Alterations in neural crest migration by a monoclonal antibody that affects cell adhesion

The possible role of a 140-kD cell surface complex in neural crest adhesion and migration was examined using a monoclonal antibody JG22, first described by Greve and Gottlieb (1982, J. Cell. Biochem. 18:221-229). The addition of JG22 to neural crest cells in vitro caused a rapid change in morphology of cells plated on either fibronectin or laminin substrates. The cells became round and phase bright, often detaching from the dish or forming aggregates of rounded cells. Other tissues such as somites, notochords, and neural tubes were unaffected by the antibody in vitro even though the JG22 antigen is detectable in embryonic tissue sections on the surface of the myotome, neural tube, and notochord. The effects of the JG22 on neural crest migration in vivo were examined by a new perturbation approach in which both the antibody and the hybridoma cells were microinjected onto neural crest pathways. Hybridoma cells were labeled with a fluorescent cell marker that is nondeleterious and that is preserved after fixation and tissue sectioning. The JG22 antibody and hybridoma cells caused a marked reduction in cranial neural crest migration, a build-up of neural crest cells within the lumen of the neural tube, and some migration along aberrant pathways. Neural crest migration in the trunk was affected to a much lesser extent. In both cranial and trunk regions, a cell free zone of one or more cell diameters was generally observed between neural crest cells and the JG22 hybridoma cells. Two other monoclonal antibodies, 1-B and 1-N, were used as controls. Both 1-B and 1-N bind to bands of the 140-kD complex precipitated by JG22. Neither control antibody affected neural crest adhesion in vitro or neural crest migration in situ. This suggests that the observed alterations in neural crest migration are due to a functional block of the 140-kD complex.     

Localization of three distinct heparin-binding domains of laminin by monoclonal antibodies

Monoclonal antibodies were utilized to localize novel heparin-binding domains of laminin. A solid-phase radioligand binding assay was designed such that [3H] heparin bound to laminin in a time- and concentration-dependent manner. Tritiated heparin binding to laminin was saturable and specific as determined by competition with unlabeled heparin, dextran sulfate, and dermatan sulfate. By Scatchard analysis, two distinct dissociation constants were calculated (Kd = 50 and 130 nM), suggesting the presence of at least two binding sites for heparin on laminin. Tritiated heparin bound to thrombin-resistant (600 kDa) and chymotrypsin-resistant (440 kDa) laminin fragments, both known to lack the terminal globular domain of the long arm. Sodium dodecyl sulfate-polyacrylamide gels of chymotrypsin- and thermolysin-digested laminin chromatographed on a heparin-Sepharose column showed multiple proteolytic fragments binding to the column. Monoclonal antibodies generated against laminin were tested for their ability to inhibit [3H]heparin binding to laminin. Four monoclonal antibodies significantly inhibited the binding of [3H]heparin to laminin in the range of 15-21% inhibition. Laminin-monoclonal antibody interactions examined by electron microscopy showed that one antibody reacted at the terminal globular domain of the long arm, domain Hep-1, while epitopes for two of these monoclonal antibodies were located on the lateral arms of laminin, domain Hep-2, and the fourth monoclonal antibody bound below the cross-region of laminin, domain Hep-3. When two monoclonal antibodies recognizing distinctly different regions of laminin were added concomitantly, the inhibition of [3H]heparin binding to laminin increased almost 2-fold. These results suggest that at least two novel heparin-binding domains of laminin may be located in domains distinct from the terminal globular domain of the long arm.     

Isolation of a tumor cell laminin receptor

BL6 murine melanoma cells contain approximately 110,000 cell surface binding sites for the basement membrane glycoprotein laminin. Treatment of isolated melanoma cell plasma membranes with detergent yields a single class of laminin receptor. The receptor was purified 900 fold by laminin affinity chromatography. The isolated receptor has a Mr of 67,000 and binds laminin with high affinity: kd = 2 nm. The binding affinity of the isolated receptor was similar to that of the plasma membranes or the whole cells. Such a laminin receptor, isolated here for the first time, could facilitate the interaction of metastasizing tumor cells with the basement membrane.     

Prostaglandin E2 receptor modulation affects tumor cell adhesion to laminin.

