chondroitin and Melanoma

Topics: Ammonium Chloride; Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Biological Transport; Chondroitin; Chondroitin Sulfates; Clinical Trials as Topic; Epitopes; Gangliosides; Humans; Killer Cells, Natural; Melanoma; Melanoma-Specific Antigens; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Neoplasm Proteins; Proteoglycans; Rats

Topics: Ammonium Chloride; Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Biological Transport; Chondroitin; Chondroitin Sulfates; Clinical Trials as Topic; Epitopes; Gangliosides; Humans; Killer Cells, Natural; Melanoma; Melanoma-Specific Antigens; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Neoplasm Proteins; Proteoglycans; Rats

Glycosaminoglycans (GAGs) are anionic polysaccharides present on cells and in the extracellular matrix (ECM). They likely play a role in tumor formation because of their capacity to bind and modulate a variety of proteins including growth factors, cytokines, and proteases. Using a panel of (human) phage display-derived anti-GAG antibodies, the location and expression of GAG epitopes in human cutaneous melanocytic lesions was studied. Antibodies EW4E1 and EW4G2 identified a melanoma-associated chondroitin sulfate/heparan sulfate epitope, whereas antibody EW4B7 recognized a melanoma-associated heparan sulfate epitope. These antibodies showed a high reactivity with blood vessels and ECM in cutaneous melanoma tumors, whereas their reactivity with nevi was very low. Using a set of defined oligosaccharides it was established that sulfate groups are of main importance in the binding to the antibodies and that glycomimetics can mimic natural oligosaccharides. In xenografts of melanoma cell line MeL57, a strong association of GAG epitopes with an injected fluorescent fluid flow tracer was observed. In uveal melanoma antibody, EW4E1 proved to be a sensitive probe for the detection of the geometry of ECM structures, known to have prognostic value. Taken together, data indicate that in melanoma a defined set and location of GAG epitopes are present with possible functional significance.

Topics: Animals; Antibodies; Chondroitin; Epitopes; Heparitin Sulfate; Humans; Melanoma; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Oligosaccharides; Peptide Library; Rats; Rats, Wistar; Skin Neoplasms; Transplantation, Heterologous; Uveal Neoplasms

Changes in glycosaminoglycan composition occurring during the cell cycle were determined in B16-F10 cells sorted flow cytometrically with respect to DNA content. Incorporation of 35S-sulfate into heparan sulfate and chondroitin sulfate of unsorted and G1,S, and G2 +M sorted cells was determined following chondroitinase ABC or nitrous acid treatment; the incorporation into surface material was measured as the difference between the radioactivity of control and trypsin-treated cells. Incorporation of 35S-sulfate and 3H-glucosamine into cetyl pyridinium chloride (CPC)-precipitable material was characterized before and after chondroitinase or nitrous acid treatment by Sephadex G50 chromatography. Long-term (48 h) and short-term (1 h) labeling studies demonstrate that (a) the amount of total cellular chondroitin sulfate is greater than that of heparan sulfate, with larger amounts of unsulfated heparan than chondroitin being present; (b) the rate of turnover of heparan sulfate is greater than that of chondroitin sulfate; (c) greatest short-term incorporation of 3H-glucosamine into CPC-precipitable material occurs during S phase; and (d) the rate of turnover of both heparan sulfate and chondroitin sulfate is decreased in S phase relative to G1 and G2 + M.

Topics: Cell Cycle; Cell Line; Chondroitin; Chondroitin Sulfates; Flow Cytometry; Glucosamine; Glycosaminoglycans; Heparitin Sulfate; Humans; Interphase; Isotope Labeling; Melanoma; Sulfur Radioisotopes; Tritium

The treatment of chondroitin sulfate isolated from cultured B16 mouse melanoma cells with 0.04 M HCl at 100 degrees C for 90 min released up to 45% of O-sulfate residues as free inorganic sulfate. In addition to the release of inorganic sulfate, extensive degradation of this polysaccharide as well as of cartilage chondroitin sulfate, pig rib cartilage proteoglycan, heparin and hyaluronic acid was also evident under these conditions. The above hydrolysis conditions are used for characterizing 35S-labeled heparan sulfates synthesized by cultured cells and to calculate ratio of N- and O-sulfates in these molecules. Our results suggest that caution is necessary in interpreting the results of mild acid hydrolysis of glycosaminoglycans.

Topics: Animals; Cell Line; Chondroitin; Chondroitin Sulfates; Heparin; Hyaluronic Acid; Melanoma; Mice; Neoplasms, Experimental; Sulfuric Acids

The mucopolysaccharides produced by B16 mouse melanoma cells have been isolated in milligram quantities from the spent media in which the cells were grown in the presence of 2-amino-2-deoxy-D-glucose-t and [35S]-sulfate. The mucopolysaccharides obtained by precipitation with cetylpyridinium chloride from the Pronase digest of the media were further purified by gel filtration, ion-exchange chromatography, and treatment with nucleases. The major components were identified as chondroitin-4-sulfates by identification of the hexosamine as 2-amino-2-deoxy-D-galactose, and by digestibility with hyaluronidases, chondroitinase AC, and chondro-4-sulfatase. The o.r.d. curve and i.r. spectra of these components also confirmed their similarity to chondroitin-4-sulfate from cartilage. The molecular weight of the polysaccharide chains was estimated to be in the range 90,000-120,000 by sedimentation equilibrium analysis.

Topics: Carbohydrates; Cell Line; Chondroitin; Chondroitin Sulfates; Hexosamines; Melanoma; Molecular Weight; Optical Rotatory Dispersion; Spectrophotometry, Infrared

A human melanoma cell line established in our laboratory was characterized in terms of tyrosinase activity and anionic polysaccharide production. Tyrosinase levels were diluted during the growth phase and increased after the cell culture became confluent. The anionic polysaccharides produced included hyaluronic acid, heparitin sulfate, and a high-molecular-weight condroitin 4-sulfate. In contrast, a primary culture of human melanocytes derived from embryonic iris produced much greater amounts of hyaluronic acid, about 30-fold less heparitin sulfate, and a mixture of chondroitin 4-sulfate and dermatan sulfate. Saccharides secreted into the culture medium were generally identical to those remaining cell associated except for the melanoma heparitin sulfate, wherein the latter fraction appeared to be of lower molecular weight.

Topics: Animals; Catechol Oxidase; Cell Line; Cells, Cultured; Chondroitin; Enzyme Activation; Glycosaminoglycans; Heparitin Sulfate; Humans; Hyaluronic Acid; Iris; Melanocytes; Melanoma; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms, Experimental; Transplantation, Heterologous

Mucopolysaccharides have been isolated, fractionated, and characterized from the nuclei of cultured B16 mouse melanoma cells grown in the presence of (3-H)-glucosamine and (35-S)sulfate. Digestion of the nuclei with DNase followed by Pronase gave a mixture of complex carbohydrates from which the mucopolysaccharides were isolated by precipitation with cetylpyridinium chloride. After fractionation by differential salt extraction and chromatography on controlled pore glass bead columns, the components were identified by chemical and enzymatic methods. The major polysaccharide components were a family of high-molