泌的功能,还是体内重要的生长因子,研究已证实它可促进正常结肠粘膜及结肠癌的增殖,与受体结合后可升高细胞内钙离子;而CacyBP/SIP具有依赖钙离子浓度的核转位。那么,胃泌素可否诱导CacyBP/SIP核转位呢?我们给予胃泌素刺激后,间接免疫荧光、Western Blot显示CacyBP/SIP转位至细胞核。我们进而构建了CacyBP/SIP的两个siRNA载体,稳定转染了结肠癌SW480和HT29细胞,发现CacyBP/SIPsi1载体能显著抑制结肠癌细胞中CacyBP/SIP的表达,命名为SW480-CacyBP/SIPsi及HT29-CacyBP/SIPsi,此细胞在胃泌素刺激后,间接免疫荧光、Western Blot显示CacyBP/SIP无转位现象发生。我们通过给予胃泌素诱导CacyBP/SIP核转位,建立了研究CacyBP/SIP入核功能的细胞模型;通过抑制CacyBP/SIP的表达,建立了研究CacyBP/SIP入核受抑的细胞模型。5 CacyBP/SIP入核可促进结肠癌增殖我们采用MTT法、平皿克隆形成试验探讨胃泌素诱导CacyBP/SIP核转位后对结肠癌细胞增殖的影响,结果发现:与未加胃泌素的对照细胞相比,给予胃泌素后促细胞增殖的作用增强（P<0.05）,促细胞集落形成明显增加（P< 0.05）;细胞周期结果表明:与未刺激组相比,给予胃泌素后,SW480与HT29细胞的G1期明显缩短。在SW480-CacyBP/SIPsi及HT29-CacyBP/SIPsi中,CacyBP/SIP入核受抑,MTT、平皿克隆形成试验表明:与未加胃泌素的对照细胞相比,给予胃泌素后细胞增殖的差别无显著性差异（P>0.05）,促细胞集落形成的能力无明显差异（P>0.05）。细胞周期结果表明:与未刺激组相比,给予胃泌素后,SW480-CacyBP/SIPsi及HT29-CacyBP/SIPsi细胞的G1期无明显变化。以上研究提示CacyBP/SIP核转位可促进结肠癌增殖,这种增殖作用可能通过促进细胞周期进展而实现的。6 CacyBP/SIP通过增强泛素介导的P27^kip1降解促结肠癌增殖为进一步明确CacyBP/SIP核转位后促进细胞周期进展的分子机制,我们首先应用Western Blot检测G1期进展中关键细胞周期蛋白的表达。应用同步化药物nocodazole处理结肠癌细胞SW480及HT29,结果表明:胃泌素刺激后,P27^kip1表达降低,Cyclin E蛋白表达升高。抑制CacyBP/SIP核转位后P27^kip1与Cyclin E的表达无变化。Cdk2激酶活性检测明显升高,同时细胞中P27^kip1结合Cdk2的量减少。以此结果提示CaycBP/SIP入核后P27^kip1蛋白量减少,Cdk2激酶活性增加,导致G1期进展。给予蛋白酶体抑制剂MG132抑制26S蛋白酶体的活性,结果发现: MG132处理细胞后,P27^kip1、Cyclin E表达无明显变化,说明P27^kip1降解的增强是通过26S蛋白酶通路。P27^kip1是SCF泛素酶的靶蛋白,研究已发现CacyBP/SIP通过其C未端与Skp1结合,我们应用CacyBP/SIP免疫共沉淀亦证实:在结肠癌细胞HT29及SW480中,CacyBP/SIP可以与Skp1结合。我们进而构建了CacyBP/SIP的截短体,CacyBP/SIP （Δ73–228）,缺失了C末端结构域,并克隆入pEGFP/C1表达融合绿色荧光蛋白的质粒pEGFP/C1- CacyBP/SIP （Δ73–228）。转染SW480-CacyBP/SIPsi细胞,激光共聚焦观察C端截短体在胃泌素刺激后亦可入核。免疫共沉淀证实,该截短体不能与Skp1结合,与此同时,P27^kip1的表达则无明显变化。这一结果说明CacyBP/SIP通过与Skp1结合,增强了泛素-26S蛋白酶体复合物对P27^kip1的降解。【结论】本研究发现胃泌素诱导CacyBP/SIP入核后通过增强泛素介导P27^kip1的降解促进结肠癌细胞的增殖。更多还原

【Abstract】 The CalCyclin Binding Protein （CacyBP） was firstly found in the cytosolic fraction of Ehrlich ascites tumor cells interacting with S100A6 （calCyclin） at a physiological range of Ca²⁺ concentration in 1998. Three years later, Siah-1 Interacting Protein （SIP） was confirmed as a human ortholog’s CacyBP. Hence, CalCyclin Binding Protein was formally named as CacyBP/SIP.Further investigation showed that it could also bind other S100 proteins such as S100A1, S100A12, S100B and S100P. Recently, CacyBP/SIP was found to be involved in the development and differentiation of cells. Au et al. reported that overexpression of CacyBP/SIP promotes the differentiation and DNA synthesis in H9C2 cells, primary rat cardiomyocytes. The study of Yang et al. also revealed that progesterone （P4） and 17h-estradiol （E2） increase the expression of CacyBP/SIP gene. Hence, CacyBP/SIP was believed to participate in the regulation of apoptosis, and play an important role in mouse endometrial events such as pregnancy establishment. Interestingly, CacyBP/SIP could be translocated into nucleus and phosphorylated when Ca²⁺ concentration was changed in neurons and neuroblastoma NB-2a cells. This phenomenon has also been observed in retinoic acid-induced neuronal differentiation of neuroblastoma SH-SY5Y cells. However, the significance of CacyBP/SIP nuclear translocation is unknown. So we investigated whether CacyBP/SIP nuclear translocation might influence the function of colon cancer cells in the present work.【Objectives】（1） Establishment and Characterization of CacyBP/SIP Monoclonal Antibody. （2） To detect CacyBP/SIP protein expression in normal and malignant human tissues. （3） To investigate the founction of CacyBP/SIP in colon cancer by expressional and functional studies. （4） To examine the possible mechanisms of colon cancer induced by CacyBP/SIP nuclear translocation.