heparitin-sulfate and Teratoma

Topics: Animals; Basement Membrane; Cell Differentiation; Chondroitin Sulfate Proteoglycans; Collagen; Gene Expression Regulation, Neoplastic; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Laminin; Mice; RNA, Messenger; Teratoma; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Antibodies were raised against a small high-density and a large low-density form of heparan sulfate proteoglycan from a basement membrane-producing mouse tumor and were characterized by radioimmunoassays, immunoprecipitation and immunohistological methods. Antigenicity was due to the protein cores and included epitopes unique to the low density form as well as some shared by both proteoglycans. The antibodies did not cross-react with other basement membrane proteins or with chondroitin sulfate proteoglycans from interstitial connective tissues. The heparan sulfate proteoglycans occurred ubiquitously in embryonic and adult basement membranes and could be initially detected at the 2-4 cell stage of mouse embryonic development. Low levels were also found in serum. Biosynthetic studies demonstrated identical or similar proteoglycans in cultures of normal and carcinoembryonic cells and in organ cultures of fetal tissues. They could be distinguished from liver cell membrane heparan sulfate proteoglycan, indicating that the basement membrane types of proteoglycans represent a unique class of extracellular matrix proteins.

Topics: Animals; Basement Membrane; Bone and Bones; Cattle; Cell Line; Cell Membrane; Chondroitin Sulfate Proteoglycans; Chondrosarcoma; Embryo, Mammalian; Female; Fluorescent Antibody Technique; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Lens, Crystalline; Liver; Mice; Nasal Septum; Pregnancy; Proteoglycans; Radioimmunoassay; Rats; Teratoma

Tumors were developed from the mouse teratocarcinoma-derived endodermal cell line PF HR-9 by subcutaneous injections in syngeneic mice of large numbers of cells previously cultured for several years at high cell density. Cell cultures were established from the tumors and the cells were cloned. The cloned sublines were highly malignant in vivo and tumor metastases were occasionally observed. The tumors contained abundant extracellular material, which was distinctly laminated and contained type IV collagen, laminin, and heparan sulfate proteoglycan. The tumorigenic sublines were also shown to have retained markers of the original cells, such as the cytoskeletal proteins Endo A and B. These cell lines should be useful for biosynthetic studies on basement membrane and cytoskeletal components and the tumors for isolation of these macromolecules and their mRNAs.

Topics: Animals; Basement Membrane; Cell Line; Clone Cells; Collagen; DNA; Endoderm; Extracellular Matrix; Fibronectins; Glycoproteins; Heparitin Sulfate; Intermediate Filament Proteins; Laminin; Mice; Proteoglycans; Teratoma

Teratocarcinoma-derived endodermal PYS-2 cells are known to synthesize an extracellular matrix containing the basement membrane molecules laminin, type IV collagen, and heparan sulfate proteoglycan as major constituents (I. Leivo, K. Alitalo, L. Risteli, A. Vaheri, R. Timpl, J. Wartiovaara, Exp Cell Res 137:15-23, 1982). Immunoferritin techniques with specific antibodies were used in the present study to define the ultrastructural localization of the above constituents in the fibrillar network. Laminin was detected in matrix network adjacent to the basal cell membrane and in protruding matrix fibrils that connect the matrix to the cell membrane. Ruthenium red-stainable heparinase-sensitive 10- to 20-nm particles were often present at the junction of the attachment fibrils and the matrix network, or along the attachment fibrils. A corresponding distribution of ferritin label was observed for basement membrane heparan sulfate proteoglycan. Type IV collagen was found in the matrix network but not in the attachment fibrils. The results suggest that the PYS-2 cells are connected to their pericellular matrix by fibrils containing laminin associated with heparan sulfate-containing particles. These results may also have relevance for the attachment of epithelial cells to basement membranes.

Topics: Animals; Basement Membrane; Cells, Cultured; Ferritins; Glycoproteins; Glycosaminoglycans; Heparitin Sulfate; Laminin; Mice; Neoplasms, Experimental; Staining and Labeling; Teratoma

Topics: Cell Line; Fibronectins; Glycosaminoglycans; Heparitin Sulfate; Humans; Neoplasm Proteins; Polysaccharides; Protein Binding; Teratoma

Topics: Animals; Chromatography, Gel; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Fibronectins; Glycopeptides; Glycosaminoglycans; Hematopoietic Stem Cells; Heparitin Sulfate; Mice; Mice, Inbred BALB C; Polysaccharides; Teratoma

A high-speed gel-permeation chromatographic system for analyzing glycosaminoglycans which uses two 0.7 X 75-cm stainless-steel columns containing Fractogel (Toyopearl) TSK HW-55(S), was developed. Glycosaminoglycans were applied and eluted with a 0.2 M sodium chloride solution and monitored by ultraviolet absorption at 210 nm or radioactivity. The best resolution of glycans was obtained at 55 degrees C at a flow rate of 1.0 ml/min. Acidic and neutral glycans in the molecular weight (Mr) range 600-60,000 eluted within 45 min. A linear relationship was found between retention time and molecular weight using standard glycosaminoglycans, chitin oligosaccharides, and a porcine thyroglobulin glycoprotide. This system was used to analyze the heparan sulfate synthesized by PYS-2 embryonic carcinoma cells and the degradation products produced by incubating it with extracted glycosidases from metastatic B16 melanoma cells. The results indicated that B16 melanoma cells contain at least two different heparan sulfate degradative activities, one of which appears to be an endoglycosidase.

