arginyl-glycyl-glutamic-acid and arginyl-glycyl-aspartic-acid

Fibronectin (FN) is an essential glycoprotein of the extracellular matrix; binds integrins, syndecans, collagens, and growth factors; and is assembled by cells into complex fibrillar networks. The RGD motif in FN facilitates cell binding- and fibrillogenesis through binding to α5β1 and αv-class integrins. However, whether RGD is the sole binding site for αv-class integrins is unclear. Most notably, substituting aspartate with glutamate (RGE) was shown to eliminate integrin binding in vitro, while mouse genetics revealed that FNRGE preserves αv-class integrin binding and fibrillogenesis. To address this conflict, we employed single-cell force spectroscopy, engineered cells, and RGD motif-deficient mice (Fn1ΔRGD/ΔRGD) to search for additional αv-class integrin-binding sites. Our results demonstrate that α5β1 and αv-class integrins solely recognize the FN-RGD motif and that αv-class, but not α5β1, integrins retain FN-RGE binding. Furthermore, Fn1ΔRGD/ΔRGD tissues and cells assemble abnormal and dysfunctional FNΔRGD fibrils in a syndecan-dependent manner. Our data highlight the central role of FN-RGD and the functionality of FN-RGE for αv-class integrins.

Topics: Animals; Mice; Mice, Mutant Strains; Mutation; Oligopeptides; Receptors, Vitronectin

Adverse cardiac remodeling and dysfunction after myocardial infarction (MI) is associated with (BioLineRx, BL-1040 myocardial implant) excessive damage to the extracellular matrix. Biomaterials, such as the in situ-forming alginate hydrogel, provide temporary support and attenuate these processes. Here, we tested the effects of decorating alginate biomaterial with cell adhesion peptides, containing the sequences RGD and YIGSR, or a non-specific peptide (RGE), in terms of therapeutic outcome soon after MI. The biomaterial (i.e., both unmodified and peptide-modified alginate) solutions retained the ability to flow after cross-linking with calcium ions, and could be injected into 7-day infarcts, where they underwent phase transition into hydrogels. Serial echocardiography studies performed before and 60 days after treatment showed that alginate modification with the peptides reduced the therapeutical effects of the hydrogel, as revealed by the extent of scar thickness, left ventricle dilatation and function. Histology and immunohistochemistry revealed no significant differences in blood vessel density, scar thickness, myofibroblast or macrophage infiltration or cell proliferation between the experimental groups BioLineRx BL-1040 myocardial implant. Our studies thus reveal that the chemical and physical traits of the biomaterial can affect its therapeutical efficacy in attenuating left ventricle remodeling and function, post-MI.

Topics: Actins; Alginates; Animals; Animals, Newborn; Biocompatible Materials; Female; Hydrogels; Immunohistochemistry; Materials Testing; Muscle, Smooth; Myocardial Infarction; Oligopeptides; Peptides; Proliferating Cell Nuclear Antigen; Rats; Rats, Sprague-Dawley; Ventricular Remodeling

Androgens have important physiological effects in women. Not only are they the precursor hormones for estrogen biosynthesis in the ovaries and extragonadal tissues, but also act directly via androgen receptors (ARs) throughout the body. Studies of the role of androgens on breast cancer development are controversial and the mechanisms involved are not fully understood. In this report we demonstrate that a non-aromatizable androgen metabolite, dihydrotestosterone (DHT), stimulated cell proliferation in vitro of both estrogen receptor-alpha (ER-alpha)-positive MCF-7 cells and ER-alpha-negative MDA-MB-231 human breast cancer cells. A contribution of ER to the proliferative effect of DHT in MCF-7 cells was supported by actions of small interfering RNA (siRNA) ER-alpha transfection and of the specific inhibitor of ER, ICI 182,780 to block DHT-induced proliferation. A contribution of the possible conversion of DHT to androstane-3alpha, 17beta-diol was not excluded in these MCF-7 cell studies. In MDA-MB-231 cells, a novel mechanism was implicated, in that anti-integrin alphavbeta3 or an Arg-Gly-Asp (RGD) peptide targeted at a small molecule binding domain of the integrin eliminated the DHT effect on cell proliferation. Anti-integrin alphavbeta3 did not affect DHT action on MCF-7 cells. A contribution from classical androgen receptor to the DHT effect in each cell line was excluded. A proliferative DHT signal is transduced in both ER-alpha-positive and ER-alpha-negative breast cancer cells, but by discrete mechanisms.

