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1.
Naoya Yoshihama Koujiro Yamaguchi Satomi Chigita Mariko Mine Masakazu Abe Kotaro Ishii Yosuke Kobayashi Naonari Akimoto Yoshihide Mori Tsuyoshi Sugiura 《PloS one》2015,10(4)
We have recently elucidated a novel function for CD82 in E-cadherin-mediated homocellular adhesion; due to this function, it can inhibit cancer cell dissociation from the primary cancer nest and limit metastasis. However, the effect of CD82 on selectin ligand-mediated heterocellular adhesion has not yet been elucidated. In this study, we focused on the effects of the metastasis suppressor CD82/KAI1 on heterocellular adhesion of cancer cells to the endothelium of blood vessels in order to further elucidate the function of tetraspanins. The over-expression of CD82 in cancer cells led to the inhibition of experimentally induced lung metastases in mice and significantly inhibited the adhesion of these cells to human umbilical vein epithelial cells (HUVECs) in vitro. Pre-treatment of the cells with function-perturbing antibodies against sLea/x significantly inhibited the adhesion of CD82-negative cells to HUVECs. In addition, cells over-expressing CD82 exhibited reduced expression of sLea/x compared to CD82-negative wild-type cells. Significant down-regulation of ST3 β-galactoside α-2, 3-sialyltransferase 4 (ST3GAL4) was detected by cDNA microarray, real-time PCR, and western blotting analyses. Knockdown of ST3GAL4 on CD82-negative wild-type cells inhibited expression of sLex and reduced cell adhesion to HUVECs. We concluded that CD82 decreases sLea/x expression via the down-regulation of ST3GAL4 expression and thereby reduces the adhesion of cancer cells to blood vessels, which results in inhibition of metastasis. 相似文献
2.
Zélia Silva ZiQiu Tong M. Guadalupe Cabral Catarina Martins Rita Castro Celso Reis Hélder Trindade Konstantinos Konstantopoulos Paula A. Videira 《Biochemical and biophysical research communications》2011,(3):459
The limited efficacy of monocyte-derived dendritic cell (mo-DC)-based vaccines is primarily attributed to the reduced mo-DC migratory capacity. One undefined aspect is the initial binding of mo-DCs to endothelial cells and vascular selectins. In this study, we investigated the role and modulation of the selectin binding determinant sialyl Lewisx (sLex) in selectin-dependent mo-DC binding. Our data reveal that sLex is required for maximal binding of mo-DCs to tumor necrosis factor (TNF)-α-activated endothelial cells under static conditions, as evidenced by the use of sialidase. Sialidase treatment also abrogated mo-DC cell tethering to immobilized, purified P-, L-, or E-selectin under flow. The requirement of sLex-dependent binding of mo-DC to selectins was further substantiated by using sLex free sugar and anti-sLex antibody, which significantly suppressed mo-DC-selectin binding. P-selectin glycoprotein ligand-1 is required for mo-DC binding to both P- and L-selectin, but it is dispensable for E-selectin recognition. Interestingly, the extent of mo-DC tethering was maximal on P-selectin, followed by E- and L- selectin. Accordingly, L-selectin mediated faster mo-DC rolling than E- or P-selectin. Interferon (IFN)-γ induces a significant increase in mo-DC surface sLex expression, which is probably due to the enhanced synthesis of C2GnT-I. These findings may contribute to improving mo-DC-based vaccination protocols. 相似文献
3.
4.
