EHD2调节乳腺上皮细胞极性的实验研究

研究EHD2基因对上皮细胞极性的调控作用及其机制,探讨EHD2基因在乳腺癌组织中表达失调的肿瘤分子生物学和细胞生物学意义。方法：以乳腺永生化正常上皮细胞的二维和三维培养为模型,建立EHD2突变体的Doxycycline外源诱导表达体系,应用荧光显微镜和激光共聚焦显微成像技术,观察诱导表达前后细胞在二维培养条件下细胞内β肌动蛋白的微丝组装及聚合情况,以及三维基质胶培养条件下对上皮细胞腺泡形成的影响,综合判断EHD2蛋白在调控上皮细胞极性中的生物学作用。结果：EHD2突变的诱导表达显著影响乳腺上皮细胞的骨架重排,在细胞表面发生密集发散状微刺,细胞内定向排列的β肌动蛋白微丝结构消失。三维培养中,EHD2突变体的引入破坏了原本规则的腺泡型生长,细胞向外侵入基质。结论：EHD2突变可导致乳腺上皮细胞的骨架重排和极性改变,可能是乳腺肿瘤发生的重要机制之一。     

癌胚抗原定旺研究在胃癌早期诊断中的作用

采用金葡菌Ａ蛋白标记辣根过氧化酶免疫组化方法，观察６２例胃癌切片和９４例例胃涂片癌胚抗原反应。结果显示：ＣＥＡ存在于胃癌细胞及办粘膜上皮细胞内，具有极性分布特征细胞分光光度计检测，胃癌细胞内ＣＥＡ含量显著高于胃上皮细胞，且与腺癌分化程度有关；胃涂片细胞ＣＥＡ阳性率高于胃癌病理检出率，二者符合确诊率亦较高。     

连环素在非小细胞肺癌患者中的表达及相关研究

连环素（ｃａｔｅｎｉｎｓ，ｃａｔ）是近年发现的一种胞浆蛋白，包括α－、β－、γ－ｃａｔ三个亚型，它们以一定的比例和上皮细胞粘附分子（Ｅ－ｃａｄｈｅｒｉｎ，Ｅ－ＣＤ）结合形成上皮细胞粘附分子复合物（Ｅ－ＣＤ／ｃａｔ）。它们通过维持Ｅ－ＣＤ正常功能，促进上皮组织形成和维持组织结构完整。目前ｃａｔｅｎｉｎｓ在肺癌中的表达情况尚不清楚，为探讨α－、β－ｃａｔ在肺癌进展中的作用，我们用免疫组化技术检测了非小细胞肺癌（ＮＳＣＬＣ）中α－、β－ｃａｔ表达情况，旨在探讨其与非小细胞肺癌（ＮＳＣＬＣ）患者临床病理…     

癌胚抗原定量研究在胃癌早期诊断中的作用

采用金葡菌Ａ蛋白标记辣根过氧化酶（ＨＲＰ－ＳＰＡ）免疫组化方法，观察６２例胃癌切片和９４例胃涂片癌胚抗原（ＣＥＡ）反应。结果显示；ＣＥＡ存在于胃癌细胞及胃粘膜上皮细胞内，具有极性分布特征；细胞分光光度计（ＣＳＭ）检测，胃癌细胞内ＣＥＡ含量显著高于胃上皮细胞，且与腺癌分化程度有关；胃涂片细胞ＣＥＡ阳性率高于胃癌病理检出率，二者符合确诊率亦较高。提示：ＣＥＡ免疫组化检测可辅助胃癌病理学早期诊断及确定分化程度。     

hMLH1蛋白在食管鳞状细胞癌和不典型增生组织中的表达

目的：探讨同一食管癌患者食管的鳞状细胞癌组织、不典型增生组织、正常鳞状上皮组织中错配修复基因1（human mutl homolog 1，hMLH1）的蛋白表达情况，深入研究它与食管鳞状细胞癌发生发展的关系。方法：取92例食管鳞状细胞癌患者的病变组织，其病理切片中鳞状细胞癌组织、不典型增生组织和正常鳞状上皮组织均存在，采用免疫组织化学的方法，检测hMLH1蛋白在3种不同组织中的表达，并分析其与性别、年龄、分化程度、浸润深度、肿瘤分期、淋巴结转移等临床病理参数之间的关系。结果：hMLH1蛋白在食管鳞状细胞癌组织、不典型增生组织、正常鳞状上皮组织细胞核中的阳性表达率分别为36．96％、56．52％、84．78％，鳞状细胞癌组织和不典型增生组织均显著低于正常食管鳞状上皮组织（P〈0．01）。鳞状细胞癌组织中hMLH1蛋白表达阴性者，年龄较阳性者大（P〈0．05）；hMLH1蛋白表达与肿瘤分期、淋巴结转移等有明显相关性（P〈0．05）。结论：hMLH1基因缺失可能在早期就参与了食管鳞状细胞癌的发生过程，hMLH1蛋白可能抑制或延缓食管鳞状细胞癌的发生和发展。     

