Susceptibility of Epithelial Cells to Pseudomonas aeruginosa Invasion and Cytotoxicity Is Upregulated by Hepatocyte Growth Factor

Normal cell polarity protects epithelial cells against Pseudomonas aeruginosa invasion and cytotoxicity. Using epithelial cell clones with selective defects in sorting of membrane constituents, and using hepatocyte growth factor pretreatment, we found that polarized susceptibility to P. aeruginosa can be altered without disrupting tight junctions. The results also showed that cellular susceptibility factors for invasion and cytotoxicity are not the same, although both are localized to the basolateral cell surface in polarized epithelial cells.     

Rho GTPases与肿瘤侵袭转移相关性的研究进展

Rho GTPases属小分子量GTPases,作为信号传导通路中重要的“分子开关”,调控细胞形态、骨架重建、迁移及细胞周期与凋亡等诸多过程.近来的研究表明,Rho GTPases家族成员在多种恶性肿瘤中异常表达,并通过调控上皮细胞极性丧失、细胞-细胞间的连接及黏附、细胞骨架重组和迁移运动、细胞外基质的重塑、细胞进入血管淋巴管的能力等方面参与肿瘤细胞的侵袭转移.深入研究Rho GTPases在肿瘤侵袭转移中的作用意义重大,Rho GT-Pases将有望成为判断肿瘤侵袭转移性发展的新指标,并为肿瘤治疗提供新靶点、新方向.     

Epithelial cell polarity affects susceptibility to Pseudomonas aeruginosa invasion and cytotoxicity.

Intact tissues are relatively resistant to Pseudomonas aeruginosa-induced disease, and injury predisposes tissue to infection. Intact epithelia contain polarized cells that have distinct apical and basolateral membranes with unique lipids and proteins. In this study, the role of cell polarity in epithelial cell susceptibility to P. aeruginosa virulence mechanisms was tested. Madin-Darby canine kidney (MDCK) cells, human corneal epithelial cells, and primary cultures of two different types of airway epithelial cells were grown on Transwell filters or in plastic tissue culture wells. P. aeruginosa invasion of cells was quantified by gentamicin survival assays with two isolates that invade epithelial cells (6294 and PAO1). Cytotoxic activity was assessed by trypan blue exclusion assays with two cytotoxic strains (6206 and PA103). Basolateral surfaces of cells were exposed by one of two methods: EGTA pretreatment of epithelial cells or growth of cells in low-calcium medium. Both methods of exposing basolateral membranes increased epithelial cell susceptibility to P. aeruginosa invasion and cytotoxicity. Migrating cells were also found to be more susceptible to P. aeruginosa invasion than confluent monolayers that had established membrane polarity. Monolayers of MDCK cells that had been selected for resistance to killing by concanavalin A were resistant to both cytotoxicity and invasion by P. aeruginosa because they were more efficiently polarized for their susceptibility to P. aeruginosa virulence factors than regular MDCK cells and not because they were defective in glycosylation. These results suggest that there are factors on the basolateral surfaces of epithelial cells that promote interaction with P. aeruginosa or that there are inhibitory factors on the apical cell surface. Thus, cell polarity of intact epithelia is likely to contribute to defense against P. aeruginosa infection.     

Methods to Examine Tight Junction Physiology in Cancer Stem Cells: TEER, Paracellular Permeability, and Dilution Potential Measurements

Our understanding of the essential role played by cancer stem cells or tumor-initiating cells in epithelial cell-derived tumor types is rapidly advancing. Nevertheless, the identification and characterization of these cells pose a considerable challenge. Among changes in the epithelium in oncogenesis are changes in the permeability barrier, a phenotypic trait based on tight junction formation and function. Tight junctions regulate the movement of solutes, ions and water across the paracellular space. On a cellular level, they maintain cell polarity by limiting the lateral diffusion of membrane components. Depending on the type of epithelial tissue, the barrier characteristics with respect to electrical resistance, size and ion charge selectivity vary quite significantly. Thus, elucidating changes in expression of Claudins, an essential component of tight junctions, has become a very active area of investigation in oncogenesis. This chapter provides detailed protocols on how to quantify three aspects of tight junction physiology using in vitro cell culture systems that are particularly applicable to analysis and comparison of cancer stem cells and their normal counterparts.     

