Profiling of the tetraspanin web of human colon cancer cells

Tetraspanins are integral membrane proteins involved in a variety of physiological and pathological processes. In cancer, clinical and experimental studies have reported a link between tetraspanin expression levels and metastasis. Tetraspanins play a role as organizers of multimolecular complexes in the plasma membrane. Indeed each tetraspanin associates specifically with one or a few other membrane proteins forming primary complexes. Thus, tetraspanin-tetraspanin associations lead to a molecular network of interactions, the "tetraspanin web." We performed a proteomic characterization of the tetraspanin web using a model of human colon cancer consisting of three cell lines derived from the primary tumor and two metastases (hepatic and peritoneal) from the same patient. The tetraspanin complexes were isolated after immunoaffinity purification using monoclonal antibodies directed against the tetraspanin CD9, and the associated proteins were separated by SDS-PAGE and identified by mass spectrometry using LC-MS/MS. This allowed the identification of 32 proteins including adhesion molecules (integrins, proteins with Ig domains, CD44, and epithelial cell adhesion molecule) (EpCAM), membrane proteases (ADAM10, TADG-15, and CD26/dipeptidyl peptidase IV), and signaling proteins (heterotrimeric G proteins). Importantly some components were differentially detected in the tetraspanin web of the three cell lines: the laminin receptor Lutheran/B-cell adhesion molecule (Lu/B-CAM) was expressed only on the primary tumor cells, whereas CD26/dipeptidyl peptidase IV and tetraspanin Co-029 were observed only on metastatic cells. Concerning Co-029, immunohistofluorescence showed a high expression of Co-029 on epithelial cells in normal colon and a lower expression in tumors, whereas heterogeneity in terms of expression level was observed on metastasis. Finally we demonstrated that epithelial cell adhesion molecule and CD9 form a new primary complex in the tetraspanin web.     

Molecular cloning and expression analysis of CD82 in pig

CD82, which was originally referred to as KAI1 (kangai 1), is a member of the tetraspanin protein family, which contains four transmembrane domains. CD82 is implicated in a variety of biological processes, including apoptosis, cell adhesion, and cell migration. In this study, the full-length cDNA of pig CD82 was cloned and sequenced. Pig Cd82 cDNA contains an open reading frame (801 bp) encoding 266 amino acids. Sequence alignment results indicated that pig CD82 cDNA evidenced 85.45%, 85.63%, 77.03%, and 77.78% identity with human, cattle, rat, and mouse, respectively. In the expression study, the constitutive expression of swine Cd82 mRNA was detected in a variety of tissues, including lymphoid tissues as well as nonlymphoid tissues. Future studies will be focused on the functional role of CD82 during the course of pig infectious diseases or tumor development.     

Molecular Cloning and Expression Analysis of CD82 in Pig

CD82, which was originally referred to as KAI1 (kangai 1), is a member of the tetraspanin protein family, which contains four transmembrane domains. CD82 is implicated in a variety of biological processes, including apoptosis, cell adhesion, and cell migration. In this study, the full-length cDNA of pig CD82 was cloned and sequenced. Pig Cd82 cDNA contains an open reading frame (801 bp) encoding 266 amino acids. Sequence alignment results indicated that pig CD82 cDNA evidenced 85.45%, 85.63%, 77.03%, and 77.78% identity with human, cattle, rat, and mouse, respectively. In the expression study, the constitutive expression of swine Cd82 mRNA was detected in a variety of tissues, including lymphoid tissues as well as nonlymphoid tissues. Future studies will be focused on the functional role of CD82 during the course of pig infectious diseases or tumor development.     