We have shown previously that murine mammary adenocarcinoma cells both synthesize prostaglandin E2 (PGE2) and have a high affinity receptor for this ligand. Modulation of either PGE synthesis or PGE receptor function changes the metastatic potential of these cells. Because of the importance of laminin and laminin receptors to the metastatic process, we asked whether or not the PGE receptor participates in tumor cell-laminin interactions. As has been reported for many other tumor cells, laminin and the laminin-derived peptide PA22-2, containing the sequence IKVAV, mediate attachment of line 410.4 mammary tumor cells in vitro. We now demonstrate that the attachment of 410.4 cells to laminin or peptide PA22-2 was significantly inhibited by three PGE receptor antagonists, LE0101, SC19220, and sodium meclofenamate. LE0101 was most active, inhibiting tumor cell adhesion in a dose-dependent manner in the absence of nonspecific toxicity. These receptor antagonists had no effect on the PA22-2-mediated attachment of a PGE receptor negative tumor cell line, except at the highest concentration of LE0101 tested. No inhibition of adhesion to Type I collagen was seen. These results indicate that the PGE2 receptor modulates tumor cell adhesion to laminin which may subsequently affect the in vivo process of metastasis.     

Localization and partial characterization of a rat ovarian granulose cell protein with a monoclonal antibody

Summry— Hybridoma cell lines were obtained from mouse splenocytes sensitized to granulosa cells collected from rat ovaries after gonadotropin stimulation. A monoclonal antibody (5G5) was obtained which reacted with granulosa cells and showed a positive reaction with serum-free conditioned medium containing granulosa cell secreted proteins. Immoblotting of the conditioned medium and light- and electron-microscopic immunocytochemistry of rat ovary show that mAb 5G5 is directed against a 59-kDa protein which is located on the plasma membrane of granulosa cells. Furthermore, the immunoreactivity of the granulosa cells depends both on the degree of follicle development and on the position of the granulosa cells within the follicles. Strong immunoreactivity was observed in the innermost granulosa cell layers, close to the oocyte and the antral cavity. The results obtained show that mAb 5G5 is a useful marker of a 59-kDa granulosa cell protein which might be of importance for the follicle and the occyte maturation.     

Platelet-collagen adhesion: inhibition by a monoclonal antibody that binds glycoprotein IIb

To identify platelet surface structures involved in adhesion to collagen, the effect of 16 murine antiplatelet membrane hybridoma antibodies were tested in a defined, in vitro assay. Four of these antibodies inhibited platelet-collagen adhesion and reacted with a polypeptide with Mr approximately 125,000, as determined by immunoblots after gel electrophoresis under reducing conditions. Through detailed studies with one of these antibodies, the monoclonal antibody PMI-1, the relevant antigen was identified as platelet glycoprotein IIb alpha, based upon (a) co-migration with this glycoprotein in two-dimensional gel electrophoresis and (b) co-purification by immunoaffinity chromatography with a protein with apparent Mr identical to that of glycoprotein III, under conditions in which glycoproteins IIb and III form a complex. Univalent antibody fragments prepared from monoclonal antibody PMI-1 inhibited greater than 80% of platelet-collagen adhesion, and inhibition was completely blocked by the immunopurified antigen. These results indicate that glycoprotein IIb participates in some aspect of platelet-collagen adhesion. In contrast, the purified antigen only partially neutralized a polyclonal antiserum that blocked platelet-collagen adhesion, to a maximum of approximately 25%, at saturating antigen concentrations. Thus, by these immunological criteria, glycoprotein IIb is not the only molecule involved in this process.     

Cell adhesion and metastasis: a monoclonal antibody approach

Cellular adhesion processes are important during the growth of tumors and the generation of metastases. We therefore expect that monoclonal antibodies directed against molecules regulating cell adhesion of tumor cells will be powerful tools for specifically interfering with these processes. In the experimental system of the mouse B16 melanoma, a series of such functional monoclonal antibodies has recently been prepared and their inhibitory effect on the formation of metastatic lesions has been explored.     