Characterization of Porphyromonas gingivalis-induced degradation of epithelial cell junctional complexes

Porphyromonas gingivalis is considered among the etiological agents of human adult periodontitis. Although in vitro studies have shown that P. gingivalis has the ability to invade epithelial cell lines, its effect on the epithelial barrier junctions is not known. Immunofluorescence analysis of human gingival epithelial cells confirmed the presence of tight-junction (occludin), adherens junction (E-cadherin), and cell-extracellular matrix junction (beta1-integrin) transmembrane proteins. These transmembrane proteins are expressed in Madin-Darby canine kidney (MDCK) cells. In addition, MDCK cells polarize and therefore serve as a useful in vitro model for studies on the epithelial cell barrier. Using the MDCK cell system, we examined the effect of P. gingivalis on epithelial barrier function. Exposure of the basolateral surfaces of MDCK cells to P. gingivalis (>10(9) bacteria/ml) resulted in a decrease in transepithelial resistance. Immunofluorescence microscopy demonstrated decreases in the amounts of immunoreactive occludin, E-cadherin, and beta1-integrin at specific times which were related to a disruption of cell-cell junctions in MDCK cells exposed to basolateral P. gingivalis. Disruption of cell-cell junctions was also observed upon apical exposure to bacteria; however, the effects took longer than those seen upon basolateral exposure. Cell viability was not affected by either basolateral or apical exposure to P. gingivalis. Western blot analysis demonstrated hydrolysis of occludin, E-cadherin, and beta1-integrin in lysates derived from MDCK cells exposed to P. gingivalis. Immunoprecipitated occludin and E-cadherin molecules from MDCK cell lysates were also degraded by P. gingivalis, suggesting a bacterial protease(s) capable of cleaving these epithelial junction transmembrane proteins. Collectively, these data suggest that P. gingivalis is able to invade the deeper structures of connective tissues via a paracellular pathway by degrading epithelial cell-cell junction complexes, thus allowing the spread of the bacterium. These results also indicate the importance of a critical threshold concentration of P. gingivalis to initiate epithelial barrier destruction.     

Apically exposed, tight junction-associated beta1-integrins allow binding and YopE-mediated perturbation of epithelial barriers by wild-type Yersinia bacteria

Using polarized epithelial cells, primarily MDCK-1, we assessed the mode of binding and effects on epithelial cell structure and permeability of Yersinia pseudotuberculosis yadA-deficient mutants. Initially, all bacteria except the invasin-deficient (inv) mutant adhered apically to the tight junction areas. These contact points of adjacent cells displayed beta1-integrins together with tight junction-associated ZO-1 and occludin proteins. Indeed, beta1-integrin expression was maximal in the tight junction area and then gradually decreased along the basolateral membranes. Wild-type bacteria also opened gradually the tight junction to paracellular permeation of different-sized markers, viz., 20-, 40-, and 70-kDa dextrans and 45-kDa ovalbumin, as well as to their own translocation between adjacent cells in intimate contact with beta1-integrins. The effects on the epithelial cells and their barrier properties could primarily be attributed to expression of the Yersinia outer membrane protein YopE, as the yopE mutant bound but caused no cytotoxicity. Moreover, the apical structure of filamentous actin (F-actin) was disturbed and tight junction-associated proteins (ZO-1 and occludin) were dispersed along the basolateral membranes. It is concluded that the Yersinia bacteria attach to beta1-integrins at tight junctions. Via this localized injection of YopE, they perturb the F-actin structure and distribution of proteins forming and regulating tight junctions. Thereby they promote paracellular translocation of bacteria and soluble compounds.     