Background
The process of metastasis involves a series of steps and interactions between the tumor embolus and the microenvironment. Key alterations in adhesion molecules are known to dictate progression from the invasive to malignant phenotype followed by colonization at a distant site. The invasive phenotype results from the loss of expression of the E-cadherin adhesion molecule, whereas the malignant phenotype is associated with an increased expression of the carbohydrate ligand-binding epitopes, (e.g. Sialyl Lewis x/a) that bind endothelial E-selectin of the lymphatics and vasculature.Methodology
Our study analyzed the expression of two adhesion molecules, E-cadherin and Sialyl Lewis x (sLex), in both a canine mammary carcinoma and human inflammatory breast cancer (IBC) model, using double labelled immunofluorescence staining.Results
Our results demonstrate that canine mammary carcinoma and human IBC exhibit an inversely correlated cellular expression of E-cadherin and sLex within the same tumor embolus.Conclusions
Our results in these two comparative models (canine and human) suggest the existence of a biologically coordinated mechanism of E-cadherin and sLex expression (i.e. molecular plasticity) essential for tumor establishment and metastatic progression. 相似文献5.
We have previously shown that P-selectin binding to Colo-320 human colon carcinoma cells induces specific activation of the alpha(5)beta(1) integrin with a concomitant increase of cell adhesion and spreading on fibronectin substrates in a phosphatidylinositol 3-kinase (PI3-K) and p38 MAPK-dependent manner. Here, we identified by affinity chromatography and characterized nucleolin as a P-selectin receptor on Colo-320 cells. Nucleolin mAb D3 significantly decreases the Colo-320 cell adhesion to immobilized P-selectin-IgG-Fc. Moreover, nucleolin becomes clustered at the external side of the plasma membrane of living, intact cells when bound to cross-linked P-selectin-IgG-Fc chimeric protein. We have also found P-selectin binding to Colo-320 cells induces tyrosine phosphorylation specifically of cell-surface nucleolin and formation of a signaling complex containing cell-surface nucleolin, PI3-K and p38 MAPK. Using siRNA approaches, we have found that both P-selectin binding to Colo-320 cells and formation of the P-selectin-mediated p38 MAPK/PI3-K signaling complex require nucleolin expression. These results show that nucleolin (or a nucleolin-like protein) is a signaling receptor for P-selectin on Colo-320 cells and suggest a mechanism for linkage of nucleolin to P-selectin-induced signal transduction pathways that regulate the adhesion and the spreading of Colo-320 on fibronectin substrates. 相似文献
6.
Catarina Gomes Hugo Osório Marta Teixeira Pinto Diana Campos Maria José Oliveira Celso A. Reis 《PloS one》2013,8(6)
Sialyl-Lewis X (SLex) is a sialylated glycan antigen expressed on the cell surface during malignant cell transformation and is associated with cancer progression and poor prognosis. The increased expression of sialylated glycans is associated with alterations in the expression of sialyltransferases (STs). In this study we determined the capacity of ST3GAL3 and ST3GAL4 sialyltransferases to synthesize the SLex antigen in MKN45 gastric carcinoma cells and evaluated the effect of SLex overexpression in cancer cell behavior both in vitro and in vivo using the chicken chorioallantoic membrane (CAM) model. The activation of tyrosine kinase receptors and their downstream molecular targets was also addressed. Our results showed that the expression of ST3GAL4 in MKN45 gastric cancer cells leads to the synthesis of SLex antigens and to an increased invasive phenotype both in vitro and in the in vivo CAM model. Analysis of phosphorylation of tyrosine kinase receptors showed a specific increase in c-Met activation. The characterization of downstream molecular targets of c-Met activation, involved in the invasive phenotype, revealed increased phosphorylation of FAK and Src proteins and activation of Cdc42, Rac1 and RhoA GTPases. Inhibition of c-Met and Src activation abolished the observed increased cell invasive phenotype. In conclusion, the expression of ST3GAL4 leads to SLex antigen expression in gastric cancer cells which in turn induces an increased invasive phenotype through the activation of c-Met, in association with Src, FAK and Cdc42, Rac1 and RhoA GTPases activation. 相似文献
7.
Minas V Mylonas I Schiessl B Mayr D Schulze S Friese K Jeschke U Makrigiannakis A 《Histochemistry and cell biology》2007,128(1):55-63
Lewis antigens belong to the blood group of antigens and mediate cellular adhesion through interaction with selectins. Invasive
trophoblasts use an array of adhesion molecules to facilitate cell–cell and cell–extracellular matrix interactions. Here,
we examined immunohistochemically the expression of Sialyl Lewis a (sLea), Sialyl Lewis x (sLex) and Lewis y (Ley) in term placentas obtained from cases of normal, intrauterine growth retardation (IUGR), preeclamptic (PE) and hemolysis,
elevated liver enzymes and low platelets syndrome (HELLP) pregnancies. We report the expression of sLex in third trimester extravillous trophoblasts (EVT). sLex was significantly decreased in IUGR and moderately decreased in PE compared to normal placentas. sLex was additionally found in syncytiotrophoblast, without however any significant differences in staining intensity between
normal and pathological cases. sLea was restricted to amnion epithelium. Finally, Ley was expressed in cytotrophoblasts and villous endothelial cells. Ley expression was significantly upregulated in IUGR and HELLP, whereas there was a trend toward increase in PE compared to normal
placentas. The present study suggests that downregulation of sLex in EVT might be associated with IUGR and PE. Furthermore, Ley, which was recently described as a potent angiogenic factor, is upregulated in placental villi in conditions associated with
placental malperfusion.
U. Jeschke and A. Makrigiannakis have contributed equally. 相似文献
8.
Detection in human blood platelets of sialyl Lewis X gangliosides, potential ligands for CD62 and other selectins 总被引:1,自引:0,他引:1
Cooling Laura L.W.; Zhang De-Sheng; Walker Katherine E.; Koerner Theodore A.W. 《Glycobiology》1995,5(6):571-581
Activated platelets are known to express P-selectin, a lectin-likeadhesion receptor (CD62), through which they bind to sialylLewis X (sLex) ligands displayed on the membranes of leukocytes.To determine whether direct platelet-platelet interactions viaP-selectin/sLex interactions are also possible, we have examinedthe ganglioside extract of human blood platelets for the presenceof sLex ligands. Using the sensitive method of high-performancethin-layer chromatography (HPTLC)-immunostaining with the monoclonalantibody (mAb) CSLEX or with sialidase followed by mAbs MC480or PM81, eight sLex bands were demonstrated at R1 0.01, 0.03,0.05, 0.06, 0.08, 0.10, 0.14 and 0.21 in the solvent 45:55:10chloroform-methanol-aqueous 0.02% CaCl2. The sensitivity ofall eight bands to sialidase or endoglycoceramidase confirmedthat they were gangliosides. Comparison of the HPTLC mobilitiesand densities of platelet bands with those from five other humantissues (granulocytes, monoblasts, kidney, aortic endotheliumand erythrocytes) in three different solvents revealed threemajor bands associated with platelets: 3 (R1 0.03), 6 (0.08)and 14 (0.21). Platelet bands were demonstrated not to haveresulted from granulocyte contamination. Partial purificationof platelet sLex gangliosides by high-performance liquid chromatographyand their reaction with 14 oligosaccharide-specific mAbs (FH4,FH5, LM112-161, LM-181, A5, 1B2, BR55-2, BE2, ES4, MC631, MH04,SH34, P001 and MC813-70) revealed that band 6 is a multifucosylatedneolacto ganglioside and band 14 is a branched, disialo neolactofucoganglioside. Platelet band 3 combined the features of bothbands 6 and 14, and reacted differently than granulocyte band3. These partial structures resemble gangliosides associatedwith adhesion in other cell systems. It is concluded that plateletsexpress tissue-specific sLex gangliosides (sLex ligands). Thus,it is possible that platelet-platelet binding may be mediatedat least partially through P-selectin/sLex interactions, especiallyafter platelet activation. gangliosides HPTLC-immunostaining platelets selectin ligands sLex 相似文献
9.
Leukocyte cell surface sialyl Lewis x (sLex) and related epitopes play an important role in cell rolling and adhesion during diapedesis via interaction with E-selectin. Here, we present evidence that Mac-1 (CD11b/CD18, CR-3) is a major neutrophil glycoprotein decorated with sLex and ligation of these carbohydrate moieties by anti-sLex antibody significantly impairs neutrophil functions. First, Western blot analysis shows that both CD11b and CD18 subunit of purified Mac-1 are decorated with sLex moieties. A significant co-localization of CD11b and sLex moieties is observed at neutrophil secondary granules. With stimulation of formyl-Met-Leu-Phe (fMLP), neutrophil surface labeling with anti-sLex antibody follows an identical up-regulation pattern of Mac-1. Second, protein-binding assays indicate that sLex moieties on Mac-1 are critical for binding interaction of Mac-1 to E-selectin. Removal of sLex moieties completely abolishes Mac-1-E-selectin binding. Finally, ligation of Mac-1 sLex by anti-sLex antibody induces a significant degranulation of neutrophil secondary granules at the absence of chemoattractant stimulation. This “dysregulated” degranulation induced by anti-sLex antibody strongly inhibits neutrophil transmigration in response to fMLP. In summary, Mac-1 sLex moieties play a critical role in regulating β2 integrin functions during neutrophil transmigration and degranulation. 相似文献
10.
Reduction of rat myocardial ischemia and reperfusion injury by sialyl Lewis x oligosaccharide and anti-rat P-selectin antibodies 总被引:4,自引:0,他引:4
Tojo Shinichiro J.; Yokota Shin-ichi; Koike Haruhiko; Schultz Jody; Hamazume Yasuki; Misugi Emi; Yamada Kazuto; Hayashi Masaji; Paulson James C.; Morooka Shigeaki 《Glycobiology》1996,6(4):463-469
Polymorphonuclear leukocytes (PMN) are directly involved indevelopment of ischemic myocardial injury. Adhesion of PMN toendothelial cells is an initial step that triggers a sequentialprocess leading to acute inflammatory responses. Interactionbetween P-selectin and its oligosaccharide ligand, sialyl Lewisx (sLex), plays an important role in the early stage of theadhesion. To examine the role of P-selectin in various animaldisease models especially in rats, we have cloned rat E- andP-selectin cDNAs and established monoclonal antibodies againstthese rat selectins. In this report, we describe the generationand characterization of anti-rat P-selectin antibodies (ARPs).These antibodies detect cell surface P-selectin on thrombin-stimulatedrat platelets. More importantly, intravenous administrationof ARP2-4 reduced infarction developed after 30 min of ischemiafollowed by 24 h of reperfusion in a rat myocardial injury model.In addition, similar protective effect was also observed byadministration of a sLex- oligosaccharide. These results indicatethat cell adhesion mediated via P-selectin is involved in thedevelopment of ischemia and reperfusion injury in rat heart. ischemia and reperfusion injury monoclonal antibodies selectins sialyl Lewis x 相似文献
11.