Topics: Androgen Antagonists; Androgens; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Estradiol; Estrogen Antagonists; Estrogen Receptor alpha; Female; Flutamide; Fulvestrant; Humans; Integrin alphaVbeta3; Oligopeptides; Receptors, Androgen; RNA, Small Interfering

Osteopontin (OPN), a phosphoprotein containing an arginine-glycine-aspartic acid (RGD) sequence, has been identified in cow oviduct epithelium and fluid. To investigate the potential role OPN in fertilization, we evaluated the ability of RGD peptide (arginine-glycine-aspartic), RGE peptide (arginine-glycine-glutamic acid), integrins alphaV and alpha5 antibodies and OPN antibody to influence bovine in vitro sperm-egg binding and fertilization. Treatment of sperm or oocytes with the RGD peptide prior fertilization significantly decreased in vitro sperm-egg binding and fertilization compared to the non-treated controls or those treated with RGE peptide. Binding and fertilization were also significantly decreased when in vitro matured bovine oocytes or sperm were pre-incubated with integrins alphaV and alpha5 antibodies at concentration ranging from 5 to 20 microg/mL. Addition of a rabbit polyclonal IgG antibody against purified bovine milk OPN with sperm or/and oocytes decreased (P<0.05) fertilization compared to the in vitro-fertilized control. These data provided evidence that integrin ligands existed on bovine oocytes and spermatozoa that contained RGD recognition sequences, and that antibody to OPN, a protein that contains that RGD sequence, was capable of reducing sperm-egg binding and fertilization in vitro.

Topics: Animals; Antibodies; Cattle; Female; Fertilization in Vitro; Integrin alpha5; Integrin alphaV; Male; Oligopeptides; Osteopontin; Proteins; Sperm-Ovum Interactions

Fibronectin (FN) is secreted as a disulfide-bonded FN dimer. Each subunit contains three types of repeating modules: FN-I, FN-II, and FN-III. The interactions of alpha5beta1 or alphav integrins with the RGD motif of FN-III repeat 10 (FN-III10) are considered an essential step in the assembly of FN fibrils. To test this hypothesis in vivo, we replaced the RGD motif with the inactive RGE in mice. FN-RGE homozygous embryos die at embryonic day 10 with shortened posterior trunk, absent tail bud-derived somites, and severe vascular defects resembling the phenotype of alpha5 integrin-deficient mice. Surprisingly, the absence of a functional RGD motif in FN did not compromise assembly of an FN matrix in mutant embryos or on mutant cells. Matrix assembly assays and solid-phase binding assays reveal that alphavbeta3 integrin assembles FN-RGE by binding an isoDGR motif in FN-I5, which is generated by the nonenzymatic rearrangement of asparagines (N) into an iso-aspartate (iso-D). Our findings demonstrate that FN contains a novel motif for integrin binding and fibril formation whose activity is controlled by amino acid modification.

Topics: Amino Acid Motifs; Amino Acid Sequence; Amino Acid Substitution; Animals; Aspartic Acid; Binding Sites; Cell Line, Transformed; Dimerization; Disulfides; Embryo, Mammalian; Extracellular Matrix; Fibroblasts; Fibronectins; Heterozygote; Integrin alphaVbeta3; Mice; Oligopeptides; Protein Binding; Protein Structure, Tertiary; Recombinant Proteins; Reticulin; Solubility

