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PubMed1993

[Establishment of monoclonal antibody mNI-11 which induces homotypic cell aggregation of LPS-treated U937 cells].

A mouse monoclonal antibody (mAb), designated as mNI-11, was produced. The reactivity of mNI-11 was assessed by immunofluorescence assay with various cells. Myelomonocytic cell line, U937, and lipopolysaccharide (LPS)-treated U937 (LPS-U937 cells) were found to bind strongly to the mAb. EBV-B cell line and peripheral blood mononuclear cells (PBMCs) were found to bind moderately to it. This mAb strongly induced homotypic cell aggregation of LPS-U937 cells. The mAbs to CD18 and CD54 completely inhibited the LPS-U937 cell aggregation induced by mNI-11. The surface antigens of the U937 and LPS-U937 cells recognized by mNI-11 had a molecular size of 95-97 kDa as determined by immunoblotting analysis.

Animals↗
PubMed1995

Cell aggregation and sedimentation.

The aggregation of cells into clumps or flocs has been exploited for decades in such applications as biological wastewater treatment, beer brewing, antibiotic fermentation, and enhanced sedimentation to aid in cell recovery or retention. More recent research has included the use of cell aggregation and sedimentation to selectively separate subpopulations of cells. Potential biotechnological applications include overcoming contamination, maintaining plasmid-bearing cells in continuous fermentors, and selectively removing nonviable hybridoma cells from perfusion cultures.

Animals↗
PubMed1980

Evaluation of flow cytometric data of human tumours. Correction procedures for background and cell aggregations.

Background signals and aggregation of cells affect flow cytometric DNA measurements to a great and mostly uncontrolled extent. These artefacts appear mainly in investigations of solid tumours leading to significant distortions in the results. Procedures are described that make possible quantitative corrections both of the background and of the cell aggregations.

Brain Neoplasms↗
PubMed1989

[Aggregate cell cultures]

Aggregate cultures are primary cell cultures prepared from dissociated fetal cells. In rotation-mediated culture under rigorously controlled conditions, the isolated cells are able to reaggregate spontaneously, and to form a large number of practically identical spheres. The three-dimensional cell structure in each aggregate provides a maximum of cell-cell interactions, and thus enables the cells to rearrange and to develop in an organotypic fashion. Relatively simple techniques are now available which permit the preparation of aggregate cultures from fetal brain and liver cells. Since they can be grown in a chemically defined medium, and because they mimic several morphogenetic events occurring in vivo, these cultures offer a unique model for developmental studies. Moreover, they may be used as well for routine testing, for example for screening purposes in toxicology, and thus contribute to the reduction of animal experiments.

Journal Article↗
PubMed1997

Neutral glycosphingolipids induce cell-cell aggregation of a variety of hematopoietic cell lines.

Exogenous neutral glycosphingolipids induced homotypic cell-cell aggregation of a variety of hematopoietic cell lines. A mouse cytotoxic T cell line, CTLL-2, was chosen to study the neutral glycosphingolipid-induced aggregation. Among neutral glycosphingolipids tested, galactosylceramide (GalCer) and glucosylceramide (GlcCer) were potent inducers, followed by lactosylceramide (LacCer); globotriaosylceramide (Gb3Cer) and neolactotetraosylceramide (nLc4Cer) were less effective. GalCer that contained a non-hydroxy fatty acid was more efficient than GalCer containing an alpha-hydroxy fatty acid. The minimum concentration of GalCer containing a non-hydroxy fatty acid that induced aggregation was 1 microM, and maximum aggregation occurred at 10-20 microM within 24 h. Cytochalasin B and a mixture of sodium azide and 2-deoxyglucose inhibited the aggregation, whereas cycloheximide, actinomycin D, and colchicine did not. Aggregated and dispersed cells, which were designated as competent cells, re-aggregated in the absence of neutral glycosphingolipids. Anti-GalCer polyclonal antibody inhibited GalCer-induced reaggregation. Furthermore, competent cells bound and aggregated non-competent cells in the absence of neutral glycosphingolipids. Cell-cell aggregatign was similar for CTLL-2 cells and the other hematopoietic cells that were tested. These findings suggest that the neutral glycosphingolipid-induced cell-cell aggregation of CTLL-2 cells was mediated by heterophilic interaction(s) between glycosphingolipids and other cell surface components. These properties are shared by a variety of hematopoietic cell lines.