ADAM9 silencing inhibits breast tumor cell invasion in vitro

ADAM9 (A Disintegrin And Metalloproteinase 9) is a member of the ADAM protein family which contains a disintegrin domain. This protein family plays key roles in many physiological processes, including fertilization, migration, and cell survival. The ADAM proteins have also been implicated in various diseases, including cancer. Specifically, ADAM9 has been suggested to be involved in metastasis. To address this question, we generated ADAM9 knockdown clones of MDA-MB-231 breast tumor cells using silencing RNAs that were tested for cell adhesion, proliferation, migration and invasion assays. In RNAi-mediated ADAM9 silenced MDA-MB-231 cells, the expression of ADAM9 was lower from the third to the sixth day after silencing and inhibited tumor cell invasion in matrigel by approximately 72% when compared to control cells, without affecting cell adhesion, proliferation or migration. In conclusion, the generation of MDA-MB-231 knockdown clones lacking ADAM9 expression inhibited tumor cell invasion in vitro, suggesting that ADAM9 is an important molecule in the processes of invasion and metastasis.     

C-kit gene product (CD117) immunoreactivity in canine and feline paraffin sections.

CD117 is a transmembrane tyrosine kinase growth factor receptor expressed by a variety of normal human cell types, including germ cells, immature myeloid cells, and mast cells. To evaluate the pattern of CD117 expression in dogs and cats, we applied a polyclonal antibody on paraffin sections from 44 samples of normal tissues and 104 tumors. In both species, strong immunoreactivity was observed in mast cells, interstitial cells of Cajal, and in mast cell tumors. Among gastrointestinal mesenchymal neoplasms, tissues from five dogs and one cat revealed strong CD117 expression, enabling us to identify them as gastrointestinal stromal tumors (GISTs).     

Immunohistochemical localization of glutathione S-transferase in preneoplastic and neoplastic lesions of the human uterine cervix

A mouse monoclonal antibody and a rabbit polyclonal antibody prepared against the placental form of the enzyme glutathione S-transferase (GST-pi) were used to immunohistochemically stain normal and neoplastic human uterine cervical tissues from 88 cases. Of 65 cases of preneoplastic squamous lesions and invasive carcinomas of the cervix, 94% stained with the monoclonal antibody and 100% with the polyclonal antibody. In the 23 benign tissues, staining of ectocervical squamous epithelium was generally not observed; however, areas of reserve-cell hyperplasia, immature squamous metaplasia and adjacent endocervical cells did show staining (68% with the monoclonal antibody and 95% with the polyclonal antibody). Many of the positive tissue types showed a variety of staining patterns and intensities. These findings do not support the concept that GST-pi staining can be used to distinguish preneoplastic lesions of the cervix from benign reactive or proliferative processes. These results are of interest in the investigation of cervical carcinogenesis since GST-pi may be involved in an early stage of neoplastic transformation of the cervical epithelium. The correlation of these findings with the results of human papillomavirus testing and DNA content analysis should be of interest in determining the relationship of this enzyme to cervical neoplasia.     

Stealth siRNA against CD147 inhibits hepatocellular carcinoma cell metastatic properties

CD147 plays a critical role in the invasive and metastatic activity of hepatocellular carcinoma (HCC) cells by stimulating the surrounding fibroblasts to secrete matrix metalloproteinases (MMPs). Tumor cells adhesion to extracellular matrix (ECM) proteins is the first step to the tumor metastasis. MMPs degrade the ECM to promote tumor metastasis. The aim of this research was to investigate the inhibitory effects of stealth small interfering RNA (siRNA) against CD147 on HCC cell line (SMMC-7721) metastatic properties including invasion, adhesion to ECM, gelatinase production, focal adhesion kinase (FAK) and vinculin expression. Flow cytometry (FCM) and western blot assays were employed to detect the transfection efficiency of the stealth siRNA against CD147. Invasion assays and gelatin zymography were also used to detect the effects of stealth siRNA against CD147 on SMMC-7721 cells’ invasion and gelatinase production. The effects of stealth siRNA against CD147 on FAK and vinculiln expression in SMMC-7721 cells were also detected by western blot. The results showed that stealth siRNA against CD147 inhibited SMMC-7721 invasion, adhesion to ECM proteins, MMP-2 production, and FAK and vinculin expression. These findings indicate that CD147 is required for tumor cell invasion and adhesion. Perturbation of CD147 expression may have potential therapeutic uses in the prevention of MMP-2-dependent tumor invasion.     

