Role of IIb-IIIa-like glycoproteins in cell-substratum adhesion of human melanoma cells

The platelet fibrinogen receptor, glycoprotein complex IIb-IIIa, was isolated from human platelets by lectin and monoclonal antibody affinity chromatography and a polyclonal antiserum (anti-IIb-IIIa) was generated and used to probe for the presence and function of IIb-IIIa-like molecules in two adherent human cell lines. Both C32 melanoma cells and WI38 fibroblasts expressed a IIb-IIIa-like complex on their surface as indicated by immunoprecipitation of detergent extracts of surface radiolabeled cells. When added to cells plated in medium containing 10% serum, the anti-IIb-IIIa antiserum perturbed the adhesion of C32 melanoma cells, but not of WI38 fibroblasts. In a serum-free system, anti-IIb-IIIa antibodies inhibited attachment and spreading of C32 cells to fibrinogen, vitronectin, and fibronectin adsorbed to glass. Anti-IIb-IIIa had no effect on the attachment and spreading of WI38 cells to the extracellular matrix proteins, however. Thus, the IIb-IIIa-like complex appears to play a predominant role in cell-substratum adhesion of C32 cells, but not WI38 cells, and may result from the fact that, on a protein basis, the C32 melanoma cells express approximately 3 times more complex on their surface than do WI38 fibroblasts. The results suggest that the relative abundance of a particular adhesion receptor on the cell surface may govern its importance to cell-substratum adhesion.     

Inhibition of murine melanoma cell-matrix adhesion and experimental metastasis by albolabrin, an RGD-containing peptide isolated from the venom of Trimeresurus albolabris

Albolabrin, a 7.5-kDa cysteine-rich protein isolated from the venom of Trimeresurus albolabris, contains the arginine-glycine-aspartic acid (RGD) cell recognition sequence found in many cell adhesion-promoting extracellular matrix proteins, such as fibronectin and laminin. Albolabrin belongs to a family of RGD-containing peptides, termed disintegrins, recently isolated from the venom of various vipers and discovered to be potent inhibitors of both platelet aggregation and cell-substratum adhesion. Here we report that albolabrin inhibited the attachment of B16-F10 mouse melanoma cells to either fibronectin or laminin absorbed on plastic. When immobilized on plastic, albolabrin promoted B16-F10 melanoma cell attachment; this was inhibited by either RGD-serine (RGDS) or antibodies to integrins, suggesting that albolabrin binds via its RGD amino sequence to integrin receptors expressed on the melanoma cell surface. In an in vivo experimental metastasis system, albolabrin at a concentration of 300-600 nM inhibited C57BL/6 mouse lung colonization by tail vein-injected mouse melanoma cells and was at least 2000 times more active than RGDS in this assay. We propose that albolabrin inhibits tumor cell metastasis by inhibiting integrin-mediated attachment of melanoma cells to RGD-containing components of the extracellular matrix in the mouse lung.     

Mechanism of interaction of thrombospondin with human endothelium and inhibition of sickle erythrocyte adhesion to human endothelial cells by heparin

Thrombospondin (TSP) mediates sickle erythrocyte adhesion to endothelium, but the mechanism remains unknown. Since TSP is comprised of heterogeneously distinct domains, this adhesion may depend on the interaction of specific regions of TSP with different cell surface receptors. To examine the mechanisms of interaction of TSP with human umbilical vein endothelial cells (HUVEC), we performed binding studies using soluble [¹²⁵I]TSP. Our data showed that (i) monoclonal antibodies (MoAbs) against cell surface heparan sulfate (HS) or the heparin-binding domain of TSP, or cleavage of HS on HUVEC by heparitinase reduced TSP binding by 28–40%, (ii) the RGD peptide or MoAbs against integrin α_vβ₃ or the calcium binding region of TSP inhibited binding by 18–28%, and (iii) a MoAb against the cell-binding domain of TSP inhibited binding by 36%. Unmodified heparin inhibited the binding of TSP to endothelial cells by 70% and did so far more effectively than selectively desulfated heparins, HS or chondroitin sulfate. Heparin inhibited TSP binding to HUVEC at much lower concentrations than were required to inhibit TSP binding to sickle erythrocytes. Unmodified heparin effectively inhibited the TSP-mediated adhesion of sickle erythrocytes to HUVEC. These data imply that cell surface HS-mediated mechanisms play a key role in TSP-mediated sickle erythrocyte adhesion to endothelium, and heparin may be of use for inhibition of this adhesion.     