Localization of a major nidogen-binding site to domain III of laminin B2 chain

The large pepsin fragments P1 and P1X, which comprise most of the rod-like domains III of the three short arms of laminin from the mouse Engelbreth-Holm-Swarm tumor, possess full binding activity for nidogen in radioligand assays. Partial reduction (70-80%) of disulfide bonds in P1 did not reduce binding activity and allowed the separation of domain III segments originating from the A, B1 and B2 chains of laminin as demonstrated by sequence analysis. Only the B2 chain segment consisting of seven cysteine-rich repeats with similarity to epidermal growth factor showed substantial nidogen-binding activity. Further degradation of this component to an active 28-kDa fragment was achieved by a second pepsin digestion of partially reduced P1. This indicates that a major binding structure for nidogen is located within three or four cysteine-rich repeats occupying sequence positions 755 to about 920 in the B2 chain. The data also show that fragments P1 and P1X differ by the absence or presence of a large portion, domain IIIb, of the laminin A chain but are indistinguishable in nidogen binding.     

Identification of asparagine-linked oligosaccharides involved in tumor cell adhesion to laminin and type IV collagen

MDW4, a wheat germ agglutinin-resistant nonmetastatic mutant of the highly metastatic murine tumor cell line called MDAY-D2 has previously been shown to attach to fibronectin and type IV collagen, whereas MDAY- D2 and phenotypic revertants of MDW4 attached poorly to these substrates. The increased adhesiveness of the mutant cells appeared to be closely related to a lesion in cell surface carbohydrate structures. In an effort to identify the carbohydrates involved in cell attachment, glycopeptides isolated from mutant and wild-type cells as well as from purified glycoproteins were tested for their ability to inhibit the attachment of MDW4 cells to plastic surfaces coated with fibronectin, laminin, or type IV collagen. The addition of mannose-terminating glycopeptide to the adhesion assay inhibited MDW4 cell attachment to type IV collagen. In contrast, a sialylated poly N-acetyllactosamine- containing glycopeptide, isolated from wheat germ agglutinin-sensitive MDAY-D2 cells but absent in MDW4 cells, inhibited MDW4 attachment to laminin. None of the glycopeptides used in this study inhibited attachment of MDW4 cells to fibronectin-coated plastic. Peptide N- glycosidase treatment of the cells to remove surface asparagine-linked oligosaccharides inhibited MDW4 adhesion to type IV collagen, but not to laminin, and the same treatment of the wheat germ agglutinin- sensitive cells enhanced attachment to laminin. Tumor cell attachment to, and detachment from, the sublaminal matrix protein laminin and type IV collagen are thought to be important events in the metastatic process. Our results indicate that tumor cell attachment to these proteins may be partially modulated by the expression of specific oligosaccharide structures associated with the cell surface.     

Amyloid fibril formation by a domain of rat cell adhesion molecule

Amyloid fibrils are protein aggregates implicated in several amyloidotic diseases. Cellular membranes with local decrease in pH and dielectric constant are associated with the amyloid formation. In this study, domain 1 of cell adhesion molecule CD2 (CD2-1) is used for studying amyloid fibril formation in a water/trifluoroethanol (TFE) mixture. CD2-1 is an all beta-sheet protein similar in topology to the amyloidogenic light chain variable domain, which deposits as amyloid in light chain amyloidosis at acidic pH. When incubated at pH 2.0 in the presence of 18% TFE, CD2-1 initiates the process to assemble into amyloid fibrils. It has been shown that TFE induces CD2-1 conformational change with a chemical transition (C(m)) of 18% (v/v). ANS (1-anilinonapthalene-8-sulfonic acid) binding was used to show that the hydrophobic surface becomes exposed under these solvent conditions. Our studies indicate that partial formation of a non-native conformation and the exposure of the hydrophobic interior could be the origins of oligomerization and fibril formation of CD2-1.     

Identification of a novel adhesion molecule in human leukocytes by monoclonal antibody LB-2

Monoclonal antibody LB-2 to a surface antigen on human B cells, lymphoblast, monocytes and vascular endothelial cells largely inhibited adhesion among Epstein Barr virus-immortalized normal B cells (EBV-B) and concanavalin A-stimulated blood mononuclear cells (Con A-BMC) before and after phorbol ester treatment. The antibody inhibited to a lesser extent phorbol ester-induced aggregation of monocytes, U937 cells and fresh BMC and had virtually no inhibitory effect on the adhesion among enriched T cells and granulocytes. A surface glycoprotein band of 84 kDa was obtained from EBV-B cells by immunoprecipitation and gel electrophoresis. Immunological and biochemical studies clearly distinguished this molecule from gp90 and associated glycoproteins which also mediate leukocyte adhesion.     