Identification of tissue transglutaminase as a novel molecule involved in human CD8+ T cell transendothelial migration

During inflammation, T lymphocytes migrate out of the blood across the vascular endothelium in a multistep process. The receptors mediating T cell adhesion to endothelium are well characterized; however, the molecules involved in T cell transendothelial migration (TEM) subsequent to lymphocyte adhesion to the endothelium are less clear. To identify receptors mediating TEM, mAbs were produced against human blood T cells adhering to IFN-gamma-activated HUVEC in mice and tested for inhibition of lymphocyte TEM across cytokine-activated HUVEC. Most of the mAbs were against beta(1) and beta(2) integrins, but one mAb, 6B9, significantly inhibited T cell TEM across IFN-gamma, TNF-alpha, and IFN-gamma plus TNF-alpha-stimulated HUVEC, and did not react with an integrin. 6B9 mAb did not inhibit T cell adhesion to HUVEC, suggesting that 6B9 blocked a novel pathway in T cell TEM. The 6B9 Ag was 80 kDa on SDS-PAGE, and was expressed by both blood leukocytes and HUVEC. Immunoaffinity purification and mass spectrometry identified this Ag as tissue transglutaminase (tTG), a molecule not known to mediate T cell TEM. Treatment of HUVEC with 6B9 was more effective than treatment of T cells. 6B9 blockade selectively inhibited CD4(-), but not CD4(+), T cell TEM, suggesting a role for tTG in recruitment of CD8(+) T lymphocytes. Thus, 6B9 is a new blocking mAb to human tTG, which demonstrates that tTG may have a novel role in mediating CD8(+) T cell migration across cytokine-activated endothelium and infiltration of tissues during inflammation.     

Immunohistochemical analysis of beta3 integrin (CD61): expression in pig tissues and human tumors

CD61 is a membrane glycoprotein that associates with CD41 (alphaIIb) to form the heterodimeric complex gpIIb/IIIa (CD41/CD61), predominantly expressed in platelets and megakariocytes. CD61 or beta3 integrin also associates with alpha v (CD51) to form the vitronectin receptor, which is expressed in many tissues. We have used a monoclonal antibody against the porcine gpIIIa or CD61 (JM2E5) to study the distribution of this molecule in different normal pig tissues. As in humans, CD61 was broadly expressed in all tissues examined. In the kidney, strong expression of CD61 was observed in epithelial cells from renal tubules. In the testis, CD61 expression was detected in the Leydig cells. However, in liver, CD61 was weak or not detected. Many integrins are particularly involved in tumogenicity and in tumor progression mediating cell-cell interaction. Immunofluorescence experiments using cultured human tumor HeLa cells showed nuclear and cytoplasmic staining of mAb JM2E5. Immunohistochemical analysis of human tumor sections from several organs showed a heterogeneus distribution in metastatic cases from colon and breast carcinoma. However, no staining was found in metastasis from melanoma.     

Human natural killer cell activity against porcine targets: modulation by control of the oxidation-reduction environment and role of adhesion molecule interactions