Topics: Animals; Chromatography, Gel; Chromatography, High Pressure Liquid; Glucuronidase; Glycosaminoglycans; Glycoside Hydrolases; Heparitin Sulfate; Mice; Neoplasms, Experimental; Teratoma

The murine embryonal carcinoma derived cell line M1536-B3 secretes the basement membrane components laminin and entactin and, when grown in bacteriological dishes, produces and adheres to sacs of basement membrane components. Heparan sulfate proteoglycans have been isolated from these sacs, the cells, and the medium. At least three different heparan sulfate proteoglycans are produced by these cells as determined by proteoglycan size, glycosaminoglycan chain length, and charge density. The positions of the N- and O-sulfate groups in the glycosaminoglycan chains from each proteoglycan appear to be essentially the same despite differences in the size and culture compartment locations of the heparan sulfate proteoglycan. Additionally, small quantities of chondroitin sulfate proteoglycans are found in each fraction and copurify with each heparan sulfate proteoglycan. Because this cell line appears to synthesize at least three different heparan sulfate proteoglycans which are targeted to different final locations (basement membrane, cell surface, and medium), this will be a useful system in which to study the factors which determine final heparan sulfate proteoglycan structures and culture compartment targeting and the possible effects of the protein core(s) on heparan sulfate carbohydrate chain synthesis and secretion.

Topics: Animals; Basement Membrane; Cell Line; Chondroitin Sulfate Proteoglycans; Chromatography, Gel; Embryonal Carcinoma Stem Cells; Glucosamine; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Kinetics; Mice; Molecular Weight; Neoplastic Stem Cells; Proteoglycans; Stem Cells; Teratoma; Tritium

The effect of biochemically purified basement membrane components as mediators of cell attachment is studied in vitro using an endodermal PYS-2 cell line known to produce a basement membrane-like insoluble matrix. Fibronectin is shown to be as effective as laminin as an attachment-promoting protein, although the latter is a major product of these cells and the former is not produced by them in any detectable amount. Fibronectin also increases the attachment of the cells to type IV collagen-coated plates, but laminin lacks this effect. Protein synthesis-blocking agents such as cycloheximide totally abolish the attachment-promoting effect of extracellularly supplied laminin, but not that of extracellular fibronectin. Type IV collagen alone is no better a substratum for these cells than type I collagen or the plastic surface of the dish itself. The importance of an intact tertiary structure for the attachment is obvious in the case of both fibronectin and laminin. Denatured molecules or smaller fragments of these molecules do not promote cell attachment.

Topics: Animals; Basement Membrane; Cell Adhesion; Cell Line; Collagen; Cycloheximide; Endoderm; Fibronectins; Glycoproteins; Heparitin Sulfate; Laminin; Macromolecular Substances; Mice; Teratoma

Topics: Animals; Basement Membrane; Cell Line; Endoderm; Glycoproteins; Glycosaminoglycans; Heparitin Sulfate; Membrane Proteins; Mice; Mice, Inbred C3H; Molecular Weight; Sulfates; Teratoma; Yolk Sac

Human teratocarcinoma-derived cells of line PA 1, which are capable of differentiating in vitro [Zeuthen, J. et al. (1980) Int J. Cancer, 25, 19-32], incorporate label from radioactive sulfate and/or glucosamine into several large-sized glycosaminoglycans including hyaluronate, chondroitin sulfate/dermatan sulfate co-polymers, heparan sulfate and keratan-sulfate-like molecules. All these polysaccharide fractions were identified by specific degradation methods. The labeled hyaluronate was degraded into a mixture of unsaturated octa-, hexa- and tetra-saccharides by a treatment with Streptomyces hyaluronidase (EC 4.2.2.1). The chondroitin sulfate/dermatan sulfate co-polymers were cleaved with chondroitin AC lyase (EC 4.2.2.5) into unsaturated disaccharides and a series of unsaturated oligosaccharides; the latter were degraded by a treatment with chondroitin ABC lyase (EC 4.2.2.4) into unsaturated disaccharides. Heparan sulfate was degraded with nitrous acid into free inorganic [35S]sulfate and a series of [35S]sulfate-labeled oligosaccharides and/or glycopeptides. The keratan-sulfate-like molecules were hydrolyzed by a treatment with endo-beta-galactosidase from Escherichia freundii into a series of distinct [35S]sulfate-labeled oligosaccharides; small oligosaccharides were liberated also from [3H]galactose-labeled molecules. The smallest one of the liberated oligosaccharides was tentatively identified as a sulfated disaccharide.

Topics: beta-Galactosidase; Cell Line; Chondroitin Lyases; Chondroitin Sulfates; Dermatan Sulfate; Glycosaminoglycans; Glycoside Hydrolases; Heparitin Sulfate; Humans; Hyaluronic Acid; Hyaluronoglucosaminidase; Keratan Sulfate; Nitrous Acid; Teratoma