【Methods】（1） The monoclonal antibodies against CacyBP/SIP were established with the lymphocyte hybridoma technology, and identified the corresponding MAbs of specificity and sensitivity with Western Blot and ELISA. （2） Purify the MAbs with caprylic acid-saturated ammonium sulfate and detecting the expression of CacyBP/SIP in normal and tumor tissue using immunohistochemistry. （3） The subcellular location and expression of CacyBP/SIP in normal colon tissues, colon cancer tissues, colon adjacent tissues and colon cancer cells were determined by immunohistochemistry assay and Western Blot. （4） Full-length vector, deletion mutants and siRNA vector of CacyBP/SIP were constructed. （5） The subcellular location of CacyBP/SIP in colon cancer cells induced by gastrin was observed by immuneofluorescence and Western Blot. （6） The effects of CacyBP/SIP nuclear translocation on the proliferation of colon cancer cell lines were respectively investigated by MTT assay, colony formation assays and cell cycle analysis. （7） The expression levels and the activity of cell cycle proteins after CacyBP/SIP nuclear translocation were determined by Western Blot, Cdk2 kinase assays and co-IP. （8） Detecting the changing of cell cycle protein with protease inhibitor MG132. （9） The location of truncation mutant of CacyBP/SIP induced by gastrin with confocal laser microscope. （10） The expression level of cell cycle protein of colon cancer cell transfecting truncation mutant of CacyBP/SIP with Western Blot and co-IP.【Results】1.Establishment and Characterization of CacyBP Monoclonal Antibody.Three hybridoma clones secreted MAb specific to the CacyBP/SIP protein were obtained. Immunoglobulin subclass determination showed that the MAbs were IgG1/κtype. Their ascites potency was 1×10^-7. Western blot and ELISA showed that the MAbs against CacyBP/SIP could recognize CacyBP/SIP protein in both native and denatured forms. These MAbs would act as a usefull tool for the detection of CacyBP/SIP protein in future studies.2. Expression of CacyBP/SIP protein in normal and malignant human tissues.CacyBP/SIP protein expression profiles in a broad range of human normal tissues and carcinomas were analyzed by immunohistochemistry staining with anti-CacyBP/SIP monoclonal antibody produced in our laboratory. CacyBP/SIP was generally localized in the cytoplasm/nucleus. Positive staining of CacyBP/SIP was found in brain, heart and lymph node. Weak stain was shown in rectum, kidney, prostate and esophagus. No CacyBP/SIP was detected in other normal tissues. However, CacyBP/SIP was ubiquitously detected in all kinds of tumor tissues, especially highly expressed in nasopharyngeal carcinoma, osteogenic sarcoma and pancreatic adenocarcinoma. Our results suggested that CacyBP/SIP may play important roles in tumorigenesis of human tumors.3. CacyBP/SIP expression in colon adencarcinoma tissues.To assess the biological roles of CacyBP/SIP in tumor progression, we firstly did immunohistochemistry on surgically removed colon tumors and their benign counterparts. CacyBP/SIP expressions were detected in the cytoplasm/nuclear of colon cancer tissues, with the positive rate 51% of colon adencarcinoma via 26% of colon adjacent tissues （p<0.05）. In contrast, CacyBP/SIP stain was not detectable in 10 cases of normal colon tissues. The specificity of CacyBP/SIP immunoreactivity in tissues was validated by Western Blot analysis in the colon cancer and adjacent normal tissues taken from 4 patients. The result showed that CacyBP/SIP was overexpressed in colon cancerous tissues but not detectable in adjacent normal tissues. CacyBP/SIP protein was also present in the colon cancer cell lines HT29 and SW480. These results provide evidence that CacyBP/SIP expression level may be positively correlated with colon cancer onset or progression of the cancer.4. Nuclear translocation of CacyBP/SIP in colon cancer cells.It was well known that gastrin is a carcinogen in triggering colon cancer and it induces the intracellular Ca²⁺ mobilization. Others studies showed that CacyBP/SIP could be translocated into nucleus when Ca²⁺ concentration was changed. So we surmised that gastrin may induce CacyBP/SIP nucleus translocation by increasing the intracellular Ca²⁺ concentration. To elucidate the effect of gastrin on the intracellular distribution of CacyBP/SIP, we analyzed cultured colon cancer cells before and after gastrin stimulation. In unstimulated cells, CacyBP/SIP was distributed throughout the cytoplasm and CacyBP/SIP could translocate to the perinuclear region upon stimulation of gastrin. These phenomena were also observed with Western