Tenascin-C in the extracellular matrix promotes the selection of highly proliferative and tubulogenesis-defective endothelial cells

The extracellular matrix (ECM) contains important cues for tissue homeostasis and morphogenesis. The matricellular protein tenascin-C (TN-C) is overexpressed in remodeling tissues and cancer. In the present work, we studied the effect of different ECM—which exhibited a significant diversity in their TN-C content—in endothelial survival, proliferation and tubulogenic differentiation: autologous (endothelial) ECM devoid of TN-C, but bearing large amounts of FN; fibroblast ECM, bearing both high TN-C and FN contents; and finally, glioma-derived matrices, usually poor in FN, but very rich in TN-C. HUVECs initially adhered to the immobilized matrix produced by U373 MG glioma cells, but significantly detached and died by anoikis (50 to 80%) after 24 h, as compared with cells incubated with endothelial and fibroblast matrices. Surviving endothelial cells (20 to 50%) became up to 6-fold more proliferative and formed 74–97% less tube-like structures in vitro than cells grown on non-tumoral matrices. An antibody against the EGF-like repeats of tenascin-C (TN-C) partially rescued cells from the tubulogenic defect, indicating that this molecule is responsible for the selection of highly proliferative and tubulogenic defective endothelial cells. Interestingly, by using defined substrata, in conditions that mimic glioma and normal cell ECM composition, we observed that fibronectin (FN) modulates the TN-C-induced selection of endothelial cells. Our data show that TN-C is able to modulate endothelial branching morphogenesis in vitro and, since it is prevalent in matrices of injured and tumor tissues, also suggest a role for this protein in vascular morphogenesis, in these physiological contexts.     

TN-C与MDA-7在增生性瘢痕中表达与调控

增生性瘢痕是以皮肤损伤后成纤维细胞过度增殖为特征的一种病理改变,其发病机制尚不明确,目前没有有效的治疗方法。当皮肤组织损伤时,腱糖蛋白C(Tenascin-C,TN-C)具有多种不同的作用介导炎症和纤维化进程,并使组织有效修复。TN-C是细胞外基质中一个具有独特的六聚体结构的寡聚糖蛋白家族,TN-C一过性表达在器官形成期,在大多数成人组织不表达或表达极少。然而,在病理条件下TN-C表达增加,诸如炎症,伤口愈合和纤维化。TN-C参与胚胎形成、肿瘤发生及损伤修复过程有关,参与细胞黏附、增殖、迁徙、分化、细胞间相互作用以及细胞凋亡。黑色素瘤分化相关基因7/白介素24(MDA-7/IL-24)能选择性抑制瘢痕疙瘩中成纤维细胞的增殖,并诱导瘢痕疙瘩中成纤维细胞的凋亡,而对正常细胞无任何作用。MDA-7/IL-24很可能与瘢痕的形成有关。     

Distinct Sites on Tenascin-C Mediate Repellent or Adhesive Interactions with Different Neuronal Cell Types

In this study we have determined the binding specificities of four different neuronal cell types to tenascin-C (TN-C) and larninin using a cell adhesion assay. TN-C was repulsive for small cerebellar neurons and PC12 phaeochromocytoma cells, since after short-term adhesion to the substrate-bound molecule with a maximum of cell binding at 45 min, the cells detached from the substrate and after 22 h only about 25% of the originally adherent cells were still bound. For N2A neuroblastoma cells and retinal cells TN-C was an adhesive substrate, since the number of adherent cells did not decrease after the initial attachment period. All four cell types adhered well to larninin at all time points studied. For short-term adhesion of small cerebellar neurons and PC12 cells two binding sites were identified on TN-C, one being localized within the epidermal growth factor-like repeats three to five and the second within fibronectin type III-like repeats three and four. One binding site for N2A and retinal cells was localized within fibronectin type III-like repeat seven. Binding of small cerebellar neurons to TN-C was dependent on Ca²⁺, but not on Mg²⁺and was inhibitable by polyclonal antibodies to β1 integrin. Short-term adhesion of small cerebellar neurons was also inhibitable with a mixture of recombinant fragments of TN-C encompassing the whole molecule, although the specific inhibitory activity of this mixture was ten-fold lower on a molar basis when compared to the native molecule. Our observations indicate that different neuronal cell types use distinct binding sites on TN-C for repellent or adhesive interactions and that β1 integrin is involved in the recognition event leading to repulsion of small cerebellar neurons.     