Occluding Junctions in an Epithelioid Mesothelioma: A Freeze-Fracture Study

The presence of a well-developed tight junction system in a case of diffuse, papillary, malignant mesothelioma of the peritoneum is documented using the freeze-fracture methodology. The mean number of strands within the tight junction network was determined to be 3.83 i 0.16. In addition, the tight junctions were specifically located in regions of the plasma membrane adjacent to recognizable microvilli. Gap junctions and desmosomes were found in close association with the occluding complexes; however, they were also seen in areas of the plasma membrane quite distinct from those that exhibit tight junction differentiation. The possible significance of the presence of tight junctions in neoplastic epithelial cells is also discussed.     

Orchestration of Neutrophil Movement by Intestinal Epithelial Cells in Response to Salmonella typhimurium Can Be Uncoupled from Bacterial Internalization

Intestinal epithelial cells respond to Salmonella typhimurium by internalizing this pathogen and secreting, in a polarized manner, an array of chemokines which direct polymorphonuclear leukocyte (PMN) movement. Notably, interleukin-8 (IL-8) is secreted basolaterally and directs PMN through the lamina propria, whereas pathogen-elicited epithelial chemoattractant (PEEC) is secreted apically and directs PMN migration across the epithelial monolayer to the intestinal lumen. While most studies of S. typhimurium pathogenicity have focused on the mechanism by which this bacterium invades its host, the enteritis characteristically associated with salmonellosis appears to be more directly attributable to the PMN movement that occurs in response to this pathogen. Therefore, we sought to better understand the relationship between S. typhimurium invasion and epithelial promotion of PMN movement. First, we investigated whether S. typhimurium becoming intracellular was necessary or sufficient to induce epithelial promotion of PMN movement. Blocking S. typhimurium invasion by preventing, with cytochalasin D, the epithelial cytoskeletal rearrangements which mediate internalization did not reduce the epithelial promotion of PMN movement. Conversely, bacterial attainment of an intracellular position was not sufficient to induce model epithelia to direct PMN transmigration, since neither basolateral invasion by S. typhimurium nor apical internalization of an invasion-deficient mutant (achieved by inducing membrane ruffling with epidermal growth factor) induced this epithelial cell response. These results indicate that specific interactions between the apical surface of epithelial cells and S. typhimurium, rather than simply bacterial invasion, mediate the epithelial direction of PMN transmigration. To further investigate the means by which S. typhimurium induces epithelia to direct PMN movement, we investigated whether the same signaling pathways regulate secretion of IL-8 and PEEC. IL-8 secretion, but not PEEC secretion, was activated by phorbol myristate acetate and blocked by an inhibitor (mg-132) of the proteosome which mediates NF-κβ activation. Further, secretion of IL-8, but not PEEC, was activated by an entry-deficient (HilΔ) S. typhimurium mutant or by basolateral invasion of a wild-type strain. Together, these results indicate that distinct signaling pathways mediate S. typhimurium invasion, induction of IL-8 secretion, and induction of PEEC secretion in model intestinal epithelia.     

Tumor Suppressor Scribble Regulates Assembly of Tight Junctions in the Intestinal Epithelium