Exposure of binding sites for vitronectin on platelets following stimulation

Vitronectin is a glycoprotein that mediates cell adhesion and spreading in a number of cell culture systems. Liposomes containing platelet glycoproteins IIb-IIIa complex have been shown to bind vitronectin-coated surfaces through an Arg-Gly-Asp cell attachment mechanism. We examined the expression of the binding sites for vitronectin on the surface of intact, resting platelets and following stimulation. 125I-Labeled vitronectin bound specifically in a saturable manner to platelets treated with physiological concentrations of thrombin. The binding reached saturation at 100 nM concentration, and, at saturation, approximately 5000 specific binding sites were detected per platelet. The binding was divalent cation-dependent and only partially reversible after complete saturation. A synthetic hexapeptide containing the Arg-Gly-Asp sequence inhibited vitronectin binding to platelets. A monoclonal antibody against platelet glycoprotein IIb-IIIa complex also inhibited the binding of vitronectin to stimulated platelets. These data suggest that platelets possess an inducible divalent cation-dependent receptor for vitronectin and that the glycoprotein IIb-IIIa complex is involved in the expression of the vitronectin receptor.     

Inhibition of in vitro tumor cell invasion by Arg-Gly-Asp-containing synthetic peptides [published erratum appears in J Cell Biol 1989 Jun;108(6):following 2546]

The interaction of cells with extracellular matrix components such as fibronectin, vitronectin, and type I collagen has been shown to be mediated through a family of cell-surface receptors that specifically recognize an arginine-glycine-aspartic acid (RGD) amino acid sequence within each protein. Synthetic peptides containing the RGD sequence can inhibit these receptor-ligand interactions. Here, we use novel RGD-containing synthetic peptides with different inhibition properties to investigate the role of the various RGD receptors in tumor cell invasion. The RGD-containing peptides used include peptides that inhibit the attachment of cells to fibronectin and vitronectin, a peptide that inhibits attachment to fibronectin but not to vitronectin, a cyclic peptide with the opposite specificity, and a peptide, GRGDTP, that inhibits attachment to type I collagen in addition to inhibiting attachment to fibronectin and vitronectin. The penetration of two human melanoma cell lines and a glioblastoma cell line through the human amniotic basement membrane and its underlying stroma was inhibited by all of the RGD-containing peptides except for the one that inhibits only the vitronectin attachment. Various control peptides lacking RGD showed essentially no inhibition. This inhibitory effect on cell invasion was dose-dependent and nontoxic. A hexapeptide, GRGDTP, that inhibits the attachment of cells to type I collagen in addition to inhibiting fibronectin- and vitronectin-mediated attachment was more inhibitory than those RGD peptides that inhibit only fibronectin and vitronectin attachment. Analysis of the location of these cells that were prevented from invading indicated that they attached to the amniotic basement membrane but did not proceed further into the tissue. These results suggest that interactions between RGD-containing extracellular matrix adhesion proteins and cells are necessary for cell invasion through tissues and that fibronectin and type I collagen are important for this process.     

Effect of cyclic RGD peptide on cell adhesion and tumor metastasis.