Distinct Sites on Tenascin-C Mediate Repellent or Adhesive Interactions with Different Neuronal Cell Types

In this study we have determined the binding specificities of four different neuronal cell types to tenascin-C (TN-C) and larninin using a cell adhesion assay. TN-C was repulsive for small cerebellar neurons and PC12 phaeochromocytoma cells, since after short-term adhesion to the substrate-bound molecule with a maximum of cell binding at 45 min, the cells detached from the substrate and after 22 h only about 25% of the originally adherent cells were still bound. For N2A neuroblastoma cells and retinal cells TN-C was an adhesive substrate, since the number of adherent cells did not decrease after the initial attachment period. All four cell types adhered well to larninin at all time points studied. For short-term adhesion of small cerebellar neurons and PC12 cells two binding sites were identified on TN-C, one being localized within the epidermal growth factor-like repeats three to five and the second within fibronectin type III-like repeats three and four. One binding site for N2A and retinal cells was localized within fibronectin type III-like repeat seven. Binding of small cerebellar neurons to TN-C was dependent on Ca²⁺, but not on Mg²⁺and was inhibitable by polyclonal antibodies to β1 integrin. Short-term adhesion of small cerebellar neurons was also inhibitable with a mixture of recombinant fragments of TN-C encompassing the whole molecule, although the specific inhibitory activity of this mixture was ten-fold lower on a molar basis when compared to the native molecule. Our observations indicate that different neuronal cell types use distinct binding sites on TN-C for repellent or adhesive interactions and that β1 integrin is involved in the recognition event leading to repulsion of small cerebellar neurons.     

TN—C与MDA-7在增生性瘢痕中表达与调控

增生性瘢痕是以皮肤损伤后成纤维细胞过度增殖为特征的一种病理改变,其发病机制尚不明确,目前没有有效的治疗方法。当皮肤组织损伤时,腱糖蛋白C（Tenascin-C,TN-C）具有多种不同的作用介导炎症和纤维化进程,并使组织有效修复。TN—C是细胞外基质中一个具有独特的六聚体结构的寡聚糖蛋白家族,TN—C一过性表达在器官形成期,在大多数成人组织不表达或表达极少。然而,在病理条件下TN—C表达增加,诸如炎症,伤口愈合和纤维化。TN—C参与胚胎形成、肿瘤发生及损伤修复过程有关,参与细胞黏附、增殖、迁徙、分化、细胞间相互作用以及细胞凋亡。黑色素瘤分化相关基因7／白介素24（MDA-7／IL-24）能选择性抑制瘢痕疙瘩中成纤维细胞的增殖,并诱导瘢痕疙瘩中成纤维细胞的凋亡,而对正常细胞无任何作用。MDA-7／IL-24很可能与瘢痕的形成有关。     

Cell surface annexin II is a high affinity receptor for the alternatively spliced segment of tenascin-C

We have investigated the binding of soluble tenascin-C (TN-C) to several cell lines using a radioligand binding assay. Specific binding was demonstrated to U-251MG human glioma cells and to a line of bovine aortic endothelial cells, but hamster fibroblasts showed no specific binding. Recombinant proteins corresponding to specific domains of TN-C were used to map the binding site(s) in TN-C. The alternatively spliced segment (TNfnA-D) inhibited the binding of native TN-C most strongly, and itself bound to glioma and endothelial cells. Scatchard analysis of TNfnA-D binding indicated 2-5 x 10(5) binding sites per cell, with an apparent 2 nM dissociation constant. The cell surface receptor for TNfnA-D was identified as a 35-kD protein on the basis of blot binding assays and affinity chromatography of membrane extracts on native TN-C and TNfnA-D columns. Protein sequencing indicated that this 35-kD receptor was annexin II. Annexin II is well characterized as a cytoplasmic protein, so it was surprising to find it as a presumably extracellular receptor for TN-C. To confirm that it was the 35-kD receptor, we obtained purified annexin II and demonstrated its binding to TNfnA-D and TN-C at nM concentrations. Antibodies to annexin II prominently stained the external surface of live endothelial cells and blocked the binding of TNfnA-D to the cells. Thus annexin II appears to be a receptor for the alternatively spliced segment of TN-C, and may mediate cellular responses to soluble TN-C in the extracellular matrix.     

