Search

We report the effect of the stable expression of a 13 amino acid human fibronectin (FN) peptide (FN13) on the organization of the FN extracellular matrix (ECM) and of FN integrin receptors (FNRs), in relationship with the inhibition of cellular invasion, in three FN-ECM defective human tumor-derived cell lines: SK-Hep1C3, hepatoma, ACN, neuroblastoma, and SK-OV-3, ovary carcinoma. All these cell lines stably expressing the FN13 peptide, organized an FN-ECM, disorganized alpha v beta 1 integrins and inactivated the ILK pathway, with the loss of secretion of MMP-9. This was associated with the inhibition of cell invasion in Matrigel matrix only in SK-Hep1C3 and ACN, but not in SK-OV-3 cells. Analysis of the integrin receptors organization showed that the FN13 expressing cells SK-Hep1C3 and ACN organized alpha v beta 3 integrins, whereas SK-OV-3 organized alpha v beta 5 dimers. The functional block of alpha v beta 5 integrins, with an inactivating anti-alpha v beta 5 antibody, led to the induction of alpha v beta 3 integrins also in SK-OV-3 cells, and to the inhibition of cell invasion. These data show that in the human tumor cells studied FN13 inhibits the in vitro invasion through the dissociation of alpha v beta 1 dimers, leading to ILK pathway inactivation, only when the organization of alpha v beta 3 integrins is induced in the plasma membrane.

MDC-9 is a widely expressed member of the metalloproteinase/disintegrin/cysteine-rich protein family. The disintegrin domain of MDC-9 lacks an RGD motif but has recently been reported to bind the alpha(6)beta(1) integrin; however, it is unclear whether MDC-9 can bind other integrins. In the present study myeloma cells, but not lymphoblastoid cells, were shown to bind to immobilised, recombinantly expressed MDC-9 disintegrin domain (A9dis). Binding was divalent cation-dependent, being supported by Mn(2+) and Ca(2+). Adhesion of myeloma cells to A9dis was completely inhibited by an antibody to the alpha(v)beta(5) integrin but not by antibodies to other subunits. RGD-containing peptides had no effect on binding, suggesting that MDC-9 interacts with alpha(v)beta(5) in an RGD-independent manner. Flow cytometric analyses demonstrated that myeloma cells, but not lymphoblastoid cells, expressed alpha(v)beta(5) on the cell membrane. These data indicated that the disintegrin domain of MDC-9 can function as an adhesion molecule by interacting with an alpha(v)beta(5) integrin.

The migratory behaviour of cells is fundamental to diverse biologic processes such as tumor metastasis, development of atherosclerotic plaques, embryonic development and wound healing. We have examined the effects of IGF-I and IGFBPs on the migration of Chinese Hamster ovary (CHO) cells, smooth muscle cells (SMC) and human breast cancer cells (HBC) and have studied the involvement of integrin receptors in migration induced by IGF-I and by IGFBPs. Using a monolayer wounding assay, we determined the effect of IGFBP-1 on SMC to be qualitatively similar to its effect we reported earlier on CHO cells, in that there is a direct stimulation of migration mediated by the alpha 5 beta 1 integrin. IGFBP-2 has no direct effect on SMC migration, and although it also contains the Arg-Gly-Asp sequence, we can detect no integrin binding. Unlike CHO cells, SMC are stimulated to migrate by IGF-I. IGFBP-2 and IGFBP-1 both inhibit this IGF-I receptor-mediated stimulation. We have also studied the migration of HBC using a Boyden chamber apparatus and have shown a potent chemotactic effect of IGF-I. We have investigated the mechanisms for IGF-I stimulation of SMC and HBC migration. IGF-I stimulation of SMC migration requires the presence of either 0.2% serum or vitronectin, because of a requirement for ligand binding by the alpha V beta 3 integrin (vitronectin receptor). MCF-7 HBC migrate toward a concentration gradient of IGF-I, the only growth factor that was able to stimulate these cells to migrate. Integrin ligand binding was also necessary for MCF-7 cells to migrate in response to IGF-I; alpha V beta 5 integrin was required for migration on vitronectin and alpha 2 beta 1 was required on collagen. These studies demonstrate that the stimulation of cell migration by IGFBP-1 and IGF-I involves signaling by members of the integrin family of receptors. The mechanisms by which the IGF-I receptor and integrin receptors interact are not yet known.

A baculovirus system was used to express full-length recombinant mouse thrombospondin 2 (rTSP2) as a disulfide-bonded homotrimer with an NH2 terminus beginning with Asp20.rTSP2, like TSP1, was more sensitive to trypsin digestion if depleted of calcium ion. The trypsin digestion pattern of rTSP2 and TSP1 differed in that trypsin cut between the first and second type 1 modules of rTSP2. For bovine aortic endothelial cells adhering to TSP-coated polystyrene plates, reduction after coating caused both TSPs to be much more adhesive; these adhesions were blocked completely by RGDS peptide or antibody to alpha v beta 3 integrin.rTSP2 and TSP1 also mediated the adhesion of HT-29 human colon adenocarcinoma cells that carry alpha v beta 5 but not alpha v beta 3 integrin. Antibody to alpha v beta 5 did not inhibit adhesion of HT-29 cells to TSP1 or rTSP2. Rather, adhesion of HT-29 cells was decreased by treatment of TSPs with EDTA, abolished by reduction of the TSPs, and, in the case of rTSP2, blocked by heparin. Adhesion of MG63 cells to both TSPs was complex. Treatment with EDTA enhanced the adhesive activity of rTSP2 but decreased the adhesive activity of TSP1. These results show that TSP2 can be processed and secreted when overexpressed using baculovirus, TSP1 and rTSP2 differ in protease susceptibility in the type 1 module region, and TSP1 and rTSP2 mediate cell adhesion by complex and similar but not identical mechanisms.

Vitronectin is a soluble serum factor that is known to promote epiboly of keratinocytes in explant cultures and enhance cell spreading and attachment to matrix. Recently, vitronectin was demonstrated to promote human keratinocyte locomotion. The mechanism(s) by which vitronectin enhances keratinocyte migration is unknown. In this study, we quantitated the vitronectin-driven migration of human keratinocytes in the presence of antibodies to vitronectin receptors. We found that vitronectin's effect of promoting human keratinocyte migration was inhibited by antibody-directed against the alpha v beta 5 receptor. In addition, we surface-labeled human keratinocytes, chromatographed extracts of the cell membranes on a vitronectin column, and then immunoprecipitated the bound and eluted proteins with antibodies to specific vitronectin receptors. We identified the vitronectin receptors on human keratinocytes as bands of 150,000 and 100,000 daltons without reduction and as 125,000 and 110,000 daltons under reducing conditions. Immunoprecipitation with specific antibodies identified the major receptor to be the alpha v beta 5 integrin. In addition, we quantitated vitronectin-driven migration of human keratinocytes in the presence of Arg-Gly-Asp (RGD) and control peptides. We found that the presence of RGD, but not control peptide, inhibited vitronectin-driven migration of human keratinocytes. These studies demonstrate that human keratinocytes express vitronectin receptors and use the alpha v beta 5 receptor for cellular locomotion.

The integrin supergene family includes receptors for a variety of extracellular matrix as well as cell surface proteins. Integrin alpha 4 has been shown to play an important role in leukocyte adhesion and extravasation during immune and inflammatory reactions. One recognition sequence known to interact with alpha 4 is the Leu-Asp-Val (LDV) site contained in the connecting segment 1 region of fibronectin. Here we present evidence that shows that a conformationally restricted RGD-containing peptide is a potent inhibitor of cell adhesion mediated by alpha 4 beta 1, a receptor not convincingly documented to interact with RGD peptides. This peptide, 1-adamantaneacetyl-Cys-Gly-Arg-Gly-Asp-Ser-Pro-Cys (disulfide bridge between residues 1-8), blocks Jurkat cell adhesion to connecting segment 1-containing peptides as well as cell adhesion to cytokine-activated endothelial cells. Adhesion of Jurkat cells to either vascular cell adhesion molecule-expressing cells or recombinant vascular cell adhesion molecule-coated plates was likewise inhibited by this peptide. Furthermore, alpha 4 beta 1 can bind directly to a cyclic RGD peptide immobilized to Sepharose. Integrins, alpha 5 beta 1, alpha v beta 3, alpha IIb/beta IIIa, alpha 2 beta 1, alpha v beta 1, alpha v beta 5, alpha v beta 6, and alpha 3 beta 1, have been shown to recognize the Arg-Gly-Asp (RGD) sequence present in a variety of extracellular matrix proteins, and our data support the addition of alpha 4 beta 1 to this group. Further studies using molecular modeling of such cyclic RGD peptides could help in the design of more potent peptides or nonpeptide mimetics that could effectively block alpha 4-mediated activity and have potential application in a number of inflammatory diseases.