Several kinds of cyclic peptides containing an L-arginine-glycine-L-aspartic acid RGD sequence were synthesized by the liquid phase method, and we investigated their effects on the attachment of mouse B16 melanoma cells onto fibronectin-coated well. Cyclo (GRGDSPA) inhibited the cell attachment at a 20-fold lower concentration than the linear form. The cell adhesion was inhibited by the synthetic peptides with the following relative order of activity: cyclo (GRGDSPA) much greater than cyclo (GRGD) greater than cyclo (RGDS), cyclo (GRGDSP) greater than cyclo (GRGDS) greater than cyclo (RGDSP), cyclo (RGDSPA). Cyclo (GRGDSPA) was more effective at inhibiting cell attachment to vitronectin than it was at competing with fibronectin attachment, as reported in the case of GRGDSP. Moreover, cyclo (GRGDSPA) significantly reduced the formation of colonies in mice injected with B16-FE7 melanoma cells.     

重组七鳃鳗富含半胱氨酸RGD蛋白rLj-RGD1对人肝癌HepG2细胞的抑制作用

rLj-RGD1为源于日本七鳃鳗口腔腺的基因重组蛋白,其富含半胱氨酸并具有一个RGD(Arg-Gly-Asp)模体.前期工作表明,rLj-RGD1具有抑制血小板聚集及血管新生的RGD毒素蛋白典型功能.为了研究rLj RGD1是否具有RGD毒素蛋白的另一典型抗肿瘤功能,以人肝癌HepG2细胞为模型,对rLj RGD1进行了活性研究.MTT法结果显示,rLj-RGD1呈剂量依赖方式抑制HepG2细胞的增殖,其半抑制浓度(IC50)为36 μmol/L;细胞迁移与浸润实验结果显示,rLj-RGD1能以剂量依赖方式抑制HepG2细胞碱性成纤维生长因子（bFGF）诱导的迁移与浸润.Hoechst染色和DNA Ladder等细胞凋亡实验表明,rLj-RGD1能够以剂量依赖方式诱导HepG2细胞发生凋亡.细胞黏附实验表明,rLj-RGD1以剂量依赖方式抑制HepG2细胞与玻连蛋白(vitronectin, VN)的黏附.上述结果表明,rLj-RGD1具有抑制人肝癌HepG2细胞增殖、迁移和浸润的功能,并可诱导其发生失巢凋亡. 本研究结果提示,rLj-RGD1具有典型的RGD毒素蛋白抗肿瘤功能,其未来具有成为抗肿瘤药物的潜力.     

Characterization of the integrin alpha v beta 6 as a fibronectin-binding protein.

Integrins are a complex family of divalent cation-dependent cell adhesion receptors composed of one alpha and one beta subunit noncovalently bound to one another. A subset of integrins contains the alpha v subunit in association with one of several beta subunits (e.g. beta 3, beta 5, beta 1). We have recently identified a novel integrin beta subunit, beta 6, that is present in a number of epithelial cell lines. Using a polyclonal antibody raised against the carboxyl-terminal peptide of beta 6, we have now identified the integrin heterodimer, alpha v beta 6, on the surface of two human carcinoma cell lines. Using affinity chromatography of lysates from the pancreatic carcinoma cell line, FG-2, we demonstrate that alpha v beta 6 binds to fibronectin, but not to vitronectin or collagen I. In contrast, the alpha v beta 5 integrin, which is also expressed on FG-2 cells, binds exclusively to vitronectin. Immobilized collagen I does not interact with alpha v integrins, but binds beta 1-containing integrins. Both alpha v beta 6 and alpha v beta 5 are eluted from their respective immobilized ligands by a hexa-peptide containing the sequence Arg-Gly-Asp (RGD). RGD is highly effective in the presence of Ca2+, somewhat less effective in Mg2+, and virtually inactive in Mn2+. These results suggest that alpha v beta 6 functions as an RGD-dependent fibronectin receptor in FG-2 carcinoma cells. In agreement with this notion, cell adhesion assays show that FG-2 cell attachment to fibronectin is only partially inhibited by anti-beta 1 integrin antibodies, implying that other fibronectin receptors may be involved. Taken together with recent reports on the vitronectin receptor function of alpha v beta 5, our results suggest that the previously described carcinoma cell integrin, alpha v beta x (Cheresh, D. A., Smith, J. W., Cooper, H. M., and Quaranta, V. (1989) Cell 57, 59-69), is a mixture of at least two different receptors: alpha v beta 5, mediating adhesion to vitronectin, and alpha v beta 6, mediating adhesion to fibronectin.     