Govind Ragupathi Payal Damani Geeta Srivastava Om Srivastava Steven J. Sucheck Yoshi Ichikawa Philip O. Livingston 《Cancer immunology, immunotherapy : CII》2009,58(9):1397-1405
Sialyl Lewisa (sLea), also termed CA19-9 antigen, is recognized by murine mAb19-9 and is expressed on the cancer cell surface as a glycolipid
and as an O-linked glycoprotein. It is highly expressed in a variety of gastrointestinal epithelial malignancies including
colon cancer and pancreatic cancer, and in breast cancer and small cell lung cancer, but has a limited expression on normal
tissues. sLea is known to be the ligand for endothelial cell selectins suggesting a role for sLea in cancer metastases and adhesion. For these reasons, sLea may be a good target for antibody mediated immunotherapy including monoclonal antibodies and tumor vaccines. However, sLea is structurally similar to sLex and other blood group related carbohydrates which are widely expressed on polymorphonucleocytes and other circulating cells,
raising concern that immunization against sLea will induce antibodies reactive with these more widely expressed autoantigens. We have shown previously both in mice and
in patients that conjugation of a variety of carbohydrate cancer antigen to keyhole limpet hemocyanin (KLH) and administration
of this conjugate mixed with saponin adjuvants QS-21 or GPI-0100 are the most effective methods for induction of antibodies
against these cancer antigens. We describe here for the first time the total synthesis of pentenyl glycoside of sLea hexasaccharide and its conjugation to KLH to construct a sLea-KLH conjugate. Groups of five mice were vaccinated subcutaneously four times over 6 weeks. Sera were tested against sLea-HSA by ELISA and against sLea positive human cell lines adenocarcinoma SW626 and small cell lung cancer (SCLC) DMS79 by FACS. As expected, mice immunized
with unconjugated sLea plus GPI-0100 or unconjugated sLea mixed with KLH plus GPI-0100 failed to produce antibodies against sLea. However, mice immunized with sLea-KLH conjugate without GPI-0100 produced low levels of antibodies and mice immunized with sLea-KLH plus GPI-0100 produced significantly higher titer IgG and IgM antibodies against sLea by ELISA. These antibodies were highly reactive by FACS and mediated potent complement mediated cytotoxicity against sLea positive SW626 and DMS79 cells. They showed no detectable cross reactivity against a series of other blood group-related
antigens, including Ley, Lex, and sLex by dot blot immune staining. This vaccine is ready for testing as an active immunotherapy for treating sLea positive cancer in clinical settings.
Govind Ragupathi and Philip O. Livingston are paid consultants and shareholders in MabVax Therapeutics, Inc., San Diego, CA
92121. The sLea vaccine is licensed to MabVax. 相似文献
12.
M.T. Goodarzi J.S. Axford S.S. Varanasi A. Alavi G. Cunnane O. Fitzgerald G.A. Turner 《Glycoconjugate journal》1998,15(12):1149-1154
Both infiltrating leukocytes and soluble immunoglobulin form aggregates in synovial fluid during the inflammatory process in rheumatoid arthritis (RA). Some of these changes are probably mediated by the adhesion molecule, E-selectin, which increases its expression with disease activity. As glycosylation changes in IgG in RA are well established, the current study was undertaken to measure the expression of the carbohydrate antigen sialyl Lewis x (sLex), on IgG in RA. sLex is a major ligand for E-selectin. Using a recently developed ELISA, sLex expression was determined in IgG isolated from 8 healthy individuals, 20 RA sufferers (10 early and 10 with more long-standing disease) and 20 patients with other rheumatic conditions (osteoarthritis, ankylosing spondylitis, systemic lupus erythematosus). S Lexexpression on IgG was elevated above the reference range in all but one of the RA patients and this change was highly significant (P < 0.0006). Expression of this antigen on IgG was also significantly different from normal in the other arthritic groups (P < 0.02), but the changes were much less than that observed for RA. In early RA, sLex was inversely correlated with parameters used to measure disease activity. This was not observed with the established RA, where there was weak positive association. These preliminary results indicate that a change in sLex expression on IgG is an early finding in the development of RA, which may be important in the development of the disease or for predicting its outcome. 相似文献
13.
14.
Inhibition of P-selectin-mediated cell adhesion by a sulfated derivative of sialic acid 总被引:2,自引:0,他引:2
Shodai T Suzuki J Kudo S Itoh S Terada M Fujita S Shimazu H Tsuji T 《Biochemical and biophysical research communications》2003,312(3):787-793
P-selectin, a carbohydrate-binding cell adhesion molecule expressed on activated endothelial cells and platelets, plays a key role in the recruitment of leukocytes to inflammatory and hemorrhagic sites. It simultaneously recognizes a sialic acid-containing carbohydrate chain and the sulfated tyrosine residues of a specific counter-receptor expressed on the leukocyte surface. We examined the inhibitory effects of a synthetic sulfated derivative of sialic acid (NMSO3) on P-selectin-mediated cell adhesion and found the following: (1) P-selectin/IgG chimera bound to immobilized NMSO3. (2) The binding of P-selectin/IgG chimera to purified P-selectin glycoprotein ligand-1 was inhibited by soluble NMSO3. (3) The adhesion of HL60 cells to P-selectin-expressing CHO cells was inhibited by NMSO3. (4) NMSO3 inhibited P-selectin-induced tumor necrosis factor-alpha production in monocytes and activated platelet-induced generation of reactive oxygen species in neutrophils. In conclusion, NMSO3 acts as a specific inhibitor for P-selectin-mediated cell adhesion and for adhesion-dependent leukocyte activation. 相似文献
15.