Cloning, expression, and characterization of TNFSF14 (LIGHT) gene in mefugu, Takifugu obscurus

Tumor necrosis factor receptor-associated factor 6 (TRAF6), which plays an important role in inflammation and immune response, is an essential adaptor protein for the NF-κB (nuclear factor κB) signaling pathway. Recent studies have shown that TRAF6 played an important role in tumorigenesis and invasion by suppressing NF-κB activation. However, up to now, the biologic role of TRAF6 in glioma has still remained unknown. To address the expression of TRAF6 in glioma cells, four glioma cell lines (U251, U-87MG, LN-18, and U373) and a non-cancerous human glial cell line SVG p12 were used to explore the protein expression of TRAF6 by Western blot. Our results indicated that TRAF6 expression was upregulated in human glioma cell lines, especially in metastatic cell lines. To investigate the role of TRAF6 in cell proliferation, apoptosis, invasion, and migration of glioma, we generated human glioma U-87MG cell lines in which TRAF6 was either overexpressed or depleted. Subsequently, the effects of TRAF6 on cell viability, cell cycle distribution, apoptosis, invasion, and migration in U-87MG cells were determined with 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) assay, flow cytometry analysis, transwell invasion assay, and wound-healing assay. The results showed that knockdown of TRAF6 could decrease cell viability, suppress cell proliferation, invasion and migration, and promote cell apoptosis, whereas overexpression of TRAF6 displayed the opposite effects. In addition, the effects of TRAF6 on the expression of phosphor-NF-κB (p-p65), cyclin D1, caspase 3, and MMP-9 were also probed. Knockdown of TRAF6 could lower the expression of p-p65, cyclin D1, and MMP-9, and raise the expression of caspase 3. All these results suggested that TRAF6 might be involved in the potentiation of growth, proliferation, invasion, and migration of U-87MG cell, as well as inhibition of apoptosis of U-87MG cell by abrogating activation of NF-κB.     

The function of multiple extracellular matrix receptors in mediating cell adhesion to extracellular matrix: preparation of monoclonal antibodies to the fibronectin receptor that specifically inhibit cell adhesion to fibronectin and react with platelet glycoproteins Ic-IIa

We have identified monoclonal antibodies that inhibit human cell adhesion to collagen (P1H5), fibronectin (P1F8 or P1D6), and collagen and fibronectin (P1B5) that react with a family of structurally similar glycoproteins referred to as extracellular matrix receptors (ECMRs) II, VI, and I, respectively. Each member of this family contains a unique alpha subunit, recognized by the antibodies, and a common beta subunit, each of approximately 140 kD. We show here that ECMR VI is identical to the fibronectin receptor (FNR), very late antigen (VLA) 5, and platelet glycoproteins Ic-IIa and shall be referred to as FNR. Monoclonal antibodies to FNR inhibit lymphocyte, fibroblast, and platelet adhesion to fibronectin-coated surfaces. ECMRs I, II, and FNR were differentially expressed in platelets, resting or activated lymphocytes, and myeloid, epithelial, endothelial, and fibroblast cell populations, suggesting a functional role for the receptors in vascular emigration and selective tissue localization. Tissue staining of human fetal skin localized ECMRs I and II to the basal epidermis primarily, while monoclonal antibodies to the FNR stained both the dermis and epidermis. Experiments carried out to investigate the functional roles of these receptors in mediating cell adhesion to complex extracellular matrix (ECM) produced by cells in culture revealed that complete inhibition of cell adhesion to ECM required antibodies to both the FNR and ECMR II, the collagen adhesion receptor. These results show that multiple ECMRs function in combination to mediate cell adhesion to complex EMC templates and predicts that variation in ECM composition and ECMR expression may direct cell localization to specific tissue domains.     

