Microtubule disruption does not prevent intracellular transport and secretory processes of cultured fibroblasts

The effect of microtubule disruption on intracellular protein translocation and secretion of cultured human fibroblasts was studied. Experiments with fluorochrome-labeled wheat germ agglutinin showed that treatments with demecolcine or vinblastine sulfate rapidly brought about dispersal of Golgi organization. Similarly, monoclonal tubulin antibodies revealed in immunofluorescence a complete disappearance of microtubules under these conditions. Cells exposed to demecolcine or vinblastine sulfate appeared to secrete both fibronectin and other polypeptides similar to the control cells, as judged by electrophoretic analysis of the culture medium. In line with this, immunofluorescence studies of the cells, treated with the antimitotic drugs and then exposed to puromycin, showed a rapid depletion of intracellular fibronectin and collagen type III-specific staining. On the contrary, cells exposed first to monensin and then to demecolcine or vinblastine sulfate and puromycin, showed an accumulation of these proteins in vesicles in the Golgi region. The results suggest that in cultured fibroblasts microtubules do not have a direct permissive role in the intracellular translocation of the secreted proteins although they are involved in the maintenance of the Golgi apparatus. The secretory processes, however, appear to continue also in cells with dispersed Golgi organization and lacking microtubules.     

Human-human hybridomas secreting antibodies specific to human lung carcinoma

Summary Human Namalwa cells were screened in serum-free medium and in 6-thioguanine, then fused with human lymphocytes from lymph nodes of lung adenocarcinoma cancer patients. Extensive testing using 14 lung cancer cell lines, 11 other cancer cell lines and 4 normal fibroblast lines identified monoclonal antibodies produced by 4 hybridoma clones that reacted specifically with lung adenocarcinoma cells. These monoclonal antibodies also reacted with lung adenocarcinoma tissues and not normal tissues or erythrocytes of any blood type. These hybridoma clones grew and stably secreted the antibodies in serum-free medium as well as in serum-containing medium. Editor's Statement Identification of monoclonal antibodies that recognize human lung adenocarcinoma cells with reasonable specificity represents a potentially important development that may prove useful in diagnosis and therapy of neoplastic disease. The selection procedures and methods for propagation of the human-human hybridomas described in this paper also represent some novel approaches that may be of general application. David W. Barnes     

Hybridoma cells containing intracellular anti-ricin antibodies show ricin meets secretory antibody before entering the cytosol

Hybridoma cells which synthesize monoclonal antibodies (mAb) that block ricin toxicity were 50-300-fold resistant to ricin compared with other hybridomas. Two of the mAb blocked two isozymes of ricin, D and E, to different and opposite extents, and the hybridoma cell resistance to the two forms of ricin closely corresponded with the mAb reactivity. The hybridoma cell resistance to ricin was therefore due to the binding activity of the mAb produced by the cells. Neither rabbit polyclonal antibodies, which neutralized extracellular anti-ricin mAb, nor quantitative removal of hybridoma cell surface IgG with papain affected the cellular resistance to ricin. Therefore, neither extracellular or cell surface antibodies contributed to the resistance of the hybridoma cells. In contrast, inhibition of protein synthesis by cycloheximide or puromycin, which selectively decreased levels of intracellular secretory IgG, decreased the hybridoma cell resistance to ricin. We conclude that intracellular mAb, synthesized de novo for subsequent secretion, block ricin toxicity. Ricin therefore must meet intracellular secretory antibodies before reaching the cytosol. The monoclonal antibodies can also be used to study toxin function within intracellular compartments. An antibody specific for the galactose-binding site of ricin blocks ricin intracellularly, showing that the ricin galactose-binding activity is required in an intracellular compartment for transport of ricin A chain to the cytosol.     

Manipulation of heterogeneous hybridoma cultures for overproduction of monoclonal antibodies.