Formation of the epithelial barrier and apico-basal cell polarity represent two characteristics and mutually dependent features of differentiated epithelial monolayers. They are controlled by special adhesive structures, tight junctions (TJs), and polarity protein complexes that define the apical and the basolateral plasma membrane. The functional interplay between TJs and polarity complexes remains poorly understood. We investigated the role of Scribble, a basolateral polarity protein and known tumor suppressor, in regulating TJs in human intestinal epithelium. Scribble was enriched at TJs in T84 and SK-CO15 intestinal epithelial cell monolayers and sections of normal human colonic mucosa. siRNA-mediated knockdown of Scribble in SK-CO15 cells attenuated development of epithelial barrier and inhibited TJ reassembly independently of other basolateral polarity proteins Lgl-1 and Dlg-1. Scribble selectively co-imunoprecipitated with TJ protein ZO-1, and ZO-1 was important for Scribble recruitment to intercellular junctions and TJ reassembly. Lastly, Scribble was mislocalized from TJs and its expression down-regulated in interferon-γ-treated T84 cell monolayers and inflamed human intestinal mucosa in vivo. We conclude that Scribble is an important regulator of TJ functions and plasticity in the intestinal epithelium. Down-regulation of Scribble may mediate mucosal barrier breakdown during intestinal inflammation.The epithelial cell layer in the gut plays two crucial physiological functions. One function involves the formation of the physical barrier that separates body compartments from the gut lumen and protects underlying tissues from pathogen invasion and other harmful external stimuli.^1,2 Another function involves the regulation of bidirectional passages of solutes and macromolecules, which is essential for nutrients supply and removal of body waste.^3–5 Both barrier integrity and vectorial transport in the intestinal epithelium are regulated by specialized cellular structures known as tight junctions (TJs). TJs represent a complex network of protein fibrils within the plasma membrane, which encircle the apical region of the epithelial cell perimeter in close proximity to the gut lumen.⁶ TJ fibrils are composed of adhesive transmembrane proteins, which associate with ensembles of scaffolding proteins at the cytosolic face of the membrane. The paracellular barrier is created by homotypical interactions between transmembrane TJ components of contacting epithelial cells such as occludin, members of the claudin family and junctional adhesion molecule-A (JAM-A).^6–8 These cell-cell adhesions are enhanced and regulated by cytosolic scaffolds such as members of zonula occludens (ZO) family and AF-6/afadin, which cluster and stabilize transmembrane TJ components at the plasma membrane.^6–8 Although other junctional complexes at the plasma membrane, viz., adherens junctions (AJs) and desmosomes also mediate cell-cell adhesions, TJs play a unique role in sealing the paracellular space and creating the epithelial barrier.^1,6,7The mature TJs not only mediate barrier function of the intestinal epithelium, but also contribute to the formation and maintenance of the apico-basal cell polarity.^9–11 Such cell polarity implies that the apical and basolateral domains of the plasma membrane differ in the composition of transporters, channels and receptors; therefore ensuring directionality of secretion and adsorption processes in epithelial cells.^12,13 TJs regulate the epithelial cell polarity by creating a fence that prevents intermixing of protein and lipid constituents of the apical and basolateral plasma membrane domains.^9–11 However, TJs alone are not sufficient for the apico-basal polarization of epithelial cells. In this process apical junctions cooperate with specialized protein polarity complexes that control the “identity” of distinct plasma membrane domains.Epithelial cells have three major evolutionarily conserved polarity complexes that were initially identified and named in model invertebrates. They are known as Crumbs (composed by Crumbs, PALS, and PATJ proteins), Par (Par3, Par6, and atypical protein kinase C) and Scribble (including Scribble, Disks Large (Dlg) and Lethal Giant Larvae (Lgl)) complexes.^11,14–16 Crumbs and Par cooperate to define the apical plasma membrane, whereas Scribble is critical for establishment of the basolateral membrane domain.^11,14–16 A number of studies have demonstrated a functional interplay between epithelial junctions and the polarity complexes, where these entities mutually affect each other. Thus, several members of the Crumbs and Par complexes were shown to regulate TJ assembly via either direct binding to TJ components (ZO-1 and claudins) or indirect mechanisms, involving modulation of vesicle trafficking and actin cytoskeleton remodeling.^11,14–16 In contrast, the role of the Scribble polarity complex in the regulation of epithelial TJs is not well understood,^17,18 although such a role is supported by several lines of evidence. For example, mutations in any member of this complex in Drosophila resulted in dramatic disorganization of epithelial architecture that included loss of columnar cell shape and cell-cell adhesions.^19–21 Furthermore, several reports have linked decreased protein levels of mammalian Scribble and Lgl with progression and invasiveness of epithelial tumors,^22–24 which is also accompanied by down-regulation of TJs.²⁵ Two recent studies have addressed the role of Scribble in the regulation cell-cell adhesions in mammalian epithelia; however their results appear to be inconsistent. Indeed, siRNA-mediated depletion of this protein in Madin-Darby canine kidney (MDCK) epithelial cells resulted in altered cell morphology and disorganized E-cadherin-based AJs.²⁶ However, no changes in cell morphology or AJ structure were observed following the silencing of Scribble expression in MCF10A human mammary epithelial cells.²⁷ Such inconsistent results may reflect tissue- specific effects of Scribble depletion, and they indicate that more work is needed to establish functional links between Scribble and TJs in human epithelia under normal physiological conditions and in disease states.In this study, we examined the role of Scribble in the regulation of the intestinal epithelial barrier and reorganization of TJs. Our results demonstrate that Scribble is important for TJ barrier function and assembly, and that it may regulate junctions by interacting with the TJ scaffold, ZO-1. We also report that Scribble is mislocalized and its expression down-regulated in the intestinal epithelium by inflammatory conditions in vitro and in vivo.     