Triflavin, an Arg-Gly-Asp-containing peptide, inhibits human cervical carcinoma (HeLa) cell-substratum adhesion through an RGD-dependent mechanism

Triflavin, a 7.5-kDa cysteine-rich polypeptide purified from Trimeresurus flavoviridis snake venom, belongs to a family of RGD-containing peptides, termed disintegrins, that have been isolated from the venoms of various vipers and shown to be potent inhibitors of platelet aggregation. The interaction of tumor cells with extracellular matrices such as fibronectin, vitronectin, and collagen has been shown to be mediated through a family of cell surface receptors that specifically recognize an arginine-glycine-aspartic acid (RGD) sequence within each adhesive protein. In this study, we show that triflavin dose-dependently inhibited adhesion of human cervical carcinoma (HeLa) cells to extracellular matrices (ECMs; i.e., fibronectin, fibrinogen, and vitronectin). On the other hand, triflavin exerted a limited inhibitory effect on cell adhesion to laminin and collagen (type I and IV). On a molar basis, triflavin is approximately 800 times more potent than Gly-Arg-Gly-Asp-Ser (GRGDS) at inhibiting cell adhesion. When immobilized on plate, triflavin significantly promoted HeLa cell adhesion, and this attachment was inhibited by GRGDS. Furthermore, FITC-conjugated triflavin bound to cells in a saturable manner and its binding was inhibited by GRGDS. In addition, triflavin did not affect [³H]thymidine uptake of HeLa cells during a 3-day incubation. These results suggest that triflavin probably binds to integrin receptors expressed on HeLa cell surface via its RGD sequence within its molecule, thereby inhibiting the adhesion of extracellular matrices to HeLa cells.     

Identification of fibronectin in chicken thrombocytes.

Cell attachment and spreading of chicken embryo fibroblasts and three mammalian cell lines were promoted by using thrombocyte secretion products (TSP) prepared from chicken thrombocytes which are analogous to mammalian platelets. The cell attachment activity following TSP treatment was interfered with by the addition of a synthetic peptide containing the RGD sequence from the cell attachment region of fibronectin (FN). Cell attachment on TSP-coated plates was inhibited by the addition of anti-chicken FN rabbit antiserum, but not by anti-chicken vitronectin (VN) rabbit antiserum. By immunoblotting, immunofluorescent test and sensitive enzyme linked immunosorbent assay using an anti-chicken FN antiserum, the presence of FN in thrombocytes and the release of FN from the cells were demonstrated. The release of FN from thrombocytes was partially induced by the in vitro cultivation of the cells and was further promoted by treatment of the cells with thrombin.     

Vaccinia Virus Induces Ca2+-Independent Cell-Matrix Adhesion during the Motile Phase of Infection