The integrin alpha v beta 6 has been shown to be a fibronectin-binding protein. To determine whether the cytoplasmic and transmembrane domains of alpha v beta 6 are necessary for binding to fibronectin, a truncated, secreted form of the integrin lacking these domains was engineered and expressed in Chinese hamster ovary cells. Fibronectin affinity chromatography demonstrated that the secreted integrin, like its full-length counterpart, was capable of binding fibronectin. Monoclonal antibodies were made to secreted alpha v beta 6 and to beta 6-transfected NIH 3T3 cells. In experiments designed to determine whether alpha v beta 6 can mediate cell attachment to fibronectin, full-length human beta 6 was expressed in Chinese hamster ovary cells and in the human colon carcinoma cell line SW480. beta 6-expressing cells were identified by alpha v beta 6-specific antibodies, and the beta 6-transfectants were used in cell-adhesion assays. In Chinese hamster ovary cells, human beta 6 associated with hamster alpha v but was incapable of mediating cell attachment to fibronectin. However, expression of beta 6 in these cells had the dominant negative effect of decreasing alpha v beta 5-dependent adhesion to vitronectin. In SW480 cells, beta 6 expression conferred the ability to bind to fibronectin even in the presence of inhibitory antibodies against beta 1 integrins. In such cells, fibronectin binding ability could be blocked by an antibody to alpha v integrins. These results constitute the first direct evidence that alpha v beta 6 mediates cell attachment to fibronectin.

The alpha 5 beta 1 integrin binds fibronectin through the integrin recognition sequence Arg-Gly-Asp (RGD). We have used a 6-amino acid peptide library expressed on filamentous phage to identify peptide ligands for alpha 5 beta 1. We found that this integrin selectively binds RGD-containing peptides from the library. Of the 32 different sequences obtained, 28 had the RGD motif, 3 contained sequences related to RGD, and only 1 had a clearly different sequence. One of the RGD-containing phage encoded a potentially cyclic insert CRGDCL. The cyclic peptide GAC*RGDC*LGA (where * denotes cysteines forming a disulfide bond) was 10-fold more efficient than any of the linear RGD-containing hexapeptides in inhibiting the binding of RGD-expressing phage to alpha 5 beta 1 or the attachment of alpha 5 beta 1-expressing cells to fibronectin. This peptide also inhibited cell attachment mediated by the alpha v beta 1, alpha v beta 3, and alpha v beta 5 integrins with about 10-fold higher efficiency than linear GRGDSP. One peptide containing an RGD-related sequence, NGRAHA, was also found to inhibit phage attachment and cell adhesion, especially adhesion mediated by the alpha v beta 5 integrin. These results indicate that novel and high affinity ligands for integrins can be isolated from a random peptide library.

The study of osteoclast integrins has been previously hampered by the lack of a source of large numbers of purified osteoclasts. Osteoclastoma, a human giant cell tumor of bone, supplied a rich source of osteoclasts within a tissue containing many diverse cell types. Osteoclastoma integrin immunostaining confirmed the presence of the integrin alpha v beta 3 complex and the alpha 2 and beta 1 integrin subunits on osteoclasts. However, weak integrin expression, for example with alpha v beta 5, was difficult to interpret. Purification with magnetic beads coated with vitronectin receptor monoclonal antibody (13C2) enabled osteoclast membranes to be isolated with high purity and yield (57%) from osteoclastoma tissue. Positively (osteoclast-enriched) selected membranes were biochemically assessed for integrin expression by immunoprecipitation and visualization by non-radioactive enhanced chemiluminescence. alpha 1, alpha 4, alpha 6, alpha 8, alpha M, alpha X, gpIIb, beta 4, beta 6, and beta 8 integrin chains were undetectable at a sensitivity of 1 ng. alpha 3, alpha 5, alpha L, beta 2, and alpha v beta 5 were found in the negatively selected osteoclastoma tissue but not in the positively purified osteoclast membranes. The presence of alpha v beta 1 and alpha 2 beta 1 dimers was demonstrated biochemically on the immunoisolated osteoclast membranes. Osteoclast alpha v beta 3 isolation by Arg-Gly-Asp (RGD) affinity chromatography for NH2-terminal amino acid sequencing confirmed that the osteoclast vitronectin receptor was identical to that previously characterized on other cell types. In situ hybridization using human alpha v riboprobes in osteoclasts from human and rodent bone further demonstrated the high level and specificity of expression of alpha v vitronectin receptor in osteoclasts.