Animals↗
PubMed1982

Tissue culture of chick embryonic choroidal cells: cell aggregation and pigment accumulation.

Cells originating from the chick embryo choroid were grown in culture for up to 1 month. The cells did not form coherent monolayer colonies but rather formed large transparent cell aggregates surrounded by a random, loose network of cells. The cell aggregates avidly took up and retained pigment granules while the individual cells did not. Choroidal cell aggregates were not similar to the "lentoid" bodies described by Okada and coworkers in cultures from other ocular tissues; they did not develop fiber cells and did not synthesize delta-crystallin. Thus aggregation of ocular cells appears to be a necessary but insufficient step for the formation of a "lentoid" body.

Animals↗
PubMed1978

Biochemical and morphological comparison of two tumour-cell-aggregation factors from rat ascites hepatoma cells.

Two tumour-cell-aggregation factors, derived from rat ascites hepatoma cells, had different antigenicity; one was not absorbed by immunoadsorbent chromatography with anti-rat serum antibody and the other was. Their activities were both lost by digestion with trypsin, but remained unchanged by oxidation with periodate, suggesting the role of the protein portions in their molecules. The potency of the unabsorbed factor was inhibited specifically by alpha-methyl-D-mannoside or D-mannose, while that of the absorbed factor was inhibited specifically by N-acetyl-D-glucosamine, suggesting that these carbohydrates may be concerned with the respective receptor structures at the tumour-cell surface. The unabsorbed factor induced not only cell aggregation (as shown in the form of simple apposition) but also cell adhesiveness characterized by development of intermediate junctions, desmosomes and tight junctions, while the absorbed factor produced only simple apposition, suggesting their functional difference.

Animals↗
PubMed1996

A region of the integrin VLA alpha 4 subunit involved in homotypic cell aggregation and in fibronectin but not vascular cell adhesion molecule-1 binding.

The VLA-4 (alpha 4 beta 1) integrin is involved in the adhesion of cells to fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In order to study alpha 4 structure-function relationships, we have expressed mutated alpha 4 subunit by transfection into VLA-4-negative K562 cells. Substitutions at alpha 4 residues Arg89-Asp90, which show the highest surface probability indexes inside the N-terminal alpha 4/80 fragment, resulted in a reduction in the reactivity of all anti-alpha 4 epitope A monoclonal antibodies (mAbs) tested, compared with the reactivity with anti-alpha 4 epitopes B1, B2, and C mAb, both by transfectant flow cytometry, and by immunoprecipitation and SDS-polyacrylamide gel electrophoresis analysis of transfectant surface-iodinated proteins. In contrast, substitutions at nearby residues, Gln101, Pro102, and Ile105 did not affect the reactivity of any anti-alpha 4 mAb representing the known alpha 4 epitopes. Homotypic cell aggregation triggered by anti-alpha 4 epitope A mAb was prevented in the transfectants expressing mutated alpha 4 Arg89-Asp90Asp residues, while cell aggregation was fully achieved with either anti-alpha 4 epitope B2 or anti-beta 1 mAb. Mutations at alpha 4 residues Gln101, Pro102, and Ile108 did not affect the homotypic cell aggregation of the transfectants expressing these mutations. In addition, the adhesion of mutant Arg89-Asp90 alpha 4 transfectants to the connecting segment-1-containing fibronectin-40 (FN-40) fragment of fibronectin was diminished compared to wild type alpha 4 transfectants, as well as to other mutant alpha 4 transfectants. This adhesion to FN-40 was restored when the activating anti-beta 1 TS2/16 mAb was present in the adhesion assays. In contrast, adhesion to VCAM-1 was not affected by mutations at Arg89-Asp90, nor at Gln101, Pro102, and Ile108 alpha 4 residues. Altogether, these results indicate that alpha 4 residues Arg89 and Asp90 are included in a region involved in homotypic cell aggregation, as well as in adhesion to FN-40, but not to VCAM-1.