In searching for ways to manipulate heterogeneous hybridoma cell cultures (ATCC HB124) to obtain increased production of monoclonal antibodies (IgG2a), we have selected for a higher secreting but slower growing subpopulation using the level of fluorescent surface-associated antibodies and a fluorescence-activated cell sorter. Cell surface fluorescence was found to be correlated with specific antibody secretion rate over the short term but not with intracellular antibody content. Also, the specific secretion rate of a heterogeneous population of hybridoma cells grown in batch culture has been shown to be inversely correlated with an increase in either the initial cell concentration or the medium antibody concentration. Several experiments suggest that an upper limit exists for medium antibody concentration, above which antibody is degraded at the same rate at which it is produced. Should other cell lines behave similarly, strategies for overproduction of monoclonal antibodies suggested herein could be profitably used in industry.     

Modulation of hybridoma cell growth and antibody production by coating cell culture material with extracellular matrix proteins

The influence of coating polystyrene tissue culture plates with different proteins on murine hybridoma cell growth and antibody production was investigated. Fibronectin, collagen I, bovine serum albumin and laminin were used to coat NUNC^® and COSTAR^® cell culture plates. Cell number and antibody concentration in culture fluids were quantified as indicators for cell viability, proliferation and productivity. Adhesive behaviour, morphology, expression of surface receptors of hybridoma cells and the presence of tyrosine-phosphorylated proteins in cell lysates were characterized by cell adhesion experiments, microscopy, flow cytometry and Western Blot analysis.It was shown that coatings with fibronectin (0.2 μg/ml) lead to a substantial improvement of cell growth by 50–70% and an increase of monoclonal antibody production by 100–120%.Collagen I coatings showed an improvement in cell growth by 30–70% and by 60% for the production of monoclonal antibodies. Coatings with BSA and laminin had minor effects on these parameters. It was found that the hybridoma cell lines used in this study did not express the α₂-chain of the α₂β₁-integrin, which is responsible for binding to collagen and laminin.However, the presence of β₁-integrin on the cell surface was shown, which should enable hybridoma cells to bind fibronectin. We propose, therefore, that fibronectin adsorption to cell culture materials may be a promising approach to enhance the production of monoclonal antibodies by cultivated hybridoma cells.     

Extracellular matrix of cultured glial cells: selective expression of chondroitin 4-sulfate by type-2 astrocytes and their progenitors

We have studied the extracellular matrix composition of cultured glial cells by immunocytochemistry with different monoclonal and polyclonal antibodies. Double immunofluorescence experiments and metabolic labeling with [3H]glucosamine performed in different types of cerebellar and cortical cultures showed that bipotential progenitors for type-2 astrocytes and for oligodendrocytes (recognized by the monoclonal antibody LB1 at early stages of their development) synthesize chondroitin sulfate (CS) and deposit this proteoglycan in their extracellular matrix. The distribution of the various [3H]glucosamine-labeled glycosaminoglycans between the intracellular and the extracellular space was different. CS was present both within the cells and in the culture medium, although in different amounts. Bi-potential progenitors became also O4-positive during their development in vitro. At the stage of O4-positivity they were still stained with antibodies against CS. However, when the progenitor cells were maintained in serum-free medium and differentiated into Gal-C-positive oligodendrocytes, they became CS-negative. In the presence of fetal calf serum in the culture medium, the bipotential progenitors differentiated into GFAP-positive type-2 astrocytes. These cells still expressed CS: their Golgi area and their surface were stained with anti-CS antibodies. Staining with monoclonal antibodies specific for different types of CS (4-sulfate, 6-sulfate, and unsulfated) revealed that both bipotential progenitors and type-2 astrocytes synthesized only chondroitin 4-sulfate. Type-1 astrocytes were negative for both the polyclonal and the monoclonal anti-CS antibodies. Finally, type-2 astrocytes and their progenitors were weakly stained with anti-laminin antibodies and unstained with anti-fibronectin. Type-1 astrocytes were positive for both anti-laminin and anti-fibronectin antibodies and appeared to secrete fibronectin in the extracellular space.     

