The detection,immunofluorescent localization,and thrombin induced release of human platelet-associated fibronectin antigen

Platelets are cells which develop adhesive properties following stimulation. Since fibronectin (fn) mediates adhesive properties of several cells, we sought evidence for platelet associated fn. Lysates of suspensions of washed human platelets containing ≤50 ng soluble fn/10⁹ cells contained 2.85 μg fn antigen per 10⁹ cells. The platelet fn antigen competition curve showed a similar slope to the curve for purified plasma fn suggesting antigenic identity. Immunofluorescent staining for fn was minimal in intact cells suggesting that the majority of fn antigen is intracellular. In permeable platelets, fluorescent staining for fn was seen in a punctate distribution suggesting a granule localization. Stimulation of platelet secretion by thrombin released platelet fn antigen. Suramin, a drug which inhibits platelet secretion, inhibited fn release. The apparent secretion of platelet fn, taken with the immunofluorescent data, support the localization of a portion of platelet fn antigen in a storage granule.     

The structure of fibronectin and its role in cellular adhesion

Fibronectin is a large, adhesive glycoprotein which is found in a number of locations, most notably on cell surfaces, in extracellular matrixes, and in blood. Fibronectin has been detected in all vertebrates tested and in many invertebrates. Its presence in sponges is significant because this suggests that fibronectin may have appeared very early in evolution, possibly with the most primitive multicellular organisms. Cellular and plasma fibronectins have many striking similarities. However, the locations of the polypcptide chain differences between these two proteins indicate that plasma fibronectin cannot be derived from cellular fibronectin by means of simple post-translational proteolysis. Instead, these different types of fibronectin may be products of different genes or of differentially spliced messenger RNA molecules. Amniotic fluid fibronectin is possibly a third form of the protein. Cellular and plasma fibronectins are composed of at least six protcaseresistant domains which contain specific binding sites for actin, gelatin, heparin, Staphylococcus aureus, transglutarninase, fibrin, DNA, and a cell surface receptor. The relative locations of these domains have been mapped in the primary structure of fibronectin. The cell surface receptor for fibronectin has not been positively identified, but may be a glycoprotein, a glycolipid, or a complex of the two. Although cell-substratum adhesion is mediated by fibronectin, the locations of the areas of closest approach of the cell to the substratum (the adhesion plaques) and fibronectin are not coincident under conditions of active cell growth. Under conditions of cell growth arrest in low scrum concentrations, some fibronectin may become localized at the adhesion plaques. Models describing the domain structure of fibronectin and the molecular organization of the adhesion plaque area are presented.     

Real-time monitoring of hematopoietic cell interaction with fibronectin fragment

Real-time cell analysis (RTCA) system based on measurement of electrical microimpedance has been introduced to monitor adherent cell cultures. We describe its use for real-time analysis of hematopoietic cell adhesion to bone marrow stroma proteins. Cells growing in suspension do not generate any significant change in the microimpedance signal until the surface with embedded microelectrodes is coated with a cell-binding protein. We show that in this case, the microimpedance signal specifically reflects cell binding to the coated surface. The optimized method was used to monitor the effect of two histone deacetylase inhibitors, suberoylanilide hydroxamic acid (SAHA) and tubastatin A, on JURL-MK1 cell adhesion to cell-binding fragment of fibronectin (FNF). Both compounds were used in non-toxic concentrations and induced an increase in the cell adhesivity. The kinetics of this increase was markedly slower for SAHA although tubulin hyperacetylation occurred rapidly for any of the two drugs. The strengthening of cell binding to FNF was paralleled with a decrease of Lyn kinase activity monitored using an anti-phospho-Src family antibody. The inhibition of Src kinase activity with PP2 accordingly enhanced JURL-MK1 cell interaction with FNF. Actin filaments were present at the proximity of the plasma membrane and in numerous membrane protrusions. In some cells, F-actin formed clusters at membrane regions interacting with the coated surface and these clusters colocalized with active Lyn kinase. Our results indicate that the role of Src kinases in the regulation of hematopoetic cell adhesion signaling is similar to that of c-Src in adherent cells.     

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.     