Xenotransplantation, especially using porcine sources, has been proposed as a means to alleviate the shortage of human organs for transplantation. NK cells appear to be important mediators of the xenogeneic immune responses, including the human anti-pig response. Having previously established the redox regulation of NK cell activity against tumor target cells, we now report that the interaction of human NK cells with porcine target cells is also regulated by redox. Thiol-deprivation strongly diminished the capacity of IL-2-activated human NK cells to kill porcine endothelial cells. This inhibition correlated with reduced proliferation and interferon (IFN)-gamma production by IL-2-activated NK cells. For fresh NK cells, pretreatment with diethyl maleate (DEM), which was used to deplete intracellular thiols, reduced lysis of porcine and human targets. Because many adhesion molecules exhibit interspecies recognition, we further investigated whether changes in expression of adhesion molecules might explain our observations. DEM treatment reduced the expression of CD11b and CD29 on fresh NK cells. Monoclonal antibody blocking studies showed that the combination of mAb to CD11b and CD18 reduced lytic activity against both PAEC as well as K562, although other qualitative differences were observed between the porcine and human target cells. These findings suggest that the oxidative stress-induced downregulation of CD18 may be important in modulating cytotoxic activity of fresh NK cells against PAEC and K562 targets through reduced formation of the CD11b/CD18 heterodimer. Thus, the appropriate manipulation of redox status may provide a means to enhance survival of non-human animal tissues in humans through modulation of adhesion molecule expression/interactions.     

CD40/CD40L interaction induces E-selectin dependent leukocyte adhesion to human endothelial cells and inhibits endothelial cell migration

Background

CD40 is a receptor expressed on a wide range of cells such as leukocytes and endothelial cells (EC). As a member of the tumor necrosis factor (TNF) superfamily the activation of CD40 by CD40-ligand (CD40L) plays a crucial role for the development and progression of a variety of inflammatory processes including atherosclerosis. The aim of the present study was to investigate the effect of CD40/CD40L interaction on leukocyte adhesion to the endothelium and on endothelial cell migration.

Methods and results

Human umbilical vein endothelial cells (HUVEC) were stimulated with either stable transfectants of mouse myeloma cells expressing the CD40L or wild type cells (4 h). Subsequently adhesion of leukocytes expressing Sialyl Lewis X, the counterpart for E-selectin (HL60 cells), was measured under shear stress (2–2.6 dyne/cm²) using a flow chamber adhesion assay. Stimulation of CD40 led to a significant increase of E-selectin dependent adhesion of leukocytes to the endothelium. Incubation of cells with either the CD40L blocking antibody TRAP-1 or the E-selectin blocking antibody BBA2 during CD40 stimulation completely abolished adhesion of leukocytes to HUVEC. Similar results were found in human cardiac microvasculature endothelial cells (HCMEC). In contrast stimulation of CD40 had no effect on adhesion of l-selectin expressing NALM6-L cells. Furthermore, CD40/CD40L interaction abrogated VEGF-induced migration of HUVEC compared to non-stimulated controls. In comparison experiments, stimulation of endothelial cells with VEGF led to a significant phosphorylation of ERK1/2, Akt, and eNOS. Stimulation of endothelial CD40 had no effect on VEGF-induced phosphorylation of ERK1/2. However, VEGF-induced activation of Akt and eNOS was reduced to baseline levels when endothelial CD40 was stimulated.

Conclusion

CD40/CD40L interaction induces E-selectin dependent adhesion of leukocytes to human endothelial cells and reduces endothelial cell migration by inhibiting the Akt/eNOS signaling pathway.     

Immunochemical analysis of CD107a (LAMP-1)

CD107a, also known as the lysosome associated membrane protein-1 (LAMP-1), is expressed largely in the endosome-lysosome membranes of cells, but is also found on the plasma membrane (1-2% of total LAMP-1). LAMP-1 has been implicated in a variety of cellular functions, including cancer metastasis. It has been proposed as a therapeutic agent for some cancers, and is a marker for lysosomal storage disorders and different cell types such as cytotoxic T cells. In light of this diversity of applications, it is important to have well characterized immune-reagents for the detection and quantification of LAMP-1. We have compared a new monoclonal antibody 80280 against LAMP-1 to an existing monoclonal antibody BB6 and a rabbit polyclonal antibody. While all antibodies gave similar results by immunofluorescence, the monoclonal antibody 80280 showed no epitope reactivity to LAMP-1 peptides, suggesting the possibility of a carbohydrate epitope. Western blotting revealed a weaker activity of the monoclonal antibody 80280 relative to either the BB6 monoclonal or the polyclonal antibodies. The monoclonal antibody 80280 is distinct from BB6, providing an additional reagent for CD107a analysis.     

Characterization of a high-affinity monoclonal antibody specific for CD44v6 as candidate for immunotherapy of squamous cell carcinomas

 Variant isoforms of CD44, a family of cell-surface glycoproteins generated by alternative splicing and post-translational modifications, are expressed in a variety of human tumors and play important roles in tumor progression and metastasis formation. The murine monoclonal IgG1 antibody VFF18, specific for an epitope encoded by human CD44 variant exon 6, binds with high affinity to the recombinant protein (K _d = 1.7×10^–10 M) as well as to tumor cell lines in vitro, and is suitable for immunohistochemical analysis of human tumors. Screening of more than 500 tumor samples of different histogenesis showed that VFF18 most strongly and uniformly reacts with squamous cell carcinomas (SCC). Detailed analysis of 185 SCC (head and neck, lung, skin) confirmed reactivity of the antibody with 99% of the samples, with intense and homogeneous staining of the tumor cells in the majority of cases, whereas reactivity of VFF18 with normal tissues is limited to certain epithelia and activated lymphocytes. When radiolabelled VFF18 was administered to nude mice bearing human epidermoid carcinoma (A-431) xenograft, fast and selective tumor uptake of the radioimmunoconjugate with a maximum of 18% of the injected dose per gram of tissue was observed. Taken together, these data suggest that mAb VFF18 is a promising targeting vehicle for radioimmunotherapy of squamous cell carcinomas in humans. Received: 9 September 1996 / Accepted: 25 September 1996     

Structure,regulatory factors and cancer-related physiological effects of ADAM9

ABSTRACT

The ADAMs family belongs to the transmembrane protein superfamily of zinc-dependent metalloproteases, which consists of multiple domains. These domains have independent but complementary functions that enable them to participate in multiple biological processes. Among them, ADAM9 can not only participate in the degradation of extracellular matrix as a metalloprotease, but also mediate tumor cell adhesion through its deintegrin domain, which is closely related to tumor invasion and metastasis. It is widely expressed in a variety of tumor cells and can affect the proliferation, invasion and metastasis of related cancer cells. We provide our views on current progress, its increasing importance as a strategic treatment goal, and our vision for the future of ADAM9.     

CEACAM6 Promotes Gastric Cancer Invasion and Metastasis by Inducing Epithelial-Mesenchymal Transition via PI3K/AKT Signaling Pathway

Overexpressed CEACAM6 in tumor tissues plays important roles in invasion, metastasis and anoikis resistance in a variety of human cancers. We recently reported that CEACAM6 expression is upregulated in Gastric cancer (GC) tissues and promoted GC metastasis. Here, we report that CEACAM6 promotes peritoneal metastases in vivo and is negatively correlated with E-cadherin expression in GC tissues. Overexpressed CEACAM6 induced epithelial-mesenchymal transition (EMT) in GC, as measured by increases in the EMT markers N-cadherin, Vimentin and Slug while E-cadherin expression was decreased in CEACAM6-overexpressing GC cells; opposing results were observed in CEACAM6-silenced cells. Furthermore, E-cadherin expression was negatively correlated with depth of tumor invasion, lymph node metastasis and TNM stage in GC tissues. Additionally, CEACAM6 elevated matrix metalloproteinase-9 (MMP-9) activity in GC, and anti-MMP-9 antibody could reverse the increasing invasion and migration induced by CEACAM6. CEACAM6 also increased the levels of phosphorylated AKT, which is involved in the progression of a variety of human tumors. We further observed that {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a PI3K inhibitor, could reverse CEACAM6-induced EMT via mesenchymal-epithelial transition. These findings suggest that CEACAM6 enhances invasion and metastasis in GC by promoting EMT via the PI3K/AKT signaling pathway.     