PSGL-1 derived from human neutrophils is a high-efficiency ligand for endothelium-expressed E-selectin under flow 总被引:1,自引:0,他引:1
Zou X Shinde Patil VR Dagia NM Smith LA Wargo MJ Interliggi KA Lloyd CM Tees DF Walcheck B Lawrence MB Goetz DJ 《American journal of physiology. Cell physiology》2005,289(2):C415-C424
P-selectin glycoprotein ligand-1 (PSGL-1) has been proposed as an important tethering ligand for E-selectin and is expressed at a modest level on human leukocytes. Sialyl Lewis x (sLex)-like glycans bind to E-selectin and are expressed at a relatively high level on circulating leukocytes. It is unclear whether PSGL-1 has unique biochemical attributes that contribute to its role as an E-selectin ligand. To probe this issue, we conjugated microspheres with either sLex or PSGL-1 purified from myeloid cells (neutrophils and HL-60) and compared their adhesion to endothelial expressed E-selectin under defined shear conditions. We found that both sLex and PSGL-1 microspheres adhere to 4 h of IL-1-activated human umbilical vein endothelial cells predominantly through E-selectin. Analysis of the adhesion revealed that the rate of initial tethering of the PSGL-1 microspheres to E-selectin was significantly greater than the rate of initial tethering of the sLex microspheres despite the fact that the sLex microspheres tested had higher ligand densities than the PSGL-1 microspheres. We also found that pretreatment of the PSGL-1 or sLex microspheres with HECA-452 had no significant effect on initial tethering to E-selectin. These results support the hypotheses that 1) PSGL-1 is a high-efficiency tethering ligand for E-selectin, 2) ligand biochemistry can significantly influence initial tethering to E-selectin, and 3) PSGL-1 tethering to E-selectin can occur via non-HECA-452 reactive epitopes. adhesion; leukocyte; inflammation 相似文献
16.
Yu M Henning R Walker A Kim G Perroy A Alessandro R Virador V Kohn EC 《Journal of cellular and molecular medicine》2012,16(10):2369-2378
Recent work identified L‐asparaginase (L‐ASP) as a putative therapeutic target for ovarian cancer. We suggest that L‐ASP, a dysregulator of glycosylation, would interrupt the local microenvironment, affecting the ovarian cancer cell—endothelial cell interaction and thus angiogenesis without cytotoxic effects. Ovarian cancer cell lines and human microvascular endothelial cells (HMVEC) were exposed to L‐ASP at physiologically attainable concentrations and subjected to analyses of endothelial tube formation, invasion, adhesion and the assessment of sialylated proteins involved in matrix‐associated and heterotypic cell adhesion. Marked reduction in HMVEC tube formation in vitro, HMVEC and ovarian cancer cell invasion, and heterotypic cell‐cell and cell‐matrix adhesion was observed (P < 0.05–0.0001). These effects were associated with reduced binding to ß1integrin, activation of FAK, and cell surface sialyl LewisX (sLex) expression. No reduction in HMVEC E‐selectin expression was seen consistent with the unidirectional inhibitory actions observed. L‐ASP concentrations were non‐toxic to either ovarian cancer or HMVEC lines in the time frame of the assays. However, early changes of autophagy were observed in both cell types with induction of ATG12, beclin‐1, and cleavage of LC‐3, indicating cell injury did occur. These data and the known mechanism of action of L‐ASP on glycosylation of nascent proteins suggest that L‐ASP reduces of ovarian cancer dissemination and progression through modification of its microenvironment. The reduction of ovarian cancer cell surface sLex inhibits interaction with HMVEC and thus HMVEC differentiation into tubes, inhibits interaction with the local matrix reducing invasive behaviour, and causes cell injury initiating autophagy in tumour and vascular cells. 相似文献
17.