The aim of this study was to identify cell adhesion molecules that could serve as targets of the human follicle-associated epithelium (FAE) overlying Peyer's patches and to assess nanoparticle uptake levels across this epithelium. We first studied the expression of the mouse M-cell marker beta(1)-integrin and used a model of human FAE derived from intestinal epithelial Caco-2 cells and Raji B-cells to identify additional potential targets by cDNA array. The protein expression of potential targets in the model FAE and in human ileal FAE tissues was quantified by immunofluorescence. Integrin targeting was studied by investigating the transport of Arg-Gly-Asp (RGD)-coated (integrin-binding), Arg-Gly-Glu (RGE)-coated (nonintegrin-binding), and uncoated nanoparticles across ileal specimens mounted in Ussing chambers. Both beta(1)-integrin and the cell adhesion molecule CD9 were more abundantly expressed in the model and human FAE compared with the Caco-2 control cells or villus epithelium (VE). Uncoated nanoparticles were not taken up across either FAE or VE. General integrin targeting with RGD improved the nanoparticle transport dramatically across the FAE and to a lower extent across the VE. Compared with RGE, RGD improved transport 4-fold across the FAE. There was no difference in the transport of RGD- and RGE-coated nanoparticles across the VE. In conclusion, beta(1)-integrin and CD9 were identified as targets in human FAE. The difference in RGD- and RGE-mediated transport across the FAE, but not the VE, suggests that a specific integrin interaction was the dominating mechanism for improved nanoparticle uptake across the FAE., whereas charge interaction contributed substantially to the improved VE uptake.

Topics: Antigens, CD; Biological Transport; Caco-2 Cells; Epithelium; Humans; Integrin beta1; Membrane Glycoproteins; Nanoparticles; Oligonucleotide Array Sequence Analysis; Oligopeptides; Peyer's Patches; Tetraspanin 29

Endothelial cells in tumor angiogenesis are highly accessible, genetically stable and present unique molecular markers for targeted therapy. Neoplasia is also characterized by enhanced vascular permeability and disordered lymphatics so that both active and passive targeting strategies may play a role in localizing angiogenesis-targeted agents. To investigate the relative importance of these targeting strategies, the tissue biodistribution of both endothelial-specific and nonspecific peptides and their macromolecular peptide-copolymer conjugates were studied in 2 xenograft models of prostate cancer. Tumor-to-normal tissue background ratios (T/B) of these constructs were compared to evaluate the effect of molecular size on blood clearance and nonspecific vascular permeability.. Water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers were synthesized with side chains terminated in a doubly cyclized Arg-Gly-Asp motif KACDCRGDCFCG (RGD4C: active peptide targeting the alpha(V)beta(3) integrin) and KACDCRGECFCG (RGE4C: nonactive peptide). The bioactivity of the polymer conjugates and free peptides was characterized in vitro by endothelial cell adhesion assay. The (99m)Tc(CO)(3)-labeled compounds were injected into SCID mice bearing DU145 or PC-3 prostate tumor xenografts for scintigraphic imaging and necropsy organ counting.. HPMA copolymer-RGD4C conjugates showed similar inhibition of cell adhesion as free RGD4C attached to (99m)Tc(CO)(3) chelator N-omega-bis(2-pyridylmethyl)-L-lysine (RGD4C-DPK) and were significantly higher (P < 0.05) than RGE4C, HPMA copolymer-RGE4C, and a hydrolyzed HPMA copolymer precursor. Scintigraphic images obtained at 24 h showed specific tumor localization of HPMA copolymer-RGD4C and RGD4C compared with RGE4C conjugates in both prostate tumor models. Twenty-four-hour necropsy data in the DU145 model showed significantly higher (P < 0.001) tumor localization for HPMA copolymer-RGD4C (4.60 +/- 1.80%ID/g [percentage injected dose per gram tissue]) and RGD4C-DPK (3.37 +/- 0.32%ID/g) compared with HPMA copolymer-RGE4C (1.24 +/- 0.15%ID/g) and RGE4C-DPK (0.32 +/- 0.04%ID/g). Similar results were observed in the PC-3 model. Moreover, higher T/B for the polymer conjugates indicated reduced extravasation of the targeted polymeric conjugates in normal tissues.. Specific molecular targeting of the alpha(v)beta(3) integrin and nonspecific vascular permeability are both significant in the relative tumor localization of polymeric conjugates of RGD4C. Extravascular leak in nonspecific organs appears to be a major factor in reducing the T/B for the peptide molecules.