Interstitial collagen in alcoholic human liver

The occurrence and intensity of staining for specific antibodies against the aminoterminal propeptide of type III procollagen (PIIIP), which is indicative of the synthesis and the degradation of that collagen type, was studied in sections from normal and alcoholic livers and compared with serum PIIIP levels, serum antipyrine clearance, fibronectin distribution and morphology as revealed by conventional stains and electronmicroscopy. Positive staining for PIIIP and fibronectin was observed in the perisinusoidal space of the normal liver and in portal tracts. In alcohol-induced fatty liver positive staining increased around the central veins, in alcoholic hepatitis increased staining reaction was seen to a limited extent in areas of cell injury. Extensive reticulin and PIIIP-positive areas were found in the periportal interstitium of the cirrhotic livers and in large fibrotic areas extending into the surrounding parenchyma in cases of active disease. The results show a distinct relationship between collagen type III metabolism, morphologically detectable hepatic injury and liver cell function tests, with tissue deposition occurring later in the disease process than biochemically detectable serum collagen levels and signs of altered liver cell function.     

Type VI collagen in extracellular, 100-nm periodic filaments and fibrils: identification by immunoelectron microscopy

Filaments and fibrils that exhibit a 100-nm axial periodicity and occur in the medium and in the deposited extracellular matrix of chicken embryo and human fibroblast cultures have been tentatively identified with type VI collagen on the basis of their similar structural characteristics (Bruns, R. R., 1984, J. Ultrastruct. Res., 89:136-145). Using indirect immunoelectron microscopy and specific monoclonal and polyclonal antibodies, we now report their positive identification with collagen VI and their distribution in fibroblast cultures and in tendon. Primary human foreskin fibroblast cultures, labeled with anti-type VI antibody and studied by fluorescence microscopy, showed a progressive increase in labeling and changes in distribution with time up to 8 d in culture. With immunoelectron microscopy and monoclonal antibodies to human type VI collagen followed by goat anti-mouse IgG coupled to colloidal gold, they showed in thin sections specific 100-nm periodic labeling on extracellular filaments and fibrils: one monoclonal antibody (3C4) attached to the band region and another (4B10) to the interband region of the filaments and fibrils. Rabbit antiserum to type VI collagen also localized on the band region, but the staining was less well defined. Control experiments with antibodies to fibronectin and to procollagen types I and III labeled other filaments and fibrils, but not those with a 100-nm period. Heavy metal-stained fibrils with the same periodic and structural characteristics also have been found in both adult rat tail tendon and embryonic chicken tendon subjected to prolonged incubation in culture medium or treatment with adenosine 5'-triphosphate at pH 4.6. We conclude that the 100-nm periodic filaments and fibrils represent the native aggregate form of type VI collagen. It is likely that banded fibrils of the same periodicity and appearance, reported by many observers over the years in a wide range of normal and pathological tissues, are at least in part, type VI collagen.     

Disialoganglioside GD2 distributes preferentially into substrate- associated microprocesses on human melanoma cells during their attachment to fibronectin

Human melanoma cells (M21) actively attach and spread on a fibronectin substrate. Indirect immunofluorescence assays with specific monoclonal antibodies directed to the disialoganglioside GD2, the major ganglioside expressed on M21 melanoma cells, indicate that during the cell attachment process this molecule redistributes into microprocesses that make direct contact with the fibronectin substrate. Scanning and transmission immunoelectron microscopic studies with anti-GD2 monoclonal antibodies and immuno-gold staining demonstrate that GD2 preferentially localizes into substrate-associated microprocesses that emanate from the plasma membrane of the M21 cells. Staining with monoclonal antibodies directed to other melanoma surface antigens fails to demonstrate a similar distribution pattern on these cells. Direct evidence is provided that GD2 is involved in M21 cell attachment to fibronectin, since treatment of these cells with anti-GD2 monoclonal antibodies causes cell rounding and detachment from a fibronectin substrate. Moreover, scanning electron microscopy demonstrates that this loss of attachment of fibronectin is characterized by a perturbation of the cell attachment-promoting microprocesses that in the presence of these antibodies lose contact with the fibronectin substrate.     