L-Selectin Ligands That Are O-glycoprotease Resistant and Distinct from MECA-79 Antigen are Sufficient for Tethering and Rolling of Lymphocytes on Human High Endothelial Venules 总被引:2,自引:0,他引:2
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Rachael A. Clark Robert C. Fuhlbrigge Timothy A. Springer 《The Journal of cell biology》1998,140(3):721-731
During the process of lymphocyte recirculation, lymphocytes bind via L-selectin to sulfated sialyl-Lewisx (sLex)–containing carbohydrate ligands expressed on the surface of high endothelial venules (HEV). We have examined the expression of sLex on HEV using a panel of mAbs specific for sLex and sLex-related structures, and have examined the function of different sLex-bearing structures using an in vitro assay of lymphocyte rolling on HEV. We report that three sLex mAbs, 2F3, 2H5, and CSLEX-1, previously noted to bind with high affinity to glycolipid-linked sLex, vary in their ability to stain HEV in different lymphoid tissues and bind differentially to O-linked versus N-linked sLex on glycoproteins. Treatment of tissue sections with neuraminidase abolished staining with all three mAbs but slightly increased staining with MECA-79, a mAb to a sulfation-dependent HEV-associated carbohydrate determinant. Treatment of tissue sections with O-sialoglycoprotease under conditions that removed the vast majority of MECA-79 staining, only partially reduced staining with the 2F3 and 2H5 mAbs. Using a novel rolling assay in which cells bind under flow to HEV of frozen tissue sections, we demonstrate that a pool of O-sialoglycoprotease–resistant molecules is present on HEV that is sufficient for attachment and rolling of lymphocytes via L-selectin. This interaction is not inhibited by the mAb MECA-79. Furthermore, MECA-79 mAb blocks binding to untreated sections by only 30%, whereas the sLex mAb 2H5 blocks binding by ~60% and a combination of MECA-79 and 2H5 mAb blocks binding by 75%. We conclude that a pool of O-glycoprotease-resistant sLex-like L-selectin ligands exist on human HEV that is distinct from the mucin-associated moieties recognized by MECA-79 mAb. We postulate that these ligands may participate in lymphocyte binding to HEV. 相似文献
18.
Background
Endothelial E-selectin has been shown to play a pivotal role in mediating cell–cell interactions between breast cancer cells and endothelial monolayers during tumor cell metastasis. However, the counterreceptor for E-selectin and its role in mediating breast cancer cell transendothelial migration remain unknown.Methodology/Principal Findings
By assessing migration of various breast cancer cells across TNF-α pre-activated human umbilical vein endothelial cells (HUVECs), we found that breast cancer cells migrated across HUVEC monolayers differentially and that transmigration was E-selectin dependent. Cell surface labeling with the E-selectin extracellular domain/Fc chimera (exE-selectin/Fc) showed that the transmigration capacity of breast cancer cells was correlated to both the expression level and localization pattern of E-selectin binding protein(s) on the tumor cell surface. The exE-selectin/Fc strongly bound to metastatic MDA-MB-231, MDA-MB-435 and MDA-MB-468 cells, but not non-metastatic MCF-7 and T47D cells. Binding of exE-selectin/Fc was abolished by removal of tumor cell surface sialyl lewis x (sLex) moieties. Employing an exE-selectin/Fc affinity column, we further purified the counterreceptor of E-selectin from metastatic breast cancer cells. The N-terminal protein sequence and cDNA sequence identified this E-selectin ligand as a ∼170 kD human CD44 variant 4 (CD44v4). Purified CD44v4 showed a high affinity for E-selectin via sLex moieties and, as expected, MDA-MB-231 cell adhesion to and migration across HUVEC monolayers were significantly reduced by down-regulation of tumor cell CD44v4 via CD44v4-specific siRNA.Conclusions/Significance
We demonstrated, for the first time, that breast cancer cell CD44v4 is a major E-selectin ligand in facilitating tumor cell migration across endothelial monolayers. This finding offers new insights into the molecular basis of E-selectin–dependent adhesive interactions that mediate breast cancer cell transendothelial metastasis. 相似文献19.