Topics: Animals; Cell Line, Tumor; Drug Delivery Systems; Humans; Integrin alphaVbeta3; Male; Metabolic Clearance Rate; Methacrylates; Mice; Mice, SCID; Neovascularization, Pathologic; Oligopeptides; Organ Specificity; Prostatic Neoplasms; Radionuclide Imaging; Tissue Distribution

The use of biodegradable polymers in medicine and biomedical research is increasing. A key growth area has been the use of these materials in tissue engineering, especially for guided regeneration of bone and cartilage. Our interest has been in determining the mechanisms by which cellular attachment and growth occurs on these materials. In the current study, we examined human osteoblast cell adhesion, growth, and morphologic changes on polymeric scaffolds composed of polylactic-co-glycolic acid and polylactic acid materials. We examined these characteristics in association with measurements of levels of key adhesion integrin receptors in the presence and absence of antibodies against alpha2, alpha3, alpha4, alpha5, alpha6, and beta1 subunits, and the adhesion ligand peptides RGD (Arg-Gly-Asp) and RGE (Arg-Gly-Ser). At 2 hours, results showed initial cell adhesion was considerably decreased on polylactic-co-glycolic acid and polylactic acid in the presence of the alpha2 and beta1, antibodies with a 70% adhesion rate difference observed among the groups evaluated. Higher levels of inhibition were observed on polylactic-co-glycolic acid relative to polylactic acid, which may be correlated to a higher number of cells being able to interact with the surface initially. The presence of known competitive peptide (RGD) at 2 hours, revealed its ability to block cellular adhesion to these matrices relative to the control and noncompetitive peptide RGE on polylactic-co-glycolic acid matrices. Overall adhesion rate was affected by the presence of the integrin antibodies to the alpha2, alpha3, alpha4, alpha5, alpha6, and beta1 subunits with highest differences among polylactic-co-glycolic acid relative to its control, therefore suggesting that initial osteoblastic cell adhesion to commonly used biomaterials is regulated through integrin binding.

Topics: Biocompatible Materials; Bone Regeneration; Cell Adhesion; Humans; Integrins; Lactic Acid; Oligopeptides; Osteoblasts; Polyesters; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Polymers; Tissue Engineering

Matrix metalloproteinases are involved in the regulation of bone remodeling. The hypothesis that matrix metalloproteinase inhibitors may be useful for experimentally limiting orthodontic tooth movement, a process involving perturbations of normal bone remodeling, was tested. General matrix metalloproteinase inhibitors limited the resorption of bone slices by mouse marrow cultures stimulated by calcitriol, parathyroid hormone, and basic-fibroblast growth factor. Pre-coating dentin slices with short arginine-glycine aspartic acid (RGD) peptides, but not arginine-glycine-glutamic acid (RGE) controls, restored bone resorption in the presence of matrix metalloproteinase inhibitors. Orthodontic tooth movement was inhibited by local delivery of Ilomastat, a general matrix metalloproteinase inhibitor, with the use of ethylene-vinyl-acetate (ELVAX) 40, a non-biodegradable, non-inflammatory sustained-release polymer. This study shows that orthodontic tooth movement can be inhibited with the use of matrix metalloproteinase inhibitors, and suggests a mechanistic link between matrix metalloproteinase activity and the production of RGD peptides.

Topics: Amides; Amino Acid Sequence; Analysis of Variance; Animals; Bone Marrow Cells; Bone Remodeling; Bone Resorption; Cell Culture Techniques; Delayed-Action Preparations; Hydroxamic Acids; Indoles; Male; Matrix Metalloproteinase Inhibitors; Maxilla; Mice; Oligopeptides; Osteoclasts; Polyvinyls; Rats; Rats, Sprague-Dawley; Receptors, Immunologic; Tooth Movement Techniques; Tyrosine

We showed recently that human periodontal ligament (PDL) and gingival fibroblasts adhere and spread on enamel matrix protein (EMP) coatings. In the present study, we investigated whether this interaction can be attributed to integrin expression. Human PDL and gingival fibroblasts were cultured for periods up to 24 h on EMP coatings, in the presence of synthetic RGD-containing peptide or an antibody against the beta1 integrin subunit. The cells were first cultured for 24 h under serum-free conditions and then cultured on EMP coatings for 48 h. Integrin expression levels were assessed by flow cytometry analysis. It was found that attachment and spreading on EMP was inhibited by the synthetic RGD-containing peptide, but not by a synthetic RGE-peptide. Both PDL and gingival fibroblasts showed expression of the integrin subunits, alpha2, alpha5, beta1, and the integrin, alphavbeta3. Incubation with an antibody against the beta1 subunit significantly inhibited the attachment and spreading of PDL and gingival fibroblasts on EMP coatings. We conclude that integrins are involved in the interaction of PDL and gingival fibroblasts with EMP.