Blot.Two CacyBP/SIP-specific siRNA vectors, named CacyBP/SIPsi1 and CacyBP/SIPsi2 were designed and constructed. After cell transfection and G418 screening, CacyBP/SIPsi1 could down-regulate the expression of CacyBP/SIP in HT29 and SW480 effectively. Then cells stably transfected with CacyBP/SIPsi1, HT29-CacyBP/SIPsi1 and SW480-CacyBP/SIPsi1 were chosen for further cellular assay. In transfecting cells, after gastrin stimulation, CacyBP/SIP was distributed throughout the cytoplasm by immunofluorescent and wasn’t detected in the cell nuclear by Western Blot. It was shown that gastrin couldn’t induce CacyBP/SIP nuclear translocation after its expression was surpressed.5. CacyBP/SIP nuclear translocation promotes proliferation and cell cycle progression of colon cancer cells.To explore the effect of CacyBP/SIP nuclear translocation induced by gastrin on colon cancer cells proliferation, MTT assay and colony formation assay were used. The proliferation of HT29 and SW480 cells were enhanced by exogenous administration of gastrin（P< 0.05）. Furthermore, CacyBP/SIP nuclear translocation after stimulation by gastrin dramatically enhanced anchorage-dependent growth as indicated by colony formation in flat （P<0.05）. The cell cycle profile of colon cancer cells treated with gastrin was characterized as decreased percentage of cells in the G1 phase of the cell cycle.In HT29-CacyBP/SIPsi1 and SW480-CacyBP/SIPsi1 transfecting cells, MTT assay showed that proliferation was not significantly changed by exogenous administration of gastrin. At the same time, no enhanced anchorage-dependent growth was observed in transfected cells as indicated by colony formation in flat after stimulation by gastrin. Cell cycle analyses showed that no change appeared in the percentage of cells in the G1 phase of the cell cycle in transfected cells treated with gastrin. So, gastrin could enhance colon cancer cells proliferation via CacyBP/SIP nuclear translocation.6. CacyBP/SIP increasing expression of Cyclin E and decreasing the leval of P27^kip1.To correlate the effect of CacyBP/SIP on cell cycle progression with some molecular effectors of the restriction point, HT29 and SW480 cells were first synchronized with nocodazole. After stimulation by gastrin, the result showed that CacyBP/SIP induced a marked decrease of P27^kip1 expression level and increase of Cyclin E expression level. In HT29-CacyBP/SIPsi1 and SW480-CacyBP/SIPsi1 cells, no significant changes in protein levels of P27^kip1 and Cyclin E were observed. In comparison to cells without gastrin treatment, cells with gastrin treatment displayed less P27^kip1 bound to Cdk2 and elevated Cdk2 kinase activity. These results suggested that P27^kip1 participated in CacyBP/SIP-mediated G1-S shortening in human colon cancer cells after gastrin stimulation.Pretreatment with MG132, the 26S proteasome inhibitor, blocked the CacyBP/SIP-induced reduction of P27^kip1, suggesting the involvement of the 26S proteasome in the degradation of P27^kip1. To date, it has been reported that P27^kip1 seems to be the primary target of the SCFcomplex. Skp1 is the adaptor protein of SCF complex. Recently, based on the domain mapping studies, it has been confirmed that CacyBP/SIP’s C-terminal region is responsible for interaction with Skp1. We also confirmed that CacyBP/SIP could bind Skp1 in HT29/SW480 cells, which is consistent with the consecutive proteasomal degradation of P27^kip1. To preliminarily assess whether CacyBP/SIP increase the degradation of P27^kip1through interaction with Skp1, we constructed the truncated mutation of CacyBP/SIP （Δ73–228） and fused it into pEGFP/C1. Using transient transfect, this mutant could be translocated into nuclear after gastrin induction but failed to interact with Skp1 by co-immunoprecipitation using transfected SW480-CacyBP/SIPsi cells. At the same time, no change was detected in the protein level of P27^kip1 after gastrin induction.【Conclusion】In conclusion, these results suggested that CacyBP/SIP promotes proliferation of colon cancer cells through enhancing ubquitin-mediated degradation of P27^kip1.更多还原