Continuous production of large amounts of monoclonal immunoglobulins in hollow fibers using protein-free medium

The performance of a protein-free medium was compared in culture flasks with a serum-supplemented medium and with a serum free medium in terms of cell growth and monoclonal antibody production by a murine hybridoma. We present results of continuous production in hollow fiber culture systems using serum-free medium and protein-free medium. In protein-free medium, it has been possible to produce large quantities of monoclonal antibody with a productivity similar to that obtained in serum-free medium. After a two steps purification process, monoclonal antibodies were characterized by SDS-PAGE, High Performance Size Exclusion Chromatography and Free Solution Capillary Electrophoresis. SDS-PAGE and high performance chromatography analysis have showed that purified monoclonal antibodies produced in serum-free medium or protein-free medium were similar. Furthermore, Capillary Electrophoresis characterization revealed that both MAbs were constituted by three isoforms with equivalent electrophoretic mobilities.Abbreviations CHES 2-(N-Cyclohexylamino)ethane-sulfonic acid - ECS Extracapillary Space - FSCE Free Solution Capillary Electrophoresis - HPSEC High Performance Size Exclusion Chromatography - ICS Intracapillary Space - MAb Monoclonal Antibody - PFM Protein-Free Medium - SFM Serum-Free Medium - SSM Serum-Supplemented Medium     

A new serum-free medium for monoclonal antibody production

A new serum-free, defined-protein, medium for the growth of murine hybridoma cells and the production of monoclonal antibodies has been developed. Designated WRC 935 medium, this formulation supports the growth of hybridoma cells in higher numbers, and promotes better cell viabilities and increased monoclonal antibody levels compared to growth in DMEM supplemented with 10% fetal bovine serum or in a DMEM/F-12 serum-free mixture. In suspension cultures, WRC 935 medium typically promoted cell growth to densities over two million cells per milliliter. This medium also promoted the rapid growth of cells following their transfer from liquid nitrogen storage. WRC 935 medium is especially useful for high density cell culture production methods using hollow-fiber bioreactors. Hollow-fiber bioreactors using this medium produced antibody at an average rate of 11 mg/day, and the antibody concentration ranged from 10 to 40 mg/ml.     

Production and preliminary characterization of monoclonal antibodies to rat plasma fibronectin

We have produced several monoclonal antibodies which appear to be directed against different antigenic determinants of rat plasma fibronectin. Fibronectin was purified from rat plasma by affinity chromatography on gelatin-Sepharose and arginine-Sepharose columns. Mice were immunized and hybridomas were prepared by fusing spleen cells with Sp2/0-Ag14 myeloma cells using poly(ethylene glycol). Three hybridomas (RFN1, RFN2 and RFN3) were selected for characterization. All are IgG molecules, one is IgG2a, one IgG2b and one IgG1. Titers of ascites fluids produced using these hybridomas range from 102 400 to greater than 409 600. The antibodies cross-reacted to different degrees with human fibronectin. Rat fibronectin was radioactively labeled and cleaved using human polymorphonuclear leukocyte elastase. Four major peptides, Mr approx. 160 000, 140 000, 60 000 and 30 000 were produced. Each of the hybridoma antibodies immunoprecipitated different elastase peptides. RFN1 precipitated the Mr 160 000 peptide, RFN2 precipitated the Mr 160 000 and the Mr 140 000 peptide and RFN3 precipitated the Mr 60 000 peptide as well as low molecular weight material migrating at the buffer front. These antibodies will be useful in studies of structure/function relationships of rat fibronectin.     

Generation and characterization of mouse monoclonal antibodies to the myelin-associated glycoprotein (MAG)

A panel of mouse monoclonal antibodies to rat and human myelin-associated glycoprotein (MAG) was developed. Normal mice were unresponsive to rat MAG, and successful immunization with rat MAG was only achieved in autoimmune NZB mice. By contrast, all strains of mice were responsive to human MAG. The monoclonal antibodies developed differ with respect to immunoglobulin type, their specificity for human and/or rat MAG, and their recognition of protein or carbohydrate epitopes in MAG. In general, the antibodies that react with the protein backbone recognize both rat and human MAG, whereas a large number of the monoclonal antibodies recognize a carbohydrate determinant in human MAG that is not in rat MAG. Immunocytochemical staining of adult human spinal cord with the monoclonal antibodies resulted in periaxonal staining of myelin sheaths, similar to that produced by well-defined, rabbit, polyclonal anti-MAG serum. In addition, the antibodies recognizing, carbohydrate determinants in human MAG strongly stained oligodendrocyte cytoplasm. These monoclonal antibodies will be of value for the further chemical and biological characterization of MAG.     