Chromosome analyses and anchorage-independent growth of SV40-induced morphologically transformed epithelial cells from amniotic fluids

Summary Epithelial cells from amniotic fluid cell cultures are morphologically transformed by simian virus 40, 20 to 30 d after infection. The cells of the transformed colonies are highly basophilic, have a high nuclear-to-cytoplasmic ratio, and show a dense growth pattern. The cells are virus producers, and ultimately, after continuous passage, the cell lines reach a crisis situation with no growth. Twelve morphologically transformed cell colonies were isolated from five different individuals for chromosome analyses after approximately 18 population doublings (second bottle passage). For all cell lines diploid cells were observed. Banding of the chromosomes revealed normal morphology of euchromatic and heterochromatic regions. The suggestion is made that chromosome alteration is not necessary, nor a prerequisite, for the morphologically transformed phenotype to be expressed and that the transformation process per se causes chromosomal instability. Tests for colony formation of the 12 cell lines in semisolid medium showed that different transformed colony isolates from the same individual donor of the cells either formed or did not form colonies in agar. The size of the colonies was also consistent within individuals as compared to between individuals. These limited results are suggestive of a dependence upon the genetic constitution of the individual donor of the cells for colony formation in soft agar. Supported by National Science Foundation Grant PCM77-15876.     

Proteolytic E-cadherin activation followed by solution NMR and X-ray crystallography

Cellular adhesion by classical cadherins depends critically on the exact proteolytic removal of their N-terminal prosequences. In this combined solution NMR and X-ray crystallographic study, the consequences of propeptide cleavage of an epithelial cadherin construct (domains 1 and 2) were followed at atomic level. At low protein concentration, the N-terminal processing induces docking of the tryptophan-2 side-chain into a binding pocket on the same molecule. At high concentration, cleavage induces dimerization (KD=0.72 mM, k(off)=0.7 s(-1)) and concomitant intermolecular exchange of the betaA-strands and the tryptophan-2 side-chains. Thus, the cleavage represents the switch from a nonadhesive to the functional form of cadherin.     

An Examination of Focal Adhesion Formation and Tyrosine Phosphorylation in Fibroblasts Isolated from src¯, fyn¯, and yes¯ Mice

As cells adhere to extracellular matrix proteins, several focal adhesion proteins become tyrosine phosphorylated. One of the most prominent of these has been identified as the tyrosine kinase p125^FAK (focal adhesion kinase, FAK). An interaction between FAK and members of the Src family tyrosine kinases p59^fyn, pp60^v-src, and activated pp60^c-src (527F) has been demonstrated, raising the possibility that these kinases may regulate FAK activity. To explore the role of Src family kinases in focal adhesions and in the regulation of FAK activity, we isolated fibroblasts from transgenic mice that lack either pp60^c-src p59^fyn, or pp62^c-yes. These primary fibroblasts, and those of a control mouse, were passaged numerous times and resulted in spontaneously immortalized cell lines without the addition of transforming agents. After confirming the absence of the appropriate nonreceptor tyrosine kinases in the fyc¯, srn¯ and yes¯ fibroblasts, the ability of these fibroblasts to form focal adhesions and stress fibers was assessed by immunofluorescence microscopy and found to be comparable to that of normal fibroblasts. We investigated phosphotyrosine levels in response to adhesion to fibronectin and identified the pp60^src substrate p130 as the one major protein with reduced levels of tyrosine phosphorylation in the cells lacking p59^fyn and pp62^c-yes, and particularly in those lacking pp60^c-scr. We examined FAK phosphorylation and kinase activity and found that there were no significant differences between these cells.     

Characterization of a membrane-associated glycoprotein complex implicated in cell adhesion to fibronectin

We have characterized a 140-kDa glycoprotein complex purified by a monoclonal antibody and implicated in cell adhesion to the extracellular molecule fibronectin. Three major polypeptide components were purified by monoclonal antibody JG22E, which had apparent molecular weights of 155,000 (band 1), 135,000 (band 2), and 120,000 (band 3). In two-dimensional gel electrophoresis, each subunit migrated as either a broad band or a series of spots at acidic isoelectric points. After treatment with neuraminidase, the spots became focused around pH 6.2 (band 1), pH 5.6 (band 2), and pH 5.3 (band 3). These three major bands were compared by two-dimensional peptide mapping in a series of pairwise combinations and were found to be distinct proteins. In sucrose gradients, these proteins co-migrated as a complex sedimenting at approximately 8.4 S either before or after affinity purification, whereas separated subunits migrated at 4.7 to 5.8 S. Amino acid analysis revealed no detectable hydroxyproline and a composition characterized by a substantial number of cysteine residues compared to the average protein. Our results suggest that a noncovalent complex of structurally distinct glycoproteins is involved in adhesive interactions of fibronectin with cells.     