The alpha chain of the vitronectin receptor, alpha v, has been found in association with the integrin subunits beta 1, beta 3, or beta 5 on different cell types. We show here that cultured embryonic fibroblasts simultaneously display alpha v beta 3, alpha v beta 1, and alpha v in association with two other beta subunits, one of which is probably beta 5. Polymerase chain reaction analysis of single cells isolated by micromanipulation identified mRNA for alpha v, beta 1, beta 3, and beta 5 in six of eight clones. Immunoprecipitation of iodinated cell surface proteins with a monoclonal antibody to alpha v indicated that the relative proportions of the different beta chains in association with alpha v varied, particularly between two different cell lines. The cytokines platelet-derived growth factor, transforming growth factor beta 1, and tumor necrosis factor alpha did not appear to alter this ratio although tumor necrosis factor alpha increased the surface expression of the alpha v-associated integrins; but overnight culture in basic fibroblast growth factor caused a lower expression of alpha v beta 1 and alpha v beta 5 with no reduction in alpha v beta 3 expression. When the cell cultures were grown to complete confluence, surface expression of beta 3 was abolished, and the expression of an unknown beta chain (beta u) became more prominent. This effect was not overcome by culturing confluent cells with basic fibroblast growth factor. Affinity column chromatography showed that alpha v beta 5 bound to vitronectin but alpha v beta 1 did not, whereas alpha v beta 1 but not alpha v beta 5 bound to fibronectin. These results suggest that, on individual cells, the beta subunits found in association with alpha v may vary according to the proliferative capacity of the cell and that the promiscuous beta 3 subunit is progressively replaced by beta subunits of individual ligand specificity.

We have purified a novel member of the integrin gene family from placenta that serves as a vitronectin receptor. This integrin is composed of the alpha v subunit and a beta subunit that we designate beta 5. Purification was accomplished by immunodepleting a placental extract of integrin alpha v beta 3, allowing us to purify alpha v beta 5 from the remaining extract by monoclonal antibody affinity chromatography on LM 142-Sepharose, which binds to the alpha v subunit. Purification to homogeneity was subsequently achieved by affinity chromatography on wheat germ lectin-Sepharose. Western blot analysis with antibodies raised against alpha v beta 5 and alpha v beta 3 demonstrated that beta 3 and beta 5 were distinct but confirmed that the alpha subunit of the two integrins were immunologically identical. Similarly, antibodies that bind beta 3 proximal to the ligand-binding site failed to react with beta 5, indicating an architectural difference at the ligand-binding site of these related integrins. This structural difference apparently results in a functional distinction, since purified alpha v beta 3 bound to vitronectin, fibrinogen, von Willebrand factor, and fibronectin, whereas integrin alpha v beta 5 bound preferentially to vitronectin. Finally, we demonstrate by three criteria that beta 5 and beta x, the latter of which was identified in lung carcinoma cells (Cheresh, D. A., Smith, J. W., Cooper, H. M., and Quaranta, V. (1989) Cell 57, 59-69), are identical. First, peptide maps of beta x and beta 5 are identical. Secondly, polyclonal antibodies raised against alpha v beta 5 immunoprecipitate both beta 5 and beta x, and finally, the amino-terminal amino acid sequences of beta x and beta 5 are identical.

We describe a novel integrin heterodimer on the surface of the human embryonic kidney cell line 293. This receptor is comprised of alpha v and beta 1 subunits, each of which has been previously found in association with other integrin subunits. This alpha v.beta 1 complex was identified as the predominant vitronectin receptor (VnR) on the surface of 293 cells by immunoprecipitation with antibodies raised against the alpha v subunit. Polymerase chain reaction analysis detected mRNAs for alpha v and beta 1 subunits while no evidence was obtained for beta 2, beta 3, or alpha IIb integrin subunit mRNA. Immunoprecipitation of surface-iodinated proteins with antibodies to alpha v gave bands of 150 and 120 kDa. The 120-kDa band reacted with antibodies to beta 1 in immunoblotting experiments. 293 cells adhere to vitronectin, fibronectin, laminin, and collagen IV, while von Willebrand factor and fibrinogen, known ligands of the VnR (alpha v.beta 3), did not support adhesion. A polyclonal antibody directed against both subunits of the VnR (alpha v, beta 3) inhibits attachment of 293 cells to vitronectin but not to other adhesive proteins. A beta 1-specific monoclonal inhibited attachment to fibronectin, laminin, and collagen IV, known ligands of beta 1 integrins, as well as vitronectin. This novel (alpha v. beta 1) VnR thus appears to mediate cell adhesion exclusively to vitronectin, in contrast to previously described VnRs which have multiple ligands.