The membrane carbohydrate antigen, sialyl Lewis x (sLex), is involved in cellular adhesive interactions in many diseases, such as cancer, inflammation and thrombosis. This antigen is also found on soluble macromolecules, such as serum glycoproteins, but the precise role of soluble sLex in modifying disease processes, or reflecting the pathological changes is still unclear. Although methods were previously reported for the measurement of soluble sLex, many of these were not well characterised, measurements were mainly made on mixtures of molecules, and the anti-sLex antibodies were used at concentrations that made the assay expensive. In this study an ELISA has been devised that detects sLex in purified soluble glycoconjugates using the anti-sLex antibody, CSLEX 1. Commercially-available haptoglobin (Hp) and synthetic complexes of Lewis antigens with polyacrylamide were used as model substances in developing the procedure. Key steps were washing the antibody/antigen complex with ten times diluted salt solution to prevent dissociation of the complex and the use of bovine serum albumin for blocking non-specific interactions. The assay was shown to be very specific, its precision was in the range 6–12%, and it could detect less than a pmol of sLex. It could also distinguish between different densities of sLex on the same amount of glycoconjugate. Determination of sLex in Hp isolated from small groups of healthy individuals, cancer patients, and rheumatoid arthritis sufferers suggested that the antigen expression is increased in disease. This method, which is an improvement on those previously described, will be useful for determining sLex in many different types of soluble glycoconjugate, and used in combination with synthetic carbohydrate polyacrylamide complexes, will help to standardize measurements of soluble sLex in the future. 相似文献
20.
《Cell Adhesion & Migration》2013,7(3):115-123
Endothelial and platelet P-selectin (CD62P) and leukocyte integrin αMβ2 (CD11bCD18, Mac-1) are cell adhesion molecules essential for host defense and innate immunity. Upon inflammatory challenges, P-selectin binds to PSGL-1 (P-selectin glycoprotein ligand-1, CD162) to mediate neutrophil rolling, during which integrins become activated by extracellular stimuli for their firm adhesion in a G-protein coupled receptor (GPCR)-dependent mechanism. Here we show that cross-linking of PSGL-1 by dimeric or multimeric forms of platelet P-selectin, P-selectin receptor-globulin, anti-PSGL-1 mAb and its F(ab’)2 induced adhesion of human neutrophils to fibrinogen (Fg) and intercellular cell adhesion molecule-1 (ICAM-1, CD54) and triggered a moderate clustering of αMβ2, but monomeric forms of soluble P-selectin and anti-PSGL-1 Fab did not. Interestingly, P-selectin did not induce a detectable interleukine-8 (IL-8) secretion (<0.1 ng/ml) in 30 minutes, whereas a high concentration of IL-8 (>50 ng/ml) was required to increase neutrophil adhesion to Fg. P-selectin-induced neutrophil adhesion was significantly inhibited by PP2 (a Src kinase inhibitor), but not by Pertussis toxin (PTX; a GPCR inhibitor). Activated platelets also increased neutrophil binding to fibrinogen and triggered tyrosine phosphorylation of cellular proteins. Our results indicate that P-selectin-induced integrin activation (Src kinase-dependent) is distinct from that elicited by cytokines, chemokines, chemoattractants (GPCR-dependent), suggesting that these two signal transduction pathways may cooperate for maximal activation of leukocyte integrins. 相似文献