Applications of monoclonal antibodies to human follicle-stimulating hormone in enzyme immunoassays

Monoclonal antibodies against human follicle-stimulating hormone (hFSH) were generated by using an improved hybridoma technique with a semisolid medium in methylcellulose for initial cloning. The generated monoclonal antibodies were characterized with respect to their subunit and epitope specificity as well as cross-reactivity to other glycoprotein hormones. Monoclonal antibodies of high affinity and high specificity to hFSH were finally selected for applications in sandwich enzyme immunoassay. The monoclonal antibody specific to the alpha-subunit of FSH was coated on microtiter wells and served as the first antibody. The other high-affinity monoclonal antibody specific to beta-subunit of FSH was labeled with horseradish peroxidase and served as the second antibody. This immunoassay can be performed within 70 min at room temperature and has a minimum sensitivity of 2 mIU/ml for serum sample.     

Monoclonal antibodies against 5'-nucleotidase from chicken gizzard. Evidence for species and tissue specific differences of 5'-nucleotidase

5'-Nucleotidase from chicken gizzard smooth muscle was purified to homogeneity and used as immunogen for generating monoclonal antibodies. From about 150 positive clones nine IgG producing hybridoma cell lines have been selected for further characterization and antibody preparation. The resulting antibodies bind 5'-nucleotidase from chicken smooth muscle, chicken skeletal muscle, and chicken heart muscle but not the enzyme from chicken liver or rat liver. It could clearly be demonstrated that the nine antibodies recognize different antigenic determinants. Four of these antibodies are strong inhibitors of the AMPase activity of 5'-nucleotidase. One antibody is a weak inhibitor and four other antibodies have no effect on its enzymic activity. One of the monoclonal antibodies was used for immunoaffinity purification of 5'-nucleotidase from chicken heart muscle and chicken skeletal muscle. Pure and active enzymes could be isolated from detergent extracts in one step with a 10 to 20-fold higher yield compared to classical purification procedures. The subcellular distribution of 5'-nucleotidase in chicken gizzard was investigated using indirect immunofluorescence. We found a staining of the plasma membrane of smooth muscle cells and endothelial cells by all of the nine antibodies with variations in the staining intensity.     

Cellular localization of insulin-degrading enzyme in rat liver using monoclonal antibodies specific for this enzyme

Although insulin-degrading enzyme (IDE) has been implicated in the intracellular degradation of insulin, the cellular localization of this enzyme is still controversial. In the present study, we have examined the cellular localization of IDE in the rat liver by three different techniques using monoclonal antibodies. First, direct immunohistochemical staining of rat liver with one of the monoclonal antibodies revealed that IDE immunoreactivity mainly exists in parenchymal cells, especially in the vicinity of the portal tract and also in the epithelium of the bile duct under light microscopy. In the electron microscopic study, IDE immunoreactivity was found in the cytoplasm near the rough endoplasmic reticulum but not in the plasma membrane, nucleus, or mitochondria. Second, immunoblotting analysis of the subcellular fraction in rat liver showed that the monoclonal antibody specifically reacted with a single polypeptide in the cytosolic fraction, of apparent Mr 110,000, which was consistent with the Mr of IDE. However, a polypeptide band corresponding to IDE could not be observed in the plasma membrane, mitochondrial, or lysosomal fraction. Third, IDE was only detectable in the cytosolic fraction by sandwich radioimmunoassay using two monoclonal antibodies. These results all suggest that IDE is a cytosolic enzyme.     

Fibronectin dependence of the contraction of collagen lattices by human skin fibroblasts

The role of fibronectin in the contraction of collagen lattices by human skin fibroblasts has been investigated. Incubation of lattice cultures in Dulbecco's modified Eagle's medium supplemented with increasing concentrations of non-dialysed or dialysed fetal calf serum demonstrated that the rate of contraction was dependent on non-dialysable serum components. The suppression of contraction observed when fibronectin was eliminated from serum, either by affinity chromatography on gelatin-agarose columns or by precipitation with anti-fibronectin antibodies, showed that fibronectin is critical for the contraction. When collagen lattices were incubated in a serum-free culture medium totally devoid of fibronectin, no contraction occurred. When fibronectin was added to this medium, their contraction was correlated with the concentration of fibronectin added. The contraction was inhibited by cycloheximide, tunicamycin, and monensin. These results demonstrate that the contraction of collagen lattices by human skin fibroblasts is dependent on fibronectin, and that other protein factors synthesized by the cells or contained in serum are also necessary.     