Immunohistological characterization of intercellular junction proteins in rhesus macaque intestine

Epithelial junctions play an important role in regulating paracellular permeability and intercellular adhesion. It has been reported that changes in the density of epithelial junctions and/or distribution pattern can contribute to various gastrointestinal (GI) disorders. In this study, we investigated the distribution of the tight junction (Claudins. 1, 3, 4, 5, 7, 10, Zonula Occludens (ZO-1), Occludin), adherens junction (E-cadherin), desmosome (Desmoglein 2, Desmocollin 2) and gap junction (Connexin 43) proteins in the jejunum, ileum and colonic epithelium of healthy rhesus macaques (RM) using immunofluorescence labeling. While proteins in these respective junctions were expressed throughout the jejunum, ileum and colon of RM, we observed differential labeling in epithelial cells from these sites. Claudins 1, 3, 4, 7, E-cadherin and Desmoglein 2 were distributed in the respective intercellular junctions with additional labeling in the lateral membrane of epithelial cells in both small and large intestine. However, claudin 5, claudin 10, ZO-1 and occludin showed uniform distribution in the intercellular junctions of crypt and surface epithelial cells of the intestine. Desmocollin 2 localized predominantly in the upper two thirds along the lateral membrane while desmoglein 2 was distributed along the entire lateral membrane of intestinal epithelial cells. In contrast, connexin 43 exhibited punctate lateral labeling in crypt epithelial cells of the small and large intestine. Our results show diverse localization of epithelial intercellular junction proteins along the intestinal tract of RM. These findings may correlate with differences in paracellular permeability and adhesion along the intestinal tract and could correlate with pathologic disease in these regions of the intestine.     

紧密连接蛋白claudins与肿瘤的研究进展

Claudins是新近发现的构成细胞紧密连接的一类完整的跨膜蛋白，维持紧密连接特有的栅栏功能和屏障功能。Claudins表达异常与肿瘤的发生发展关系密切。多种途径可能参与了claudins表达的调节。Claudins为肿瘤的诊断和治疗提供了一种新思路。     

Occludin expression decreases with the progression of human endometrial carcinoma

The tight junctions of the glandular epithelium are crucial for the maintenance of cell polarity, separating the plasma membrane into apical and basolateral domains. Thus abnormalities of the tight junctions may result in the structural disturbances of glandular epithelial neoplasia. In this study we introduced an anti-occludin monoclonal antibody for semiquantitative assay of the occludin expression in tissue sections of human normal and neoplastic endometrial epithelia using the Adobe Photoshop and NIH Image programs. Normal endometrial glands and samples of endometrial hyperplasia and endometrioid carcinoma grade 1 fully expressed occludin at the apical cell border. In endometrioid carcinomas grades 2 and 3, however, occludin disappeared in solid areas of the carcinomatous tissues. Occludin was also found at the apical borders of the cancer cells that formed glandular structures. Occludin expression decreased progressively in parallel with the increase in carcinoma grade, and the decreased occludin expression correlated with myometrial invasion and lymph node metastasis. These results suggest that the loss of tight junctions has a close relationship with structural atypia in the progression of human endometrial carcinomas and their malignant potential.     