Animals↗
PubMedDate not supplied

The growth factor inhibitor suramin reduces apoptosis and cell aggregation in protein-free CHO cell batch cultures.

We have previously shown that Chinese hamster ovary (CHO) cells capable of growing in medium free of exogenous proteins die by apoptosis during all stages of a batch culture (Zanghi et al., 1999). On the basis of the hypothesis that extracellular death factors might be important in apoptosis under these conditions, we examined the effect of the growth factor inhibitor and antitumor agent suramin on CHO cell growth and apoptosis in serum-free culture. Suramin protected against apoptosis during exponential growth, as indicated by the absence of DNA laddering and an increase in cell viability from roughly 70% to above 95%. Suramin also effectively dispersed cell aggregates so that single-cell suspension culture was possible. However, suramin did not protect against apoptosis during the death phase, in contrast to serum, suggesting that antiapoptotic factors in the serum remain to be discovered. The increased viable cell yield following suramin supplementation resulted in a 40% increase in product yield, based on results with cells expressing recombinant secreted alkaline phosphatase. Polysulfated compounds dextran sulfate and polyvinyl sulfate worked nearly as well as suramin in dispersing cell clumps and increasing viable cell yield, which implies that suramin's high sulfate group density may be responsible for its effects in cell culture. In addition, suramin was beneficial for long-term adaptation of CHO cells to protein-free media suspension culture, and the compound was synergistic with insulin in accelerating this adaptation time.

Animals↗
PubMed1979

Production of plasminogen activator and inhibition of embryonic cell aggregation by cultured human normal and neoplastic cells.

The plasminogen activator (PA) production and the capacity to inhibit embryonic neural retina (NR) cell aggregation by human normal and neoplastic cell lines have been studied. The PA production was detected by both iodinated fibrin and casein lysis assays, and by changes in cell morphology at the presence of activated PA, using dog serum. Since the casein lysis assay and morphological changes proved to be less sensitive than 125I-fibrin lysis assay, a good correlation between these three assays could be observed provided that PA production measured by fibrinolysis exceeded 10--20%. The neoplastic cell lines exhibited the PA production to quite a large extent. The highest fibrinolytic activity (78%) was found in the case of bladder carcinoma cells T24, while the B-5GT cells from giant cell tumor of bone failed to produce any detectable amount of the PA. The cells from synovial sarcoma and both glioma lines exhibited fibrinolytic activity of about 10% and four sarcoma cell lines over the range 20--50%. Out of 13 normal cell lines tested, 7 were negative or exhibited very low fibrinolysis not exceeding 3% of total radioactivity. Four cell lines derived from kidneys, lungs, intestines, and from mixed embryonic tissues showed a marked fibrinolytic activity of about 10--37%, a slightly elevated fibrinolysis being found in embryonic lung cells LEP and cells from fetal skin tissue only at the presence of dog serum. The fibrinolysis detected in the neoplastic cloned cell populations showed considerable differences in the PA production between individual cell clones isolated from the same parental cell line. Unlike the normal fibroblastic cells B-41FB derived from bone, all neoplastic cell lines tested possess the capability to inhibit embryonic NR cell aggregation significantly. The results suggest the effect not to be dependent upon the PA production.

Animals↗
PubMed1997

A novel human monoclonal antibody rapidly induces homotypic cell aggregation and promotes antibody-secreting activity by human B lymphoblastoid cell line IM-9.