Vaccinia virus (VV) induces two forms of cell motility: cell migration, which is dependent on the expression of early genes, and the formation of cellular projections, which requires the expression of late genes. The need for viral gene expression prior to cell motility suggests that VV proteins may affect how infected cells interact with the extracellular matrix. To address this, we have analyzed changes in cell-matrix adhesion after infection of BS-C-1 cells with VV. Whereas uninfected cells round up and detach from the culture flask in the presence of EGTA, infected cells remain attached to the culture flask with a stellate morphology. Ca²⁺-independent cell-matrix adhesion was evident by 10 h postinfection, after the onset of cell motility but before the formation of virus-induced cellular projections. Progression to Ca²⁺-independent adhesion required the expression of late viral genes but not the formation of intracellular enveloped virus particles or intracellular actin tails. Analyses of specific matrix proteins identified vitronectin and fibronectin as optimal ligands for Ca²⁺-independent adhesion and the formation of cellular projections. Adhesion to fibronectin was mediated via RGD motifs alone and was not inhibited by 500 μg of heparin/ml. Kistrin, a disintegrin which binds preferentially to the αvβ3 (vitronectin/fibronectin) receptor inhibited the formation of cellular projections without disrupting preformed matrix interactions. Finally, we show that Ca²⁺-independent cell-matrix adhesion is a dynamic process which mediates changes in the morphology of VV-infected cells and uninfected cells which exhibit a transformed phenotype.     

(Arg-Gly-Asp)n-albumin conjugates as a model substratum for integrin-mediated cell adhesion

We have prepared protein-peptide conjugates composed of bovine serum albumin (BSA) derivatized with short peptides containing the Arg-Gly-Asp (RGD) sequence derived from the adhesion site of fibronectin. The RGD-BSA conjugates were used to coat tissue culture plastic surfaces which then served as substrata in cell adhesion experiments. Our results indicate that the efficiency of adhesion to RGD-BSA-coated surfaces is highly dependent on the valency of the (RGD)n-BSA conjugates. For example, on surfaces with approximately equal amounts of RGD ligand, CHO cells adhered virtually 100% to the (RGD)n-BSA (n = 20.8) conjugate and not at all to the (RGD)n-BSA (n = 3.5) conjugate. Adhesion on (RGD)n-BSA-coated substrata and on fibronectin- or vitronectin-coated substrata was also examined in terms of the relationship between cell adhesion and the intermolecular distances of adsorbed proteins. It was observed that for substrata coated with relatively compact, symmetric molecules, such as RGD-BSA or vitronectin, adhesion dropped off sharply as intermolecular distances increased; by contrast, for fibronectin, a large asymmetric molecule, adhesion declined more gradually as intermolecular distances increased. Finally, we have examined the role of different cell-surface receptors in the process of adhesion to RGD-BSA substrata. Interestingly, competition and blocking experiments with antibodies and with soluble competing proteins suggest that it is the vitronectin receptor rather than the fibronectin receptor which mediates adhesion to RGD-BSA.     

Biological activities of peptides and peptide analogues derived from common sequences present in thrombospondin, properdin, and malarial proteins

Thrombospondin (TSP), a major platelet-secreted protein, has recently been shown to have activity in tumor cell metastasis, cell adhesion, and platelet aggregation. The type 1 repeats of TSP contain two copies of CSVTCG and one copy of CSTSCG, per each of the three polypeptide chains of TSP and show homology with peptide sequences found in a number of other proteins including properdin, malarial circumsporozoite, and a blood-stage antigen of Plasmodium falciparum. To investigate whether these common sequences functioned as a cell adhesive domain in TSP, we assessed the effect of peptides corresponding to these sequences and an antibody raised against one of these sequences, CSTSCG, in three biological assays which depend, in part, on the cell adhesive activity of TSP. These assays were TSP-dependent cell adhesion, platelet aggregation, and tumor cell metastasis. We found that a number of peptides homologous to CSVTCG promoted the adhesion of a variety of cells including mouse B16-F10 melanoma cells, inhibited platelet aggregation and tumor cell metastasis, whereas control peptides had no effect. Anti-CSTSCG, which specifically recognized TSP, inhibited TSP-dependent cell adhesion, platelet aggregation, and tumor cell metastasis, whereas control IgG had no effect. These results suggest that CSVTCG and CSTSCG present in the type I repeats function in the adhesive interactions of TSP that mediate cell adhesion, platelet aggregation, and tumor cell metastasis. Peptides, based on the structure of these repeats, may find wide application in the treatment of thrombosis and in the prevention of cancer spread.     