Knockdown of NOB1 expression by RNAi inhibits cellular proliferation and migration in human gliomas

NOB1 (NIN1/RPN12 binding protein 1 homolog), a ribosome assembly factor, is thought to be essential for the processing of the 20S pre-rRNA into the mature 18S rRNA. It is also reported to participate in proteasome biogenesis. However, the contribution of NOB1 gene dysfunction to the pathology of human diseases, such as gliomas, has not been addressed. Here, we detected expression levels of NOB1 mRNA in U251, U87, U373, and A172 cells by quantitative real-time PCR. To analyze the expression levels of NOB1 protein in glioma tissues, we performed immunohistochemistry on 56 pathologically confirmed glioma samples (7 Grade I cases, 19 Grade II cases, 16 Grade III cases, and 14 Grade IV cases). A recombinant lentivirus expressing NOB1 short hairpin RNA (shNOB1) was constructed and infected into U251 and U87-MG human glioma cells. We found that NOB1 mRNA was expressed in all four cell lines. The expression level of the NOB1 protein was significantly higher in high-grade gliomas than in low-grade gliomas. Knockdown of the NOB1 gene resulted in suppression of the proliferation and the colony-forming abilities of U251 and U87-MG cells, cell cycle arrest during the G₀/G₁ phase, and a significant enhancement of cell apoptosis. In addition, cell migration was significantly suppressed in U251 and U87-MG cells that were infected with the shNOB1-expressing lentivirus. These results suggest that NOB1 promotes glioma cell growth and migration and could be a candidate for molecular targeting during gene therapy treatments of glioma.     

Fibronectin-plasma membrane interaction in the adhesion of hemopoietic cells

Many hemopoietic cell lines were examined for their ability to adhere to culture dishes coated with extracellular matrix proteins. Adhesion assay was performed with murine and human leukemic cell lines representative of different stages of differentiation along both erythroid and myeloid lineages. All the hemopoietic cell lines tested adhered to fibronectin but not to laminin, types I, III, and IV collagen, serum-spreading factor, and cartilage proteoglycans. In addition to immortalized cell lines, immature erythroid and myeloid mouse bone marrow cells adhered to fibronectin. To define the fibronectin region involved in hemopoietic cell adhesion, proteolytic fragments, monoclonal antibodies, and synthetic peptides were used. Among different fibronectin fragments tested, only a 110-kD polypeptide, corresponding to the fibroblast attachment domain, was active in promoting adhesion. Moreover, a monoclonal antibody to the cell binding site located within this domain prevented hemopoietic cell adhesion. Finally, the tetrapeptide Arg-Gly-Asp-Ser, which corresponds to the fibronectin sequence recognized by fibroblastic cells, specifically and competitively inhibited attachment of hemopoietic cells to this molecule. The cell surface molecule involved in the interaction of mouse hemopoietic cells with fibronectin was identified as a 145,000-D membrane glycoprotein by adhesion-blocking antibodies. This glycoprotein was found to be antigenically and functionally related to the GP135 membrane glycoprotein involved in the adhesion of fibroblasts to fibronectin (Giancotti, F. G., P. M. Comoglio, and G. Tarone, 1986, Exp. Cell Res., 163:47-62). On the basis of these data, we conclude that interaction of hemopoietic cells with fibronectin involves a specific fibronectin sequence and a 145,000-D cell surface glycoprotein. We speculate that this property might be relevant for the interaction of hemopoietic cells with the bone marrow stroma, which represents the natural site of hemopoiesis.     

ZEB1上调神经细胞黏附分子促进胶质瘤细胞侵袭与转移

胶质瘤是一种较为常见的颅内恶性肿瘤,其侵袭转移能力强,影响临床疗效。探讨胶质瘤发生侵袭转移的分子机制,寻找新的靶点干预胶质瘤侵袭转移是目前亟待解决的重大课题。我们前期研究中发现,神经细胞黏附分子（neuronal cell adhesion molecule, NRCAM）在各种胶质瘤细胞中的表达量均显著高于其在人正常星形胶质细胞(NHA)中的表达量（NRCAM在胶质瘤A172和T98G中的表达量分别是其在NHA中的2.15和17.63倍）;且根据人类蛋白质组学数据库信息及qRT-PCR结果证实,NRCAM在胶质瘤组织中的表达量也显著高于其在正常组织中的表达。Kaplan-Meier分析提示,高表达的NRCAM与胶质瘤患者较差的预后正相关。在此基础上,通过生物信息学预测的方法结合双荧光素酶报告基因实验证实,转录因子锌指E盒结合蛋白1(ZEB1)能够增加NRCAM启动子活性,上调NRCAM mRNA和蛋白质水平的表达量。通过Transwell实验证实,在过表达ZEB1的胶质瘤细胞A172中,沉默NRCAM将抑制该细胞的侵袭能力。而在敲低ZEB1的胶质瘤细胞T98G中,过表达NRCAM将增加该细胞的侵袭能力。总之,NRCAM在胶质瘤中显著高表达且与患者较差的预后正相关。ZEB1转录上调NRCAM来增加胶质瘤细胞侵袭能力。     