Purpose: The human telomerase reverse transcriptase (hTERT) promoter is used to restrict adenoviral expression of suicide or proapoptotic genes in telomerase-positive cancer cells. Etoposide can enhance intratumoral transgene expression in mice immunized with adenoviral vectors. The aim of this study is to evaluate whether greater therapeutic benefit can be achieved by combination treatment of low-dose etoposide and replication-incompetent adenoviral vectors encoding cytosine deaminase (CD) driven by the hTERT promoter for murine bladder carcinoma.

Nonhuman adenoviruses including porcine adenovirus serotype 3 (PAd3) are emerging vectors for gene delivery. PAd3 efficiently transduces human and murine cells and circumvents preexisting humoral immunity in humans. Coxsackievirus and adenovirus receptor (CAR) is the primary receptor and |[alpha]|v|[beta]|3 or |[alpha]|v|[beta]|5 integrin is involved as a secondary receptor for various human adenovirus (HAd) subtypes including HAd5. In this study we deduced the role of CAR, |[alpha]|v|[beta]|3 or |[alpha]|v|[beta]|5 integrin in PAd3 internalization. Transduction experiments were conducted in human mammary epithelial (MCF-10A) cells using replication defective PAd-GFP (PAd3 vector expressing green fluorescent protein [GFP]) and HAd-GFP (HAd5 vector expressing GFP). MCF-10A cells were treated with or without anti-human CAR, or anti-|[alpha]|v|[beta]|3 or anti-|[alpha]|v|[beta]|5 integrin antibodies prior to infection with HAd-GFP or PAd-GFP. Significant (P

Recent reports demonstrated that GM-CSF (Granulocyte Macrophage colony-Stimulating Factor)-transduced tumor vaccines (GVAX) had substantial antitumor activity in preclinical cancer models and partially in clinical situations because of their chemotactical attraction and activation of dendritic cells. To produce GVAX efficiently, recombinant type 5 E1 deleted adenovirus encoding human GM-CSF is one of the most promising vectors (AdenoGVAX). Adenoviral gene transduction is currently considered to be mediated by CAR (Coxsackie-Adenovirus Receptor), integrin|[alpha]|v|[beta]|3 and integrin|[alpha]|v|[beta]|5. Although previous reports showed that CAR and the integrin groups could alternatively be used for adenoviral transduction in limited cells, systematical data focusing on the relationship between the expression of membrane receptors (CAR, integrin |[alpha]|v|[beta]|3 and integrin |[alpha]|v|[beta]|5) and adenoviral transduction efficiency has not been reported. As high GM-CSF production is considered to be required to obtain clinical effects, prediction of GM-CSF production from AdenoGVAX transduced tumor cells is critical to perform the clinical trial efficiently. In this study, we reported the relationship between the expression of membrane receptors and adenoviral mediated GM-CSF production rate in renal cell carcinoma (RCC) cells and non small lung carcinoma (NSCLC) cells.

Carcinoma cells express a novel integrin involved in cell adhesion to vitronectin, but not to fibrinogen or von Willebrand factor, whereas melanoma and endothelial cells express a vitronectin receptor (alpha v beta 3) that promotes cell attachment to all of these matrix components. The integrin responsible for this adhesive phenotype of carcinoma cells is composed of an alpha subunit that is indistinguishable from the alpha v of the vitronectin receptor and a beta subunit (beta x) that is distinct from any known integrin beta subunit. Accordingly, Northern blot analysis identifies an mRNA for alpha v, but not for beta 3 in carcinoma cells. This receptor appears to mediate cell adhesion to vitronectin as well as fibronectin since an antibody directed to its alpha subunit blocked carcinoma cell adhesion to both of these matrix proteins. These results suggest that homologous integrins with identical alpha subunits and structurally distinct beta subunits can account for the functional recognition of different matrixes by two cell types.