A human monoclonal antibody (mAb), designated mNKES, was generated by fusing B cells isolated from an enlarged cervical lymph node of a patient with a carotid body tumor (CBT), with human myeloma cell line KR-12 (6TG). The reactivity of mNKES was tested by the indirect immunofluorescence method. The antigen defined by mNKES was expressed on Burkitt's lymphoma cell lines Raji, Daudi, and Ramos and on B lymphoblastoid cell line IM-9. In addition, mNKES reacted with T cells stimulated with recombinant interleukin-2 (rIL-2) obtained from normal healthy donors. However, mNKES did not react with normal resting human T, B, or adherent cells (monocytes/macrophages). When the reactivity of mNKES and mouse mAbs recognizing the human adhesion-associated antigen (CD10, CD11a, CD11b, CD11c, CD14, CD16, CD18, CD23, CD28, CD29, CD31, CD43, CD44, CD45RA, CD50, CD54, CD58, CD80, CD102, CD106, and HLA class I, and HLA class II antigen) with various cell lines was compared, mNKES reactivity was found to be unique, not resembling that of any of these mouse mAbs. Interestingly, mNKES specifically and rapidly (within 2 hr) induced homotypic cell aggregation of IM-9 cells. This mNKES-induced cell aggregation was completely blocked by the addition of EDTA and when incubated at 4 degrees C. The mAbs reactive with CD11a/CD18 (leukocyte function-associated antigen-1; LFA-1) and CD54 (intercellular adhesion molecule-1; ICAM-1) completely blocked the IM-9 cell aggregation induced by mNKES, and induction of IM-9 cell aggregation by mNKES was significantly blocked in the presence of the protein kinase C inhibitors sphingosine and H-7 and completely blocked by cytochalasin B and cytochalasin D, which inhibit microfilament formation. Regarding biological function, IM-9 cells bearing surface IgG (sIgG) effectively promoted IgG-secreting activity underlying the homotypic cell aggregation induced by mNKES. The surface antigen recognized by mNKES has a molecular size of about 55 kDa, as determined by immunoblotting analysis. These findings indicate that mNKES recognizes a novel adhesion-associated antigen distinct from any previously reported adhesion-associated antigens in terms of pattern of cellular distribution and biological function and that mNKES is the first human mAb found that rapidly induces homotypic cell aggregation and effectively promotes the IgG-secreting activity of human B lymphoblastoid cell line IM-9.

Adolescent↗
PubMed1982

Teratocarcinoma cell adhesion: identification of a cell-surface protein involved in calcium-dependent cell aggregation.

Teratocarcinoma cells have a Ca2+-dependent cell-cell adhesion site (t-CDS) that is unique in being inactivated with trypsin in the absence of CA2+ but not in the presence of Ca2+. Fab fragments of antibodies raised against teratocarcinoma F9 cells dissociated by treatment with trypsin and calcium (anti-TC-F9) inhibit the aggregation of teratocarcinoma cells mediated by t-CDS. This inhibitory effect of Fab is removed when anti-TC-F9 is absorbed with F9 cells treated with trypsin and calcium (TC-F9), but not when it is absorbed with F9 cells treated with trypsin and EGTA (TE-F9). Comparisons of cell-surface antigens reactive to anti-TC-F9 in TC-F9 cells with those in TE-F9 cells reveal that only one component, with an approximate molecular weight of 140,000 (p140), is detected specifically on the surface of TC-F9 cells. When TC-F9 cells are retrypsinized in the absence of CA2+, a substance with an approximate molecular weight of 34,000 (p34) is released that can neutralize the aggregation-inhibitory effect of the Fab. This p34 interferes with the immunoprecipitation of p140 with anti-TC-F9, suggesting that p34 is a tryptic fragment of p140. Anti-TC-F9 Fab causes the dissociation of the monolayers of teratocarcinoma cells. This effect is removed by absorption of the Fab with p34 as well as with TC-F9 cells, but not with TE-F9 cells. These results suggest that p140 is essential for the function of t-CDS, and that this is an actual cell-adhesion molecule active in the establishment of monolayers of teratocarcinoma cells.