Aberrant overexpression of the cell polarity module scribble in human cancer

Human Scribble (Scrib) is an evolutionary-conserved cell polarity protein, but its potential role in human cancer is controversial. Herein, we show that Scrib is nearly universally overexpressed in cultured tumor cell lines and genetically disparate cancer patient series compared with matched normal tissues in vivo. Instead of a membrane association seen in normal epithelia, tumor-associated Scrib is mislocalized and found predominantly in the cytosol. Small-interfering RNA silencing of Scrib in model lung adenocarcinoma A549 cells inhibited cell migration in wound-healing assays, suppressed tumor cell invasion across Matrigel-coated inserts, and down-regulated the expression of cell motility markers and mediators of epithelial-mesenchymal transition. These data uncover a previously unrecognized exploitation of Scrib for aberrant tumor cell motility and invasion, thus potentially contributing to disease progression in humans.     

The structure of tight junctions in human thyroid tumors. A systematic freeze-fracture study.

Tight junctions of normal human epithelium comprise 6--8 intramembranous ridges. Tight junctions of adenomas and one case of follicular carcinoma were morphologically identical with normal controls. Another follicular carcinoma (clinically less malignant than the case mentioned above), however, showed loss of junctional polarity with spreading of ridges on lateral cell membranes. One case of papillary carcinoma (clinically low malignancy) displayed only focal attenuations of the tight junction belt. Another more malignant tumor of the same histologic type showed breaks of ridges and focal interruptions of the tight junction meshwork. Cell membranes of two anaplastic carcinomas of high malignancy were completely devoid of tight junction ridges. We conclude that tight junction alterations are not necessarily implicated in malignant transformation and that they correspond with tumor differentiation rather than directly with a single parameter of thyroid tumor malignancy.     

Par3 regulates invasion of pancreatic cancer cells via interaction with Tiam1

The conserved polarity complex, which comprises partitioning-defective proteins Par3, Par6, and the atypical protein kinase C, affects various cell-polarization events, including assembly of tight junctions. Control of tight junction assembly is closely related to invasion and migration potential. However, as the importance of conserved polarity complexes in regulating pancreatic cancer invasion and metastasis is unclear, we investigated their role and mechanism in pancreatic cancers. We first detect that the key protein of the conserved polarity complex finds that only Par3 is down-regulated in pancreatic cancer tissues while Par6 and aPKC show no difference. What is more, Par3 tissues level was significantly and positively associated with patient overall survival. Knocking-down Par3 promotes pancreatic cancer cells invasion and migration. And Par3 requires interaction with Tiam1 to affect tight junction assembly, and then affect invasion and migration of pancreatic cancer cells. Then, we find that tight junction marker protein ZO-1 and claudin-1 are down-regulated in pancreatic cancer tissues. And the relationship of the expression of Par3 and ZO-1 in pancreatic cancer tissue is linear correlation. We establish liver metastasis model of human pancreatic cancer cells in Balb/c nude mice and find that knocking down Par3 promotes invasion and metastasis and disturbs tight junction assembly in vivo. Taken together, these results suggest that the Par3 regulates invasion and metastasis in pancreatic cancers by controlling tight junction assembly.     

Neuroglian stabilizes epithelial structure during Drosophila oogenesis.

The vertebrate L1 family of cell adhesion molecules (CAMs) and their fly homolog, Neuroglian, are members of the immunoglobulin (Ig) superfamily of CAMs. In general, Ig CAMs have been found to play critical roles in mediating axon guidance. One Ig CAM, NCAM, has also been implicated in maintaining epithelial integrity and suppressing metastatic dissemination of tumor cells. Other Ig CAMs, such as Nrg, are also expressed in epithelia. We thus tested the hypothesis that, like NCAM, Nrg might also be required for maintaining epithelial integrity and for inhibiting tumor invasion. We used the Drosophila follicular epithelium to determine the function of Nrg in vivo in maintaining epithelial structure, and in regulating the motility of migrating border cells and invasive tumorous follicle cells. Nrg(167) is expressed on the lateral membrane of follicle cells. Loss of Nrg(167) causes border cells to delay delamination and causes other follicle cells to delaminate inappropriately. The delaminated cells have aberrant epithelial polarity manifested as severe mislocalization of apical and basal membrane proteins, and uniform localization of lateral membrane proteins. Furthermore, loss of Nrg(167) dramatically enhances the invasive phenotype associated with loss of Discs Large, a neoplastic tumor suppressor. These results indicate that Nrg(167) stabilizes epithelial polarity by regulating junctional adhesion and function in normal and tumorous epithelia. Our data also suggest that Ig superfamily members have significant functional redundancy in maintaining epithelial polarity, with individual members playing subtle, unique roles during epithelial morphogenesis.     