Topics: Adolescent; Adult; Antibodies; Cell Adhesion; Cell Culture Techniques; Cell Movement; Culture Media, Serum-Free; Dental Enamel Proteins; Fibroblasts; Flow Cytometry; Gingiva; Humans; Integrin alpha2; Integrin alpha5; Integrin alphaVbeta3; Integrin beta1; Integrins; Oligopeptides; Periodontal Ligament; Receptors, Immunologic; Statistics as Topic

To investigate the relationship between outflow facility and separation between the inner wall of the aqueous plexus and the juxtacanalicular connective tissue (JCT) during washout in the bovine eye.. Facility was recorded during 3 hours of anterior chamber perfusion at 15 mm Hg in eight pairs of bovine eyes. One eye of each pair was then lowered to 0 mm Hg for 1 hour, whereas the fellow eye was kept at 15 mm Hg. After a brief perfusion at 15 mm Hg, both eyes were perfusion fixed and processed for electron microscopy. Micrographs of the inner wall were analyzed for separation from the JCT. To study the role of cellular adhesion between the inner wall and JCT, 12 additional pairs were perfused with integrin-binding peptide (RGD: Arg-Gly-Asp) or sham control peptide (RGE: Arg-Gly-Glu) at 2 micro M to 2 mM, before IOP was reduced.. During the first 3 hours, facility increased in both eyes because of "washout." However, after 1 hour of 0 mm Hg, facility decreased by 13% (P < 0.006), whereas facility increased by 20% (P < 0.001) in the fellow eyes maintained at 15 mm Hg. Two types of separation were observed between the inner wall and JCT: cell-matrix separation between the endothelial cell and basal lamina and matrix-matrix separation between the basal lamina and JCT. A significant positive correlation (P = 0.042) was found between the degree of matrix-matrix separation and the change in outflow facility after 1 hour of 0 mm Hg. Compared with RGE control, RGD had no apparent effect on outflow facility (P > 0.35) or on the change in outflow facility after 1 hour at 0 mm Hg (P > 0.15).. The increase in outflow facility that occurs during washout in the bovine eye is reversible and correlates with the degree of separation between the basal lamina of the inner wall endothelium and the JCT. Therefore, adhesions tethering the inner wall to the JCT may be important ultrastructural features involved in the regulation of aqueous humor outflow resistance.

Topics: Animals; Anterior Eye Segment; Aqueous Humor; Basement Membrane; Cattle; Cell Adhesion; Connective Tissue; Intraocular Pressure; Oligopeptides; Perfusion

An inhibition assay of Candida albicans adhesion to gelatin-immobilized membranes was compared with that to intact type I collagen-immobilized membranes using an arginine-glycine-aspartic acid (RGD) containing peptide. As compared with a protein-free membrane, gelatin and collagen significantly enhanced the adherence of C. albicans. The adhesion of the yeast to gelatin was significantly inhibited by the RGD peptides, but not by arginine-glycine-glutamic acid (RGE) peptides. In contrast, attachment to collagen was not inhibited by RGD peptides. These results suggest that the RGD sequence of gelatin and the integrin-like proteins of yeasts may be involved in adherence.

Topics: Adhesiveness; Candida albicans; Collagen Type I; Gelatin; Humans; Integrins; Membranes, Artificial; Oligopeptides; Receptors, Immunologic; Surface Properties

The P2Y(2) nucleotide receptor (P2Y(2)R) contains the integrin-binding domain arginine-glycine-aspartic acid (RGD) in its first extracellular loop, raising the possibility that this G protein-coupled receptor interacts directly with an integrin. Binding of a peptide corresponding to the first extracellular loop of the P2Y(2)R to K562 erythroleukemia cells was inhibited by antibodies against alpha(V)beta(3)/beta(5) integrins and the integrin-associated thrombospondin receptor, CD47. Immunofluorescence of cells transfected with epitope-tagged P2Y(2)Rs indicated that alpha(V) integrins colocalized 10-fold better with the wild-type P2Y(2)R than with a mutant P2Y(2)R in which the RGD sequence was replaced with RGE. Compared with the wild-type P2Y(2)R, the RGE mutant required 1,000-fold higher agonist concentrations to phosphorylate focal adhesion kinase, activate extracellular signal-regulated kinases, and initiate the PLC-dependent mobilization of intracellular Ca(2+). Furthermore, an anti-alpha(V) integrin antibody partially inhibited these signaling events mediated by the wild-type P2Y(2)R. Pertussis toxin, an inhibitor of G(i/o) proteins, partially inhibited Ca(2+) mobilization mediated by the wild-type P2Y(2)R, but not by the RGE mutant, suggesting that the RGD sequence is required for P2Y(2)R-mediated activation of G(o), but not G(q). Since CD47 has been shown to associate directly with G(i/o) family proteins, these results suggest that interactions between P2Y(2)Rs, integrins, and CD47 may be important for coupling the P2Y(2)R to G(o).