Modified antigen-binding of human antibodies with glycosylation variations of the light chains produced in sugar-limited human hybridoma cultures

Summary We have characterized the effects of serum andN-acetylglucosamine in a glucose-deprived condition on the glycosylation of antibody light chains, as well as the resulting biological properties of those antibodies. We have chosen for our investigation the human hybridoma lines producing monoclonal antibodies reactive to lung adenocarcinoma. Each antibody possess aN-glycosylated carbohydrate chain in the hypervariable region of the light chains. When the cell lines were grown in the absence of glucose, variant light chains with varying molecular masses were found to be secreted. Analysis of these light chains produced in a glucose-deprived condition revealed that the changed molecular-mass of the variant light chains is due to different glycosylation. Addition ofN-acetylglucosamine or fetal calf serum to the glucose-free medium led to the creation of other light chains that exhibit increased antigen binding activity.     

Monoclonal antibody storage conditions, and concentration effects on immunohistochemical specificity

Monoclonal antibodies against a 24,000 dalton intracellular estrogen-regulated protein in human breast cancer cells were used to study storage conditions and the effects of monoclonal antibody concentrations on immunohistochemical antigen localization. Both hybridoma supernatants and ascites fluid obtained from mice injected with hybridoma cells were used as sources of monoclonal antibodies; the monoclonal antibodies in the ascites fluid were concentrated and purified. Both antibody preparations were stored at 4, -20, or -70 degrees C and periodically tested for activity at these storage conditions. There was no difference in activity for the antibodies between storage at -20 and -70 degrees C. However, when highly diluted antibody was stored at 4 degrees C, the activity was lost within 2 weeks if carrier proteins were not added. These monoclonal antibodies were applied to immunohistochemical staining of different mouse and human tissues processed for routine paraffin sections, using the avidin-biotin-peroxidase procedure. A monoclonal antibody of unrelated specificity was used as control. When these antibodies were used at high concentrations, all the different tissues examined were immunostained. With reduction of the antibody concentration, an immunohistochemical dissection of the tissues was seen until specific immunostaining was reached. When even more highly diluted monoclonal antibody was used, heterogeneity in the staining pattern became very high. On the basis of these results, certain immunohistochemical criteria are proposed for the selection of the optimum concentration of monoclonal antibodies for specific antigen detection.     

Fibronectin in cultured rat keratinocytes: distribution, synthesis, and relationship to cytoskeletal proteins

The aim of this study was to investigate whether epidermal cells can synthesise fibronectin and whether the distribution of this glycoprotein is related to the adhesion and cytoskeletal organisation of these cells. The production of fibronectin by newborn rat epidermal cells was shown by indirect immunofluorescence staining of cultures grown in the absence of a feeder layer using an antiserum which had been cross-adsorbed with foetal calf serum proteins to remove antibodies which recognised serum fibronectin. The distribution of fibronectin in areas of cell-cell and cell-substratum contact, characteristically in the form of short radial stitches, was examined in more detail using immunoelectron microscopy with colloidal gold as marker. This showed the close proximity of fibronectin to the cell membrane, with the ventral surface and fine cellular processes showing the heaviest labelling, and also revealed evidence of a relationship between external fibronectin and internal structure in epidermal cells. Immunofluorescence showed that tonofilaments (keratin) and microtubules were present as fibrillar arrays but were not related to fibronectin distribution. Vimentin and desmin were absent. Actin was distributed as a circumferential bundle of filaments, with finer stands running radially to the edge. The latter were reminiscent of the radial fibronectin stitches and a spatial correspondence between fibronectin and actin was confirmed by double-label immunofluorescence which revealed many instances of overlap and colinearity of actin and fibronectin filaments. The ability of keratinocytes to produce fibronectin suggests that these cells can contribute to the formation of the basement membrane in skin. The localisation of fibronectin and its close association with actin also suggests that it is involved in keratinocyte adhesion and is related to the internal organisation of these cells.