Platelet-activating factor regulates cadherin-catenin adhesion system expression and beta-catenin phosphorylation during Kaposi's sarcoma cell motility

In the present study, we evaluated whether motility of Kaposi's sarcoma (KS) cells induced by platelet-activating factor (PAF) is dependent on the regulation of adherens junctions components. The results obtained indicate that PAF dose and time dependently reduced the endogenous expression of the main components of the adherens junctions: VE-cadherin, alpha-catenin, and beta-catenin. In addition, PAF initiated events that directly or indirectly up-regulated both the tyrosine and serine/threonine phosphorylation pathways, and both types of phosphorylation of beta-catenin were involved in the motility of KS cells. This motility was abrogated by addition of the tyrosine kinase inhibitor genistein, suggesting that this phosphorylation is an important signal responsible for breaking down the adherens junctions and diminishing the ability of neighboring cells to interact. Furthermore, immunofluorescence analysis showed that beta-catenin and VE-cadherin staining changed from a uniform distribution along the membrane of controls to a diffuse pattern with gap formation in PAF-treated KS cells. In conclusion, the data presented here indicate that PAF induces tumor cell motility by altering cell-cell adhesion through beta-catenin phosphorylation.     

Downregulation of E-cadherin in the reparative epithelium of the human gastrointestinal tract.

E-cadherin, an epithelial adhesion molecule, is critical for the maintenance of cell polarity and differentiation. We studied the distribution of E-cadherin in normal gut and in enteric ulceration to test the hypothesis that the motility of regenerative epithelium over ulcers is associated with a decrease in E-cadherin expression. Sections of normal stomach, small intestine, and colon were examined for E-cadherin distribution using the antibody HECD-1 and compared with the pattern seen in peptic ulceration and Crohn's disease. A subset was examined by in situ hybridization using 35S radiolabeled E-cadherin riboprobes. A wounding system employing the HT-29 cell line was used as an in vitro model of early healing. In the normal gut uniform strong basolateral staining was seen. Areas of ulceration showed a patchy reduction in membrane localized E-cadherin in regenerating epithelium, even though E-cadherin mRNA was demonstrable in this population. In wounded confluent HT29 layers, migrating cells also showed reduced E-cadherin immunostaining. These data support the notion that the motility of restitutive epithelial cells may relate to altered patterns of E-cadherin and that this may play an important role in the reconstitution of epithelial integrity after mucosal injury.     

Synergistic inhibition of lung cancer cell invasion,tumor growth and angiogenesis using aptamer-siRNA chimeras

Early metastasis is one of the major causes of mortality among patient with lung cancer. The process of tumor metastasis involves a cascade of events, including epithelial-mesenchymal transition, tumor cell migration and invasion, and angiogenesis. To specifically suppress tumor invasion and angiogenesis, two nucleolin aptamer-siRNA chimeras (aptNCL-SLUGsiR and aptNCL-NRP1siR) were used to block key signaling pathways involved in lung cancer metastasis that are pivotal to metastatic tumor cells but not to normal cells under ordinary physiologic conditions. Through nucleolin-mediated endocytosis, the aptNCL-siRNA chimeras specifically and significantly knocked down the expressions of SLUG and NRP1 in nucleolin-expressing cancer cells. Furthermore, simultaneous suppression of SLUG and NRP1 expressions by the chimeras synergistically retarded cancer cell motility and invasive ability. The synergistic effect was also observed in a xenograft mouse model, wherein the combined treatment using two chimeras suppressed tumor growth, the invasiveness, circulating tumor cell amount, and angiogenesis in tumor tissue without affecting liver and kidney functions. This study demonstrates that combined treatment of aptNCL-SLUGsiR and aptNCL-NRP1siR can synergistically suppress lung cancer cell invasion, tumor growth and angiogenesis by cancer-specific targeting combined with gene-specific silencing.