Localization of human sarcoma with radiolabeled monoclonal antibody

Summary We performed a randomized controlled study of postoperative adjuvant immunochemotherapy with Nocardia rubra cell wall skeleton (N-CWS) and Tegafur for gastric carcinoma between September 1979 and March 1983. A total of 309 patients were entered into this trial. Of the 309 patients, there were 98 evaluable patients in the chemotherapy group and 115 evaluable patients in the immunochemotherapy group. In both groups, Tegafur was given as chemotherapy at a daily dose of 400 to 800 mg, starting at 24–29 days after gastrectomy. In the immunochemotherapy group, 400 g of N-CWS was injected i. d. within the 2nd postoperative week. It was given weekly during the first month and subsequently monthly for as long as practicable. The patients were surveyed for length of survival in March 1985. The postoperative survival rate was analyzed for all cases, and for patients with various histopathological stages of carcinoma for comparison between the two treatment groups. No statistical difference was detected between the two groups in terms of age, sex, surgical curabilities, or stage of carcinoma. The overall survival rate for all patients was significantly higher in the immunochemotherapy group than in the chemotherapy group (p<0.05). With stage III plus IV disease, 53 patients from the chemotherapy group and 61 patients from the immunochemotherapy group were included for the analysis. As a consequence, a highly significant survival rate was observed in patients with stage III plus IV carcinoma in the immunochemotherapy group (p<0.005) as compared to the chemotherapy group. The overall 5-year (1800 days) survival rate after surgical treatment was 60.2% for the chemotherapy group and 73.2% for the immunochemotherapy group. In patients with stage III plus IV disease, the 5-year survival rates of the two treatment groups were 28.8% and 52.4%, respectively. Accordingly, the 50% survival period of patients with stage III plus IV cancer was 1800 days or more in the immunochemotherapy group, whereas it was only 722 days in the chemotherapy group. These results emphasize the effectiveness of N-CWS as an adjuvant immunotherapeutic agent in postoperative gastric cancer patients.The main side effects of N-CWS were skin lesions in the injected sites and fever, but these were temporary and not serious.     

Tumor cytostasis mediated by a monoclonal IgM antibody promoting adhesion between macrophages and tumor cells. Evidence for a lectin-like behavior.

We have shown previously that an IgM mAb (A10) recognizing Ehrlich tumor (ET) cell surface carbohydrates, inhibits in vivo ET growth by a macrophage-dependent mechanism. The inhibition mechanism involving both IgM and macrophages was unclear because receptors for IgM on macrophages are controversial and another monoclonal IgM (E1), also recognizing ET cell surface carbohydrates, was completely unable to show any protective effect. Here we show that A10, but not E1, was able to promote adhesion between macrophages and ET cells by a receptor for IgM-independent mechanism. Immunofluorescence studies showed that A10, but not E1, did react with macrophages if these cells were preincubated with a source of Ag spontaneously released from ET cells. This Ag release appeared to be required for A10-mediated adhesion, because adhesion was not obtained when ET cells fixed with paraformaldehyde were used. Cytostasis studies performed with macrophages stimulated with L-929 conditioned medium and ET cells showed that A10, but not E1 nor unrelated IgM, was able to inhibit ET cell proliferation in vitro by a mechanism involving cell contact between both cell populations. Therefore, IgM inhibition of ET growth, both in vivo and in vitro, could be explained by a lectin-like mechanism, where IgM, recognizing Ag of tumor origin, bridges macrophages to tumor cells.     