Topics: Amino Acid Sequence; Antigens, CD; Calcium; Carrier Proteins; CD47 Antigen; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; GTP-Binding Protein alpha Subunits, Gi-Go; Heterotrimeric GTP-Binding Proteins; Humans; Integrins; Mitogen-Activated Protein Kinases; Molecular Sequence Data; Oligopeptides; Phosphorylation; Point Mutation; Protein Binding; Protein-Tyrosine Kinases; Receptors, Purinergic P2; Receptors, Purinergic P2Y1; Receptors, Purinergic P2Y2; Receptors, Vitronectin; Sequence Homology, Amino Acid; Signal Transduction

Fibrinogen interactions with vascular endothelial cells are implicated in various physiological and pathophysiological events, including angiogenesis and wound healing. We have shown previously that integrin alpha(5)beta(1) is a fibrinogen receptor on endothelial cells [Suehiro, K., Gailit, J., and Plow, E.F. (1997) J. Biol. Chem. 272, 5360-5366]. In the present study, we have characterized fibrinogen interactions with purified alpha(5)beta(1) and have identified the recognition sequence in fibrinogen for alpha(5)beta(1). The binding of fibrinogen to immobilized alpha(5)beta(1) was selectively supported by Mn(2+). Fibrinogen bound to purified alpha(5)beta(1) in a time-dependent, specific, and saturable manner in the presence of Mn(2+), and the binding was blocked completely by Arg-Gly-Asp (RGD)-containing peptides and by anti-alpha(5) and anti-alpha(5)beta(1) monoclonal antibodies. A monoclonal antibody directed to the C-terminal RGD sequence at Aalpha572-574 significantly inhibited the binding of fibrinogen to alpha(5)beta(1), whereas monoclonal antibodies directed to either the N-terminal RGD sequence at Aalpha95-97 or the C-terminus of the gamma-chain did not. Furthermore, substituting RGE for RGD at position Aalpha95-97 in recombinant fibrinogen had a minimal effect on binding, whereas substituting RGE for RGD at position Aalpha572-574 decreased binding by 90%. These results demonstrate that the C-terminal RGD sequence at Aalpha572-574 is required for the interaction of fibrinogen with alpha(5)beta(1).

Topics: Amino Acid Sequence; Antibodies, Monoclonal; Binding, Competitive; Cations, Divalent; Fibrinogen; Humans; Manganese; Molecular Sequence Data; Mutation; Oligopeptides; Peptide Fragments; Protein Binding; Receptors, Fibronectin; Recombinant Proteins; Substrate Specificity; Temperature

Cell counting, cell cycle analysis and Western immunoblotting were used to examine the effects of non-apoptotic doses of a ceramide analogue, C2, and a synthetic arginine-glycine-aspartic acid (RGD)-containing peptide, RGD, in MCF-7 and T47D cells to determine whether activation of these signalling pathways could alter the mitogenic potential of insulin-like growth factor I (IGF-I). IGF-I alone increased total cell number in both cell lines, associated with a rise in the percentage of cells in the S-phase of the cell cycle and a co-incident increase in cyclin A production. Treatments alone had no effects on cell number or cyclin A production relative to controls. C2 inhibited IGF-I-induced mitogenesis in both lines, whereas RGD was only effective in the T47D line. Despite inhibition of cell proliferation, IGF-I stimulation of cells in S-phase and of cyclin A levels were unaffected; however, an IGF-I-induced increase in cyclin B1 levels was inhibited by 30%. Low-dose induction of integrin and ceramide signalling pathways causes cells to be blocked in S-phase, thereby inhibiting the normal cycle of events associated with the IGF-I-induced mitotic signal. Activating these pathways may not only restrict tumour growth by induction of apoptosis but they may also directly inhibit IGF-I-induced cell proliferation.