Antigens, Surface↗
PubMed1995

CD23 molecule acts as a galactose-binding lectin in the cell aggregation of EBV-transformed human B-cell lines.

Epstein-Barr virus (EBV)-transformed human B-cell lines, L-KT9 and DH3 cells express CD23 antigen, and grow in a mixture of single and aggregated cells. The CD23 molecule has high amino acid sequence homology with C-type lectin and recently we have shown that the solubilized CD23 molecule can really interact with galactose residues on glycoproteins. In this study, therefore, we tested whether CD23 antigen on the cell surface really acts as a galactose-binding lectin in the aggregation of these cells. The EBV-transformed cells (L-KT9) were separated into an aggregated-cell-rich fraction and a single-cell-rich fraction. Aggregated cells disaggregated after removal of galactose by beta-galactosidase treatment, whereas single cells made large aggregation on sialidase treatment, and this aggregation was inhibited in the presence of asialo-fetuin. On the other hand, naturally aggregated cells become single cells with anti-CD23 monoclonal antibody (mAB) as well as the soluble form of CD23, but not with anti-CD21 mAB. In addition, L-KT9 and DH3 cells bound to asialo-fetuin-coupled Sepharose (ASF-Sepharose) and this binding was significantly inhibited by pre-treatment of cells with anti-CD23, but not with anti-CD21 or other anti-adhesion molecules. From these results, we conclude that the naturally aggregated state of EBV-transformed cells occurs mainly through the interaction of CD23 as a lectin molecule and galactose residues as its ligand.

Antibodies, Monoclonal↗
PubMed1982

Cell-surface discoidin in aggregating cells of Dictyostelium discoideum.

Both discoidin I and discoidin II have been detected on the surface of aggregating (10 h developmental stage) cells of Dictyostelium discoideum NC4 by radioiodination of the cell-surface followed by immunoprecipitation and sodium dodecyl sulphate/polyacrylamide-gel-electrophoretic analysis. Approx. 92% of cell-surface discoidin I and 72% of cell-surface discoidin II can be eluted with 0.5 M-galactose, showing that most of each endogenous lectin is not present as integral membrane protein but rather is bound to cell-surface discoidin receptors. Two-dimensional polyacrylamide-gel-electrophoretic analysis of discoidin I suggests that the native tetramer may be a hetero-multimer composed of both Ia and Ib subunits. Cell-surface discoidin I also contains both types of subunit, but it is not clear whether both subunits have corresponding cell-surface receptors.

Carrier Proteins↗
PubMed1997

Mouse homolog of poliovirus receptor-related gene 2 product, mPRR2, mediates homophilic cell aggregation.

Poliovirus receptor (PVR) is a cell surface glycoprotein that belongs to the immunoglobulin superfamily. Although MPH was initially reported as the mouse homolog of human PVR, recent data strongly suggest that MPH is the mouse homolog of human PRR2, a PVR-related gene 2 product, and not that of human PVR. Thus MPH is renamed mPRR2 in this study. Physiological functions of the PVR-related gene products have not been elucidated, although PVR has been well characterized as the poliovirus receptor. In this study, a possible function of mPRR2 (MPH), which is not a functional receptor for poliovirus, was investigated. Mouse L cells expressing mPRR2 were prepared. Those mouse cells showed a higher activity of cell aggregation than the parental mouse L cells. Enhancement of cell aggregation was also observed for insect Sf9 cells infected with recombinant baculovirus carrying mPRR2 cDNA. On the other hand, L cells expressing human PVR or monkey PVR (AGM alpha1 or AGM alpha2) did not show increased cell aggregation. The cell aggregation activity of L cells expressing mPRR2 was inhibited by the addition of anti-mPRR2 monoclonal antibodies or a soluble mPRR2 molecule produced by the baculovirus expression system. An immunofluorescence study revealed that mPRR2 protein was localized to the cell-cell contact sites between cells expressing mPRR2. A similar localization of mPRR2 was observed for intrinsic mPRR2 molecules of the mouse neuroblastoma cell line NS20Y. The contact site-specific localization of mPRR2 was not observed on the border between mPRR2-expressing and nonexpressing HeLa cells. Furthermore, mPRR2 proteins directly bound to each other in vitro. mPRR2 was detected on various types of cultured cells of mouse origin and in various mouse tissues. These results suggest that mPRR2 is an intercellular adhesion molecule with a homophilic binding manner.