Immunochemical and biochemical characterization of a glioma-associated extracellular matrix glycoprotein

A novel human glioma-associated extracellular matrix (ECM) glycoprotein has been identified by murine monoclonal antibody 81C6. The glycoprotein, designated GMEM, is expressed in the ECM of glioma and mesenchymal cell cultures, in the perivascular matrix of endothelial proliferations of human gliomas, and in the stroma of human glioma xenografts in athymic mice, where it has been used as a target antigen for monoclonal antibody tumor localization and radioimaging. We report here on the immunochemical and biochemical characterization of GMEM. Polyacrylamide gel analysis of immunoprecipitated [3H]-leucine- and [3H]-glucosamine-labeled ECM from the human glioma cell line U-251MG has shown that GMEM is a high-molecular-weight macromolecule (Mr approximately 1,000,000) composed of Mr approximately 230,000 disulfide-bonded glycoprotein subunits. Immunoprecipitation, immunoblot, and one-dimensional peptide map analysis have shown that GMEM is distinct from human fibroblast and plasma fibronectin. These results support previous immunohistology and absorption analysis findings, indicating that GMEM is distinct from fibronectin, laminin, and glycosaminoglycans secreted by U-251MG.     

Monoclonal anti-GD3 antibodies selectively inhibit the proliferation of human malignant glioma cells in vitro

The frequently occurring alteration of ganglioside expression in tumor cells has been implicated to play a role in the uncontrolled growth of these cells; antibodies to such gangliosides might affect tumor cell growth. We have studied the effect of IgM monoclonal antibodies to two glioma-associated gangliosides, GD3 and GM2, on cell proliferation of four human glioma cell lines and one renal tumor cell line. Of the two anti-ganglioside antibodies tested, only the anti-GD3 antibody resulted in a significant (p<0.005) inhibition of cell proliferation as measured by thymidine incorporation and Brd-U labeling, after 24[emsp4 ]h incubation. The effect was not dependent on any serum factor and no increased cell death was observed. All cell lines contained higher or similar amounts of GM2 than GD3, and both antigens were shown to be expressed on the cell surface and accessible to antibodies. The selective effect of anti-GD3 antibodies as contrasted to the inactivity of anti-GM2 antibodies suggests a possible role for ganglioside GD3 in tumor cell proliferation.     

MircoRNA‐1275 promotes proliferation,invasion and migration of glioma cells via SERPINE1

This study was designed to explore the relationship between miR‐1275 and SERPINE1 and its effects on glioma cell proliferation, migration, invasion and apoptosis. Differentially expressed miRNAs and mRNAs in glioma tissues were screened out by bioinformatic analysis. Dual‐luciferase reporter gene assay was used to validate the targeted relationship between miR‐1275 and SERPINE1. qRT‐PCR was used to detect the expression of miR‐1275 and SERPINE1 in glioma tissues. The expressions of SERPINE1 and p53 pathway‐related proteins in glioma cells were detected by western blot. Glioma cell proliferation, apoptosis, migration and invasion were respectively detected by CCK‐8 assay, flow cytometry, wound healing assay and transwell assay. Tumour xenograft model was developed to study the influence of miR‐1275 and SERPINE1 on glioma growth in vivo. The results of microarray analysis, qRT‐PCR and western blot showed that miR‐1275 was low‐expressed while SERPINE1 was high‐expressed in glioma. Dual‐luciferase assay showed that miR‐1275 could bind to SERPINE1. Overexpression of miR‐1275 could promote the p53 pathway‐related proteins’ expression. Highly expressed miR‐1275 could repress the migration, proliferation and invasion of glioma cells while highly expressed SERPINE1 had inverse effects. Tumour xenograft showed that up‐regulated miR‐1275 or down‐regulated SERPINE1 could repress glioma growth in vivo. Up‐regulation of miR‐1275 activated p53 signalling pathway via regulating SERPINE1 and therefore suppressed glioma cell proliferation, invasion and migration, whereas promoted cell apoptosis.     