上皮间质转化的分子机制研究进展

上皮间质转化(epithelial-mesenchymal transition,EMT)是上皮细胞转化为间质细胞,形成结缔组织或者形成新的上皮组织,这些细胞不会再表达上皮细胞的特性,而是具有间质细胞的形态学和转移的特性,这些特性诱使细胞转移和侵袭。EMT早在上世纪80年代在胚胎发育时发现,目前证明与癌细胞的转移、侵袭存在密切关系[1]。     

E-上皮型钙粘蛋白与肝细胞癌关系研究进展

细胞与细胞或细胞与细胞外基质之间粘附特性的异常表达是恶性肿瘤细胞最基本的生物学特征之一。而E-上皮型钙粘蛋白（E—cadherion，E—cad）作为一种钙依赖性的跨膜糖蛋白，是调节细胞与细胞之间、细胞与基质之间粘附反应的重要媒介，对维持组织结构形态起重要作用，是维护上皮细胞形态和结构完整性和极性的重要分子。近年来研究表明，E—cad与人体肿瘤的侵袭和转移密切相关，关于其异常表达在恶性肿瘤的形成、浸润及复发过程中的作用已成为研究热点。本文就E—cad与肝细胞癌的关系研究进展作一综述。     

上皮钙粘附素与肿瘤分化、浸润及转移的关系

 近几年来，人们对细胞粘附分子(Cell Adhesion Molecules，CAMs)的研究发现胚胎的发育分化和正常组织结构的维持都离不开细胞与细胞之间、细胞与间质之间的相互作用，并清楚地表明在多种疾病包括肿瘤在内，细胞间相互作用被破坏。现已有100多种细胞粘附分子((CAMS)的基因被克隆化，结构和生化性质被阐明，而钙依赖性细胞粘 附分子大家族(Cadherin)是对细胞间连接诱导和维持起重要作用的一类粘附分子，其中钙粘附分子亚群—上皮钙粘附素(E-Cadherin，E-CD)是钙依赖性具有同种上皮亲合性的粘附分子，是维持正常上皮细胞形态完整性和极性的转膜糖蛋白。     

钙粘着素E与肝细胞癌

钙粘着素（E－cadherin)是钙依赖性粘附分子的亚族成员，广泛分布在非神经上皮组织，它能与胞内的特异性分子相结合，参与同种亲和性的细胞－细胞间的粘附、胚胎发育以及正常组织中上皮细胞层的形成和维持。E-cadherin在肝细胞癌（HCC）中表达有其特点，其表达与HCC在体内的生长、浸润、转移、复发和预后关系密切。     

部分粘液素及角蛋白在腺上皮肿瘤中的表达

目前已发现13种类型粘蛋白,各型粘蛋白的表达具有组织和细胞的特征性,其表达的改变在上皮性肿瘤的发生和细胞分化等过程中起重要作用[1]。另外分化的不同阶段及不同类型的上皮细胞表达不同的细胞角蛋白。上皮组织恶变后,其表达的细胞角蛋白与正常组织基本相同,因此细胞角蛋白的     

Cell polarity proteins and cancer

Cell polarity is essential in many biological processes and required for development as well as maintenance of tissue integrity. Loss of polarity is considered both a hallmark and precondition for human cancer. Three conserved polarity protein complexes regulate different modes of polarity that are conserved throughout numerous cell types and species. These complexes are the Crumbs, Par and Scribble complex. Given the importance of cell polarity for normal tissue homeostasis, aberrant polarity signaling is suggested to contribute to the multistep processes of human cancer. Most human cancers are formed from epithelial cells. Evidence confirming the roles for polarity proteins in different phases of the oncogenic trajectory comes from functional studies using mammalian cells as well as Drosophila and zebrafish models. Furthermore, several reports have revealed aberrant expression and localization of polarity proteins in different human tumors. In this review we will give an overview on the current data available that couple polarity signaling to tumorigenesis, particularly in epithelial cells.     