Arg-Gly-Asp recognition by a cell adhesion receptor requires its 130-kDa alpha subunit

A major Arg-Gly-Asp-directed receptor on M21 human melanoma cells is a heterodimer of alpha and beta chains which under reducing conditions have molecular masses of 130 and 105 kDa, respectively. This receptor is one member of a large family of cell adhesion receptors that shares antigenic determinants with the vitronectin receptor of fibroblasts and the platelet IIb X IIIa complex. Both subunits of the M21 cell adhesion receptor acquire high mannose-type oligosaccharides that are processed before transport to the cell surface. In addition, the alpha chain undergoes a proteolytic cleavage step. Pulse-chase immunoprecipitation analysis demonstrates that, following its synthesis, the beta chain remains unbound to alpha chain for 1-2 h and in this free form is unable to bind an Arg-Gly-Asp containing heptapeptide. Conversely, the biosynthetic precursor of the alpha chain is fully capable of binding the Arg-Gly-Asp-containing peptide immediately after its synthesis. Thus, Arg-Gly-Asp recognition by one member of this cell adhesion receptor family requires its 130-kDa alpha chain which appears to be functional prior to post-translational modifications.     

Expression of recombinant platelet glycoprotein IIbIIIa results in a functional fibrinogen-binding complex

The ability of cDNAs encoding the human platelet glycoprotein IIbIIIa to be expressed and assembled into a functional integrin receptor was assessed by transient transfection into a human cell line. Transfection of full length cDNAs resulted in synthesis of high levels of integrin subunits which appear to be stable within the cell for several days. Coexpression of both subunits resulted in a proteolytically processed form of GPIIb that associated with GPIIIa as a heterodimeric complex as the cell surface. Transport to the cell surface required association of these subunits with each other or with endogenous integrin subunits. When expressed alone, the GPIIb subunit remained intracellular, while the GPIIIa subunit was found to complex with endogenous proteins and was mobilized to the cell surface. The GPIIbIIIa receptor complex facilitated attachment of cells to known ligands for GPIIbIIIa: fibrinogen, vitronectin, and von Willebrand factor. This adhesion was sensitive to inhibition by the peptide GRGDV and the monoclonal antibody AP2, known inhibitors of platelet aggregation     

Adhesion to thrombospondin by human embryonic fibroblasts is mediated by multiple receptors and includes a role for glycoprotein 88 (CD36).

Fetal embryonic fibroblasts attach and spread on thrombospondin (TSP). Adhesion is tight and focal adhesion plaques and "spots" are formed. We have investigated the receptors responsible for this adhesion. Unstimulated cells express the vitronectin receptor on their surface and this beta 3 integrin molecule contributes to adhesion. Another putative receptor for TSP, termed glycoprotein (GP) 88, which exists as a cytoplasmic pool in unstimulated cells becomes surface expressed when these cells are plated on TSP and localizes to areas of cell adhesion. Western blot analysis of cell lysate confirms GP88 as a TSP binding protein. Studies with fucoidan indicate that the heparan sulfate proteoglycan, known to function as a receptor for TSP, appears to contribute substantially to the TSP attachment of these cells and may be the receptor most important in the initial phases of TSP interaction.     