The role of gicerin, a novel cell adhesion molecule, in development, regeneration and neoplasia

Neurite outgrowth factor (NOF) is an extracellular matrix (ECM) protein in the laminin family and its ligand, gicerin, is a novel cell adhesion molecule in the immunoglobulin superfamily. Gicerin has a homophilic adhesive activity as well as a heterotypic manner to NOF. In the nervous systems, gicerin is expressed during developmental stage when neurons migrate or extend neurites to form a neural network. Gicerin promotes neurite extension and migration of embryonic neurons in vitro by its homophilic and heterophilic adhesion activities. Introduction of antigicerin antibody into early developing eyes perturbs the layer formation of neural retina. These data suggest that gicerin participates in the formation of neural tissues. Gicerin is also expressed in other non-neural tissues; in epithelia of trachea, kidney and oviduct, gicerin expression is restricted in the developmental period. In contrast, muscular tissues and endothelial cells express gicerin continuously even after maturation. Interestingly, gicerin re-appears strongly in the regenerating epithelia of trachea, kidney and oviduct, and also anti-gicerin antibody disrupts the healing process of trachea. Furthermore, gicerin and NOF are overexpressed in the chicken nephroblastomas (Wilm's tumor) and oviductal adenocarcinomas. In vitro analyses show that gicerin adhesive activities can promote binding among tumor cells and adhesion of tumor cells to NOF. A polyclonal antibody against gicerin also perturbs the re-attachment of cancer cells onto metastasizing sites. It is clear from these studies that gicerin is a potential effector for pathological tissue formation as well as for normal development.     

Immunohistochemical study of angiotensin-converting enzyme in human tissues using monoclonal antibodies

The localization of angiotensin-converting enzyme (ACE) in human tissues has been studied by the PAP-method with the use of monoclonal antibody 9 B9 against human lung ACE. The enzyme was detected on the surface of endothelial cells in lung, myocardium, liver, intestine and testis as well as in the epithelial cells of the kidney proximal tubules and intestine. The monoclonal antibody 9 B9 did not react with ACE in the epithelial cells of the testis seminiferous tubules. These data suggest that the antibody 9 B9 recognizes epitope which is shared by the ACE molecule of endothelial cells and renal and intestinal epithelial cells but is not present in testicular ACE, or is not accessible there to the antibody.     

骨桥蛋白与肿瘤转移

转移是肿瘤恶化的主要标志.骨桥蛋白被称为"肿瘤转移基因",是一种分泌型、粘附性的糖基化磷蛋白,与其主要受体整合素和CD44相互作用,参与多种器官和组织的生理病理过程,具有多种功能.近来的很多研究揭示,骨桥蛋白在肿瘤细胞的粘附、浸润、迁移及新血管生成过程中起关键作用. 骨桥蛋白在血清中的含量高低与病人的肿瘤转移情况及预后密切相关.很多研究认为,骨桥蛋白是一个很好的肿瘤转移标志物,是诊断与治疗肿瘤转移的新靶点.本文就骨桥蛋白的一般生物学特点及其在肿瘤转移过程中所起的作用和主要机制进行综述.     

Inhibition of platelets and tumor cell adhesion by the disintegrin domain of human ADAM9 to collagen I under dynamic flow conditions

This work aimed to investigate the role of the disintegrin domain of the human ADAM9 (ADAM9D) on the adhesion of breast tumor cells and platelets to collagen I, in a dynamic flow assay to simulate in vivo shear conditions. Recombinant ADAM9D was able to support tumor cell adhesion through binding to the β1 integrin subunit and also to inhibit the invasion through matrigel in vitro. In a dynamic flow assay ADAM9D inhibited about 75% and 65% of MDA-MB-231 tumor cells and platelet adhesion to collagen I, respectively. In addition, it was demonstrated that αVβ3 integrin is new interacting partner for ADAM9D. In conclusion, these results suggest a role for the disintegrin domain of ADAM9 in the metastatic process. Also, ADAM9D may be a tool for investigating the role of ADAMs in metastasis and cancer progression and for the design of selective inhibitors against the adhesion and extravasation of cancer cells.