Fibronectin‐binding proteins of Clostridium perfringens recognize the III1‐C fragment of fibronectin

The Clostridium perfringens strain 13 genome contains two genes (fbpA, fbpB) that encode putative Fbp. Both rFbpA and rFbpB were purified and their reactivity with human serum Fn was analyzed. To determine the region of the Fn molecule recognized by rFbp, a plate binding assay using N‐terminal 70‐kDa peptide, III₁‐C peptide, and 110‐kDa peptide containing III_2–10 of Fn was performed. Both rFbp bound to the III₁‐C peptide of Fn but not to the other peptides. However, the III₁‐C fragment of Fn is known to be cryptic in serum Fn. Then, rFbp‐BP from Fn were purified by rFbp‐affinity chromatography. The yield of purified proteins was approximately 1% of the applied Fn on a protein basis. Western blotting analysis of the rFbp‐BP, using four different anti‐Fn monoclonal antibodies, revealed that the rFbp‐BP carried partial Fn antigenicity. Bindings of rFbp to rFbp‐BP were inhibited by the presence of the III₁‐C peptide, suggesting that rFbp‐BP express the III₁‐C fragment. The binding of Fn to III₁‐C was inhibited by the presence of either rFbpA or rFbpB. This result that suggests C. perfringens Fbps may inhibit the formation of Fn‐matrix in vivo.     

应激抑制淋巴细胞转化的时间效应

本研究吵缚方法使大鼠接受应激刺激,然后分别取大鼠应激３、６、１２、１８ｈ和解除束缚后１２、２４、４８、７２、９６ｈ的淋巴结、脾脏提取物和血清,与刀豆素Ａ同时加入正常大鼠肠系膜淋巴结细胞悬液中育７２ｈ后用噻唑蓝（ＭＴＴ）比色分析法检测肠系膜淋巴结细胞的转化,来应激抑制淋巴细胞转化作用的出现和消失过程。结果如下：（１）应激３和６ｈ大鼠的淋巴结、脾脏提取物和血清对淋巴细胞的转化都没有明显的影响;（２）应     

Participation of Syndecan 2 in the Induction of Stress Fiber Formation in Cooperation with Integrin α5β1: Structural Characteristics of Heparan Sulfate Chains with Avidity to COOH-Terminal Heparin-Binding Domain of Fibronectin

The present study provides direct evidence that syndecan 2 participates selectively in the induction of stress fiber formation in cooperation with integrin α5β1 through specific binding of its heparan sulfate side chains to the fibronectin substrate. Our previous study with Lewis lung carcinoma-derived P29 cells demonstrated that the cell surface heparan sulfate proteoglycan, which binds to fibronectin, is syndecan 2 (N. Itano et al., 1996, Biochem. J. 315, 925–930). We here report that in vitro treatment of the cells by antisense oligonucleotide for syndecan 2 resulted in a failure to form stress fibers on fibronectin substrate in association with specific suppression of its cell surface expression. Instead, localization of actin filaments in the cytoplasmic cortex occurred. A similar response of the cells was observed when the cells were treated to eliminate functions of cell surface heparan sulfates, including exogenous addition of heparin and pretreatment with anti-heparan sulfate antibody, F58-10E4, and with proteinase-free heparitinase I. Size- and structure-defined oligosaccharides prepared from heparin and chemically modified heparins were utilized as competitive inhibitors to examine the structural characteristics of the cell surface heparan sulfates involved in organization of the actin cytoskeleton. Their affinity chromatography on a column linked with a recombinant H-271 peptide containing a C-terminal heparin-binding domain of fibronectin demonstrated that 2-O-sulfated iduronates were essential for the binding. Inhibition studies revealed that a heparin-derived dodecasaccharide sample enriched with an IdoA(2OS)–GlcNS(6OS) disaccharide completely blocked binding of the syndecan 2 ectodomain to immobilized H-271 peptide. Finally, the dodecasaccharide sample was shown to inhibit stress fiber formation, triggered by adhesion of P29 cells to a CH-271 polypeptide consisting of both the RGD cell-binding and the C-terminal heparin-binding domains of fibronectin in a fused form. All these results consistently suggest that syndecan 2 proteoglycan interacts with the C-terminal heparin-binding domain of fibronectin at the highly sulfated cluster(s), such as [IdoA(2OS)–GlcNS(6OS)]₆ present in its heparan sulfate chains, to result in the induction of stress fiber formation in cooperation with integrin α5β1.     