Animals↗
PubMed1978

Embryonic tissues as elasticoviscous liquids. I. Rapid and slow shape changes in centrifuged cell aggregates.

Certain embryonic tissue masses and cell aggregates behave like deformable solids during brief experimental manipulations but like viscous liquids in long-term organ cultures. To investigate these seemingly paradoxical physical properties, we have mechanically deformed cell aggregates derived from several embryonic chick organs by centrifuging them against solid substrata. Aggregate shapes during brief centrifugation were observed directly in a microscope-centrifuge. In addition, techniques were devised for fixing cell aggregates during prolonged centrifugation. Evidence presented here shows that these fixative-injection procedures accurately preserve the prefixation shapes of living centrifuged aggregates. According to a simple viscous-liquid model for cell aggregates, cohering cells slide past one another when external forces and/or tissue surface tensions cause gradual rearrangements in aggregate conformations. In earlier experiments, 2 types of behaviour predicted from this model were confirmed for several embryonic chick tissues subjected to prolonged centrifugation. First, initially flat aggregates rounded up against the centrifugal force to adopt the same shapes that initially round aggregates reached by flattening. Second, the relative roundness of centrifuged aggregates of different tissues at shape equilibrium correlated with the relative positions that these tissues assumed when they were combined in aggregate-spreading and cell-sorting experiments. By contrast, the brief centrifugation experiments described here provide some support for a simple elastic-solid model in which aggregate shape changes are accompanied by cell deformations rather than cell redistributions. In particular, since cell migration tends to occur quite slowly, the very rapid aggregate flattening observed during the first few minutes of centrifugation presumably requires cell stretching. Moreover, since they do also round up very rapidly following brief centrifugation, these aggregates exhibit considerable elasticity that presumably reflects the swift relaxation of cell stretching as the centrifugal force is removed. Athough both elastic-solid and viscous-liquid properties can be recognized in cell aggregates, we note that, in the prolonged centrifugation experiments described here, rapid initial aggregate flattening is followed by much more gradual, continued flattening. Similarly, after prolonged centrifugation, rapid partial aggregate rounding-up is also followed by much more gradual, continued rounding-up during subsequent culture at Ig. Such rapid-then-slow shape changes contradict both simple elastic-solid and simple viscous-liquid models for cell aggregates. These bimodal shape changes are instead consistent with both compound-viscoelastic-solid and elasticoviscous-liquid models for cell aggregates, although only the latter can also account for long-term liquid-like aggregate behaviour...

Animals↗
PubMed1985

[3H]N-Methylscopolamine binding to muscarinic receptors in intact adult rat brain cell aggregates.

Intact brain cell aggregates were dissociated from adult rat brains, by a simple sieving technique, and were used to study the binding characteristics of [3H]N-methylscopolamine to muscarinic acetylcholine receptors. The magnitude of binding of this ligand was related linearly to the amount of cell protein in the binding assay, with a high ratio of total to nonspecific binding. In addition, specific binding showed saturability and high affinity. Muscarinic receptor antagonists displaced specific [3H]N-methylscopolamine binding according to the law of mass-action, while it was possible to resolve displacement curves using receptor agonists into high- and low-affinity components. The results are discussed in terms of the usefulness of dissociated intact rat brain cells in studying muscarinic acetylcholine receptors in the central nervous system.

Animals↗