Epithelial cell polarity, stem cells and cancer

After years of extensive scientific discovery much has been learned about the networks that regulate epithelial homeostasis. Loss of expression or functional activity of cell adhesion and cell polarity proteins (including the PAR, crumbs (CRB) and scribble (SCRIB) complexes) is intricately related to advanced stages of tumour progression and invasiveness. But the key roles of these proteins in crosstalk with the Hippo and liver kinase B1 (LKB1)-AMPK pathways and in epithelial function and proliferation indicate that they may also be associated with the early stages of tumorigenesis. For example, deregulation of adhesion and polarity proteins can cause misoriented cell divisions and increased self-renewal of adult epithelial stem cells. In this Review, we highlight some advances in the understanding of how loss of epithelial cell polarity contributes to tumorigenesis.     

What is Drosophila Telling Us About Cancer?

In Drosophila, genetic loss of the tumour suppressor protein Dlg (in dlg mutants) or p127 (in lgl mutants) leads to loss of epithelial structure and excess proliferation in the imaginal discs and brain of the developing larva. These phenotypes show most of the characteristic features of human neoplasia, so study of the gene products may contribute to our understanding of cancer. Both proteins occur in high molecular-mass complexes in the membrane-associated cytoskeleton, and they both appear to play dual roles as structural proteins and active partners in signal transduction. Dlg is a membrane-associated guanylate kinase homolog (MAGUK) found at septate junctions between epithelial cells, as well as at neuromuscular junctions. Specific domains of the protein are required for membrane targeting and for localisation in junctions, and for epithelial cell proliferation control; all of these functions are probably mediated through binding to other proteins. Loss of Dlg results in the absence of septate junctions, delocalisation of several proteins including Fasciclin III, Coracle, actin and tubulin, and loss of cell polarity. p127, although mostly associated with the plasma membrane, is in most cell types also present in the cytoplasm. It shows a dynamic subcellular distribution, and its cytosolic and membrane-associated forms play distinctive roles by interacting with different binding partners, in particular the non-muscle myosin II heavy chain. Defects associated with the lgl temperature-sensitive allele include loss of the columnar organisation of epithelial cells, indicating that p127 contributes to cell structure, presumably by stabilising the plasma membrane. In addition to their organising functions, both Dlg and p127 appear to be involved in signal transduction pathways. Study of these genes shows that some proteins play both structural and functional roles, and that cancer can involve changes in the organisation of signalling pathways in addition to changes in individual pathway components.     

Nectins and nectin-like molecules: roles in cell adhesion,migration, and polarization

Nectins are a family of Ca²⁺-independent immunoglobulin-like cell-cell adhesion molecules consisting of four members, which homophilically and heterophilically trans-interact and cause cell-cell adhesion. Nectin-based cell-cell adhesion is involved in the formation of cadherin-based adherens junctions in epithelial cells and fibroblasts. The nectin-based cell-cell adhesion induces activation of Cdc42 and Rac small G proteins, which eventually regulate the formation of adherens junctions through reorganization of the actin cytoskeleton, gene expression through activation of a mitogen-activated protein kinase cascade, and cell polarization through cell polarity proteins. Five nectin-like molecules (necls), which have domain structures similar to those of nectins, have recently been identified and appear to play different roles from those of nectins. One of them, named necl-5, which does not homophilically trans -interact, but heterophilically trans -interacts with nectin-3, regulates cell migration and adhesion. In this article, the roles and modes of action of nectins and necls in cell adhesion, migration, and polarization are reviewed.     