Inhibition of neutrophil adhesion by pectic galacturonans

The inhibition of the adhesion of neutrophils to fibronectin by the fragments of the main galacturonan chain of the following pectins was demonstrated: comaruman from the marsh cinquefoil Comarum polustre, bergenan from the Siberian tea Bergenia crassifolia), lemnan from the duckweed Lemna minor), zosteran from the eelgrass Zostera marina), and citrus pectin. The parent pectins, except for comaruman, did not affect the cell adhesion. Galacturonans prepared from the starting pectins by acidic hydrolysis were shown to reduce the neutrophil adhesion stimulated by phorbol 12-myristate 13-acetate (1.625 μM) and dithiothreitol (0.5 mM) at a concentration of 50–200 μg/ml. The presence of carbohydrate chains with molecular masses higher than 300, from 100 to 300, and from 50 to 100 kDa in the galacturonan fractions was proved by membrane ultrafiltration.     

Combined effects of 60 Hz electromagnetic field exposure with various stress factors on cellular transformation in NIH3T3 cells

Epidemiological studies have suggested that extremely low‐frequency magnetic fields (ELF‐MF) are associated with an increased incidence of cancer. Studies using in vitro systems have reported mixed results for the effects of ELF‐MF alone, and the World Health Organization (WHO) Research Agenda published in 2007 suggested that high priority research should include an evaluation of the co‐carcinogenic effects of ELF‐MF exposure using in vitro models. Here, the carcinogenic potential of ELF‐MF exposure alone and in combination with various stress factors was investigated in NIH3T3 mouse fibroblasts using an in vitro cellular transformation assay. NIH3T3 cells were exposed to a 60 Hz ELF‐MF (1 mT) alone or in combination with ionizing radiation (IR), hydrogen peroxide (H₂O₂), or c‐Myc overexpression, and the resulting number of anchorage‐independent colonies was counted. A 4 h exposure of NIH3T3 cells to ELF‐MF alone produced no cell transformation. Moreover, ELF exposure did not influence the transformation activity of IR, H₂O₂, or activated c‐Myc in our in vitro assay system, suggesting that 1 mT ELF‐MF did not affect any additive or synergistic transformation activities in combination with stress factors such as IR, H₂O₂, or activated c‐Myc in NIH3T3 cells. Bioelectromagnetics 33:207–214, 2012. © 2011 Wiley Periodicals, Inc.     

cDNA表达文库转染NIH3T3-从高转移人肺腺癌细胞系Anip973中克隆新肿瘤转移相关基因

培养人高转移肺腺癌细胞系Anip973,构建其cDNA表达文库并转染小鼠成纤维细胞NIH3T3,将经药物筛选后的转染细胞克隆消化为单细胞,接种到软琼脂中培养2周,根据细胞明显的形态学变化挑选出有意义的细胞克隆,扩增再培养,提取DNA,PCR扩增插入片段并进行测序分析。结果表明:软琼脂中挑选出克隆100多个,PCR测序后,得到3个已知基因包括人类核糖体蛋白L23、人类假定蛋白FLJ22104和人类丝氨酸蛋白酶抑制因子6亚型以及一些氨基末端截短的核酸序列。进一步的研究表明转染人类核糖体蛋白L23的细胞与转染空载体细胞相比具有较高的侵袭能力(P<0.02)。利用cDNA文库在NIH3T3细胞中的表达,随后筛查鉴定在软琼脂中发生形态学变化的细胞,是一种寻找恶性转化和癌转移相关基因的有效方法。人类核糖体蛋白L23基因在细胞的运动和转移中发挥重要作用。     

Use of XTT for quantitating clonogenic growth in soft agar

The purpose of this study was to investigate the use of the tetrazolium salt. XTT, in a microassay for quantitation of anchorage-independent growth of cells in soft agar, using two human ovarian tumour cell lines (OAW 42 and OAW 28). The response of OAW 42 to Cis-platinum, EGF, TGF and insulin, and of OAW 28 to EGF, TGF and insulin were determined both by visual colony counting and use of the XTT assay. Drug inhibition and growth factor inhibition and stimulation were clearly demonstrated, with increases or decreases in visually counted colony numbers being paralleled by increases or decreases in optical density values obtained by using XTT. The XTT colorimetric assay was simple to perform and reproducible with low variability, and was found to be an acceptable alternative to visual colony counting for the quantitation of cell line response to drugs and growth factors under anchorage-independent growth conditions.