Mapping the homotypic binding sites in CD31 and the role of CD31 adhesion in the formation of interendothelial cell contacts

CD31 is a member of the immunoglobulin superfamily consisting of six Ig- related domains. It is constitutively expressed by platelets, monocytes, and some lymphocytes, but at tenfold higher levels on vascular endothelial cells. CD31 has both homotypic and heterotypic adhesive properties. We have mapped the homotypic binding sites using a deletion series of CD31-Fc chimeras and a panel of anti-CD31 monoclonal antibodies. An extensive surface of CD31 is involved in homotypic binding with domains 2 and 3 and domains 5 and 6 playing key roles. A model consistent with the experimental data is that CD31 on one cell binds to CD31 on an apposing cell in an antiparallel interdigitating mode requiring full alignment of the six domains of each molecule. In addition to establishing intercellular homotypic contacts. CD31 binding leads to augmented adhesion via beta 1 integrins. The positive cooperation between CD31 and beta 1 integrins can occur in heterologous primate cells (COS cells). The interaction is specific to both CD31 and beta 1 integrins. Neither intercellular adhesion molecule-1 (ICAM- 1)/leukocyte function-associated antigen-1 (LCAM-1) nor neural cell adhesion molecule (NCAM)/NCAM adhesion leads to recruitment of beta 1 integrin adhesion pathways. Establishment of CD31 contacts have effects on the growth and morphology of endothelial cells. CD31(D1-D6)Fc inhibits the growth of endothelial cells in culture. In addition, papain fragments of anti-CD31 antibodies (Fab fragments) disrupt interendothelial contact formation and monolayer integrity when intercellular contacts are being formed. The same reagents are without effect once these contacts have been established, suggesting that CD31- CD31 interactions are critically important only in the initial phases of intercellular adhesion.     

PAICS is related to glioma grade and can promote glioma growth and migration

Glioma is a common malignant tumour of the brain. In this study, we aimed to investigate diagnostic biomarkers and its role in glioma. Weighted gene co-expression network analysis (WGCNA) and Cytoscape software were used to screen the marker genes in glioma. RT-qPCR and Western blotting methods were performed to determine the expression of PAICS, ERCC1 and XPA genes in glioma tissues. Expression level of PAICS in different grades of glioma was examined by immunohistochemistry. CCK8 and Colony formation assays were used to detect cell proliferation. Cell adhesion assay was used to detect adhesion ability. Wound healing and transwell tests were used to detect cell migration ability. Flow cytometry was used to detect cell cycle and apoptosis. According to the predicted co-expression network, we identified the hub gene PAICS. Furthermore, we observed that PAICS expression level was up-regulated in glioma tissues compared with normal tissues, and the expression level was correlated with the grade of glioma. Moreover, we found PAICS can promote glioma cells proliferation and migration in vitro. Flow cytometry results showed that si-PAICS cells were stalled at the G1 phase compared with the si-NC cells and knocking down PAICS expression can increase apoptotic rate. PAICS can regulate the mRNA and protein levels of nucleotide excision repair pathway core genes ERCC1 and XPA. l -aspartic acid can affect the expression of PAICS and then inhibit glioma cell proliferation. Our results indicated that PAICS can promote glioma proliferation and migration. PAICS may act as a potential diagnostic marker and a therapeutic target for glioma.     

Tumor cell surface alpha 4 beta 1 integrin mediates adhesion to vascular endothelium: demonstration of an interaction with the N-terminal domains of INCAM-110/VCAM-1.