Topics: Breast Neoplasms; Cell Cycle; Cell Division; Cyclin A; Cyclin B; Cyclin B1; Female; Humans; Insulin-Like Growth Factor I; Kinetics; Oligopeptides; Recombinant Proteins; S Phase; Signal Transduction; Sphingosine; Tumor Cells, Cultured

Keratocytes are useful in the study of locomotion because they move rapidly (up to 1 micron/second) while maintaining an almost uniform shape, speed and direction. The smooth gliding motion of the keratocyte requires a precise coordination between adhesion, contractility, and retraction. To ask what role integrins play in keratocyte adhesion and locomotion, either RGD peptides or an anti-beta1 integrin mAb that binds to an ectodomain epitope and inhibits adhesion formation was added to the culture media of moving keratocytes. The response to these reagents depended on three interrelated factors: the dose of RGD/mAb, the apparent adhesion strength of the keratocyte to the substratum and the cell speed. High doses cause keratocytes to quickly and irreversibly round up. At intermediate RGD/mAb doses, keratocytes reestablish adhesion after treatment and briefly resume locomotion until partial detachment recurs. At the lowest doses, disruption of beta1 integrin-mediated adhesion formation destabilizes the lamella, temporarily preventing lamellar extension and forward movement of the cell. With increasing culture time, there is an increase in apparent adhesion and a corresponding marked decrease in locomotory velocity. Under these conditions, high doses of RGD/mAb do not cause keratocytes to detach or even produce detectable lamellar instabilities. We postulate that RGD/mAb competitively inhibits new beta1 integrin mediated adhesion formation that is required to support the rates of lamellar extension necessary for rapid locomotion.

Topics: Animals; Antibodies, Monoclonal; Binding, Competitive; Cell Adhesion; Cell Movement; Cell Size; Cells, Cultured; Cellular Senescence; Dose-Response Relationship, Drug; Epitopes; Integrin beta1; Keratinocytes; Microscopy, Interference; Models, Biological; Oligopeptides; Pseudopodia; Vitronectin; Xenopus laevis

Several reports have suggested that encapsulation of orthopaedic and dental implants with fibrous tissue can lead to implant failure. The binding of cells to the surface of the implants is specific to amino-acid sequences, typically RGD. The specific objective of this study was to investigate the interaction of cultured human peripheral macrophages with specific amino-acid sequences to determine if adherence is due to the specificity of such sequence. Macrophages were seeded at a density of 1 x 10(5) cells on ultra high molecular weight polyethylene (UHMWPE) coated with either amino-acid heteropolymers of RGE, RGD, or amino-acid homopolymer Poly-L-Lysine. Cells were observed daily and morphology was recorded. The results showed that cells growing in the presence of RGD had significantly (p < 0.05) higher numbers of cells adhering and remaining viable, in comparison to cells growing on Poly-L-lysine or RGE. Cells growing on UHMWPE coated with RGE appeared irregularly (elongated and spindle) shaped and unevenly spaced. The cells growing in the presence of Poly-L-Lysine showed cellular disruption and lysis, whereas cells growing on the RGD appeared intact, regularly spaced and began fusing into giant cells. Lactate dehydrogenase activity was used as a measure of membrane integrity, and cells grown on UHMWPE coated with Poly-L-lysine showed a two-fold increase in activity over control and peptide treated groups. Immunochemical analysis for cytokine (IL-1) release as a measure of cellular reactivity revealed an increase level in the experimental groups after 24 hours and remained measurable over the duration of the experiment. Cells incubated on uncoated polyethylene showed no evidence of increased cytokine response. Overall, the results show macrophages can interact with specific coating on the material surface, and these surfaces can affect the adhesion process adherence. Use of RGE, which inhibits binding of the cells, may be a factor that can be used to coat implants to increase their longevity.

Topics: Amino Acid Sequence; Biocompatible Materials; Cell Adhesion; Cell Survival; Cells, Cultured; Cytokines; HL-60 Cells; Humans; Implants, Experimental; L-Lactate Dehydrogenase; Macrophages; Oligopeptides; Polyethylenes; Polylysine