Human papillomavirus (HPV)‐18 E6 oncoprotein interferes with the epithelial cell polarity Par3 protein

High‐risk human papillomavirus (HPV) infection is the principal risk factor for the development of cervical cancer. The HPV E6 oncoprotein has the ability to target and interfere with several PSD‐95/DLG/ZO‐1 (PDZ) domain‐containing proteins that are involved in the control of cell polarity. This function can be significant for E6 oncogenic activity because a deficiency in cell polarisation is a marker of tumour progression. The establishment and control of polarity in epithelial cells depend on the correct asymmetrical distribution of proteins and lipids at the cell borders and on specialised cell junctions. In this report, we have investigated the effects of HPV E6 protein on the polarity machinery, with a focus on the PDZ partitioning defective 3 (Par3) protein, which is a key component of tight junctions (TJ) and the polarity network. We demonstrate that E6 is able to bind and induce the mislocalisation of Par3 protein in a PDZ‐dependent manner without significant reduction in Par3 protein levels. In addition, the high‐risk HPV‐18 E6 protein promotes a delay in TJ formation when analysed by calcium switch assays. Taken together, the data presented in this study contribute to our understanding of the molecular mechanism by which HPVs induce the loss of cell polarity, with potential implications for the development and progression of HPV‐associated tumours.     

Junctional recruitment of mammalian Scribble relies on E-cadherin engagement

Members of the LAP protein family, LET-413 in Caenorhabditis elegans, Scribble in Drosophila melanogaster, and Erbin, Lano, Densin-180 and hScrib in mammals, have conserved structural features. LET-413 and Scribble are junctional proteins involved in establishing and maintaining epithelial cell polarity. scribble also behaves as a neoplastic tumor suppressor gene. We show here that, in epithelial cells, hScrib is recruited at cell-cell junctions in an E-cadherin-dependent manner as shown by calcium switch assays in MDCK cells, re-expression of E-cadherin in MDA-231 cells treated by 5-Aza-2'-deoxycytidine (5Aza), and siRNA experiments. hScrib is restricted at the basolateral membrane of epithelial cells by its LRR domain, and is enriched in Triton X-100-insoluble fractions. In breast cancers, most lobular tumors did not express hScrib and E-cadherin while ductal tumors had a less frequent downregulation of hScrib. Our data provide additional insights on the modalities of recruitment of hScrib at the cell-cell junctions, and establish a potential link between the E-cadherin and hScrib tumor suppressors.     

Mechanisms of cell adhesion and migration]

In order to understand the molecular mechanisms underlying cancer metastasis, it is important to clarify the mechanisms of cell adhesion and cell migration. When epithelial cells start to migrate, cell-cell junctions are first disrupted. During migration, membrane protrusions, such as lamellipodia and filopodia, are observed externally at the cell front, and retraction at the cell rear. In addition, dynamic reorganization of the actin cytoskeleton, such as disassembly and reassembly of stress fibers at cell-cell and cell-matrix junctions and membrane protrusions, is observed internally. Our experiments have demonstrated that the Rho and Rab family small G proteins coordinately regulate cell adhesion and migration of cultured MDCK cells. The Rho family consists of the Rho, Rac, and Cdc 42 subfamilies. The Rho subfamily regulates stress fiber formation, and integrin-based cell matrix adhesion. Furthermore, the Rac and Cdc 42 subfamilies regulate lamellipodia and filopodia formation, respectively, as well as cadherin-based cell-cell adhesion. The detailed modes of action of these small G proteins remain to be clarified; however, it is known that these proteins regulate cell adhesion and migration through reorganization of the actin cytoskeleton. The Rab family has over thirty members. We have found that some Rab family members are involved in HGF- or phorbol ester-induced endocytosis and exocytosis (recycling) of adhesion molecules such as integrin and cadherin. Endocytosis and exocytosis of these adhesion molecules are accompanied by disassembly and reassembly of the actin cytoskeleton, respectively, in a well coordinated manner. Thus, we propose the cooperative roles of the Rho and Rab families in cell adhesion and migration.     

Tensional homeostasis and the malignant phenotype

Tumors are stiffer than normal tissue, and tumors have altered integrins. Because integrins are mechanotransducers that regulate cell fate, we asked whether tissue stiffness could promote malignant behavior by modulating integrins. We found that tumors are rigid because they have a stiff stroma and elevated Rho-dependent cytoskeletal tension that drives focal adhesions, disrupts adherens junctions, perturbs tissue polarity, enhances growth, and hinders lumen formation. Matrix stiffness perturbs epithelial morphogenesis by clustering integrins to enhance ERK activation and increase ROCK-generated contractility and focal adhesions. Contractile, EGF-transformed epithelia with elevated ERK and Rho activity could be phenotypically reverted to tissues lacking focal adhesions if Rho-generated contractility or ERK activity was decreased. Thus, ERK and Rho constitute part of an integrated mechanoregulatory circuit linking matrix stiffness to cytoskeletal tension through integrins to regulate tissue phenotype.