Hematogenous metastasis involves adhesive interactions between blood-borne tumor cells and the vessel wall. By the use of in vitro assays, the adhesion of human melanoma, osteosarcoma, and kidney carcinoma (but not colon carcinoma) cell lines was shown to involve the cytokine-inducible endothelial cell surface protein inducible cell adhesion molecule 110 (INCAM-110) and the alpha 4 beta 1 integrin, molecules normally involved in endothelial-leukocyte interactions. Tumor adhesion to human endothelial cell monolayers was increased 1.9- to 8.2-fold by endothelial activation with the cytokine tumor necrosis factor (TNF) and inhibited by the anti-INCAM-110 monoclonal antibody (mAb) E1/6. Each of these tumor cells expressed members of the beta 1 integrin family of adhesion molecules, and antibodies to the alpha 4 and beta 1 integrin subunits inhibited tumor-endothelial adhesion (48-87% inhibition). A cDNA encompassing the three N-terminal Ig-like domains of vascular cell adhesion molecule 1 (VCAM-1) encoded a protein recognized by the anti-INCAM-110 mAb E1/6 and, when captured onto plastic, supported melanoma cell adhesion by an alpha 4 integrin-dependent mechanism. In contrast to mAb E1/6, a second anti-INCAM-110 mAb Hu8/4 neither inhibited adhesion to activated endothelium nor bound the first three Ig-like domains of INCAM-110/VCAM-1. These data indicate that the adherence of several human tumors to activated endothelium is mediated by an interaction of alpha 4 beta 1 integrin and the N-terminal Ig-like domains of endothelial INCAM-110/VCAM-1. Tumor acquisition of the alpha 4 integrin subunit and endothelial expression of INCAM-110 may affect the frequency and distribution of metastasis.     

Matrix valency regulates integrin-mediated lymphoid adhesion via Syk kinase

Lymphocytes accumulate within the extracellular matrix (ECM) of tumor, wound, or inflammatory tissues. These tissues are largely comprised of polymerized adhesion proteins such as fibrin and fibronectin or their fragments. Nonactivated lymphoid cells attach preferentially to polymerized ECM proteins yet are unable to attach to monomeric forms or fragments of these proteins without previous activation. This adhesion event depends on the appropriate spacing of integrin adhesion sites. Adhesion of nonactivated lymphoid cells to polymeric ECM components results in activation of the antigen receptor-associated Syk kinase that accumulates in adhesion-promoting podosomes. In fact, activation of Syk by antigen or agonists, as well as expression of an activated Syk mutant in lymphoid cells, facilitates their adhesion to monomeric ECM proteins or their fragments. These results reveal a cooperative interaction between signals emanating from integrins and antigen receptors that can serve to regulate stable lymphoid cell adhesion and retention within a remodeling ECM.     

The mitogenic activity of human T-cell leukemia virus type I is T-cell associated and requires the CD2/LFA-3 activation pathway.

The presence of a high number of activated T cells in the bloodstream and spontaneous proliferation of peripheral blood mononuclear cells in vitro are striking characteristics of human T-cell leukemia virus type I (HTLV-I) infection. The HTLV-I regulatory protein Tax and the envelope protein gp46 have been implicated in mediating the activation process. In this study, HTLV-I-producing cell lines and purified virus from the cell lines were examined for the ability to activate peripheral blood lymphocytes (PBLs) and Jurkat cells. Antisera and monoclonal antibodies against several cellular adhesion proteins involved in T-cell activation and against viral proteins were used to identify which molecules may be participating in the activation process. First, neither virus from a T-cell line, MT2, nor virus produced from the human osteosarcoma cell line HOS/PL was able to induce PBLs to proliferate. In contrast, both fixed and irradiated HTLV-I-producing T-cell lines induced proliferation of PBLs; HOS/PL cells did not activate PBLs. Second, HTLV-I-positive T-cell lines were capable of activating interleukin-2 mRNA expression in Jurkat cells. Induction of interleukin-2 expression was inhibited by anti-CD2 and anti-lymphocyte function-associated antigen 3 (LFA-3) monoclonal antibodies but not anti-human leukocyte antigen-DR, anti-CD4, anti-LFA-1, or anti-intercellular adhesion molecule 1. Similar results were obtained with PBLs as the responder cells. Furthermore, monoclonal antibodies and antisera against various regions of the HTLV-I envelope proteins gp46 and gp21 as well as p40tax did not block activation. These data indicate that HTLV-I viral particles are not intrinsically mitogenic and that infection of target T cells is not necessary for activation. Instead, the mitogenic activity is restricted to virus-producing T cells, requires cell-to-cell contact, and may be mediated through the LFA-3/CD2 activation pathway.