应用活体骨髓瘤细胞制备单克隆抗体

【目的】用活体骨髓瘤细胞SP2/0做融合提高融合率制备单克隆抗体,并与常规方法比较效果。【方法】将SP2/0细胞打到8周龄的SPF级BALB/c小鼠皮下,待实体瘤生长到直径达2～3cm时无菌解剖取实体瘤,分离出骨髓瘤细胞进行融合。同时用培养基培养SP2/0细胞进行融合做比较,分两组进行。比较两种方法的融合率以及两种方法制备出来的单克隆抗体的相对亲和力。【结果】做了6次融合,实体瘤融合组融合率为70.4％,常规法融合组44.6％,两种方法制备单抗的相对亲和力均达到1:100000以上。【结论】利用活体实体瘤细胞进行融合能明显提高细胞融合率。     

Inhibition of embryonic neural retina cell-substratum adhesion with a monoclonal antibody

The C1H3 monoclonal antibody recognizes two distinct developmentally regulated cell surface antigens, with molecular masses of 170,000 and 140,000 daltons, in embryonic chick neural retina (Cole, G. J., and Glaser, L. (1984) Proc. Natl. Acad. Sci. U. S. A., in press). In vitro, the 170,000-dalton polypeptide is released by retinal cells into the surrounding culture medium and is present in material sedimentable at 100,000 X g. This pelletable material contains particles designated as adherons (Schubert, D., LaCorbiere, M., Klier, F. G., and Birdwell, G. (1983) J. Cell Biol. 96, 990-998) which promote cell-substratum adhesion of chick neural retina cells. In the present study, evidence is provided that the C1H3 monoclonal antibody inhibits cell adhesion to adheron-coated dishes when bound either to cells or to the adherons. The failure of other monoclonal antibodies, that bind to retinal cells with equal abundance, to disrupt adhesion demonstrates that the effect is specific. These data suggest that the neural-specific 170,000-dalton C1H3 polypeptide is the neural cell-adhesion molecule which is responsible for the ability of adherons to bind to cells.     

A monoclonal antibody identifies a glycoprotein complex involved in cell-substratum adhesion

The monoclonal antibody CSAT has been reported to perturb the adhesion of chick embryo cells to their substratum (Neff et al. [19]). Evidence is presented here that the antigen recognized by this monoclonal antibody is comprised of three membrane glycoproteins. The antigen is released from cells with non-ionic detergent and purified by monoclonal antibody affinity chromatography. When analysed by SDS-PAGE under non-reducing conditions, the antigen resolves into three components of apparent molecular weights 160 000 (band 1), 135000 (band 2), and 110 000 (band 3). Following reduction of each component, bands 1 and 2 migrate at slightly lower apparent molecular weights, while band 3 migrates at a higher apparent molecular weight, suggesting that band 3 has an internal disulfide bond. All three bands differ from one another as determined by peptide mapping and by immunologic cross-reactivity. It is postulated that the three glycoproteins function as a complex that plays a central role in cell-substratum adhesion.     

Regulation of cell adhesion by the cadherin cytoplasmic domain

Juxtacrine cell interactions associated to cadherin-mediated cell-cell adhesion play a major role in the organization and homeostasis of tissues. Here, we review the intracellular molecules and regulations controlling the formation of cell-cell contacts initiated by homophilic interactions of cadherin ectodomain. These regulations involve proteins associated to cadherin cytoplasmic tail, named catenins, their association to the actin cytoskeleton and the stability of these complexes at the cell membrane. The underlying molecular mechanisms, which participate in the formation of dynamic cell-cell contacts, are intensively investigated.     

Localization of human sarcoma with radiolabeled monoclonal antibody — a follow-up report

Summary A group of 16 sarcoma patients with suspected advanced disease were studied with a radiolabeled anti-sarcoma monoclonal antibody (mAb 19–24) in an attempt to localize tumor deposits. All 16 patients received¹²⁵I-mAb 19–24 and then had external-probe analysis and imaging performed. Confirmation of tumor deposits was done at surgery or by autopsy. Tissues were studied in surgical patients when possible and analyzed for radioactivity, and tumor-to-blood ratios ranged from 0.6 to 36.8. In conjunction with the patients previously studied, probe results had an overall sensitivity of 83.3% and an overall specificity of 100%; scintigraphic results showed an overall sensitivity of 78.9% and an overall specificity of 100%. Radiolabeled mAb 19–24 may be developed into a useful tool for clinical immunodetection of sarcoma deposits.This study is supported by American Cancer Society (Illinois Division) grant 88-53