RAM, an RGDS analog, exerts potent anti-melanoma effects in vitro and in vivo

Peptides containing the RGD sequence are under continuous investigation given their ability to control cell adhesion and apoptosis. Since small peptides are quickly metabolized and degraded in vivo, developing analogs resistant to serum-induced degradation is a challenging task. RGD analogs developed so far are known as molecules mostly inhibiting cell adhesion; this feature may reduce cell proliferation and tumor development but may not induce regression of tumors or metastases already formed. In the current study, carried out in melanoma in vitro and in vivo models, we show that RAM, an RGD-non-peptide Analog-Molecule, strongly inhibits cells adhesion onto plastic, vitronectin, fibronectin, laminin and von Willebrand Factor while it does not inhibit cell adhesion onto collagen IV, similarly to the RGDS template peptide. It also strongly inhibits in vitro cell proliferation, migration and DNA-synthesis, increases melanoma cells apoptosis and reduces survivin expression. All such effects were observed in collagen IV seeded cells, therefore are most likely independent from the anti adhesive properties. Further, RAM is more stable than the template RGDS; in fact it maintains its anti-proliferation and anti-adhesion effects after long serum exposure while RGDS almost completely loses its effects upon serum exposure. In a mouse metastatic melanoma in vivo model, increasing doses of RAM significantly reduce up to about 80% lung metastases development, while comparable doses of RGDS are less potent. In conclusion these data show that RAM is a potent inhibitor of melanoma growth in vitro, strongly reduces melanoma metastases development in vivo and represents a novel candidate for further in vivo investigations in the cancer treatment field.     

Triflavin,an Arg-Gly-Asp-containing peptide,inhibits B16-F10 mouse melanoma cell adhesion to matrix proteins via direct binding to tumor cells

Triflavin, an Arg-Gly-Asp (RGD)-containing snake venom peptide, inhibits B16-F10 mouse melanoma cell adhesion to extracellular matrices, e.g., fibronectin, vitronectin, fibrinogen, and collagen type I. In this study, GRGDS inhibits B16-F10 mouse melanoma cell adhesion to immobilized triflavin in a dose-dependent manner. In addition, flow-cytometric analysis and the fluorescence staining method in which FITC-triflavin is utilized as a binding ligand were used. GRGDS inhibits the binding of FITC-triflavin to B16-F10 cells. Additionally, the above results suggest that triflavin directly binds to its receptors expressed on B16-F10 cell surface primarily via its RGD sequence, there-by inhibiting B16-F10 cell adhesion to extracellular matrices.     

Computerized analysis of tumor cell interactions with extracellular matrix proteins, peptides, and endothelial cells under laminar flow

Arrest and formation of stable adhesive interactions between circulating cells and the endothelium or exposed subendothelial matrix are important processes in many biological situations. We have developed a highly sensitive hydrodynamic assay that utilizes a parallel-plate flow chamber, video microscopy, and digital image processing to separate and measure the primary arrest and adhesion stabilization of flowing cells. Our data indicate that primary cell contact triggers secondary adhesion stabilization, and the secondary events are likely to be critical to metastasis formation. To study the relationship between tumor cell adhesion stabilization and organ-specific blood-borne metastasis, we investigated the adhesion stabilization of metastatic murine RAW117 large-cell lymphoma cells to the extracellular matrix proteins fibronectin and vitronectin, several Arg-Gly-Asp (RGD) containing peptides, and microvascular endothelial cells from the liver or lung. The highly liver metastatic RAW117-H10 subline showed the fastest stabilization to fibronectin, vitronectin, and RGD peptides. Poorly metastatic RAW117-P cells had stabilization times 3-10 times longer than for RAW117-H10 cells, while the lung- and liver-metastatic RAW117-L17 subline failed to stabilize at all. The adhesion stabilization of the RAW117-H10 cells to the extracellular matrix proteins and RGD peptides was inhibited by anti-beta(3) integrin monoclonal antibodies and RGD peptides. In contrast, the RAW117-L17 subline had the shortest stabilization time to unstimulated microvascular endothelial cells of the lung and hepatic sinusoids, followed by RAW117-H10 cells and RAW117-P cells. Monoclonal antibodies against the beta(3) integrin subunit and RGD peptides did not inhibit adhesion stabilization of RAW117-H10 cells to endothelial cells, suggesting that different metastatic variants of large-cell lymphoma cells use differing mechanisms to adhere to organ-specific endothelial cells. (c) 1996 John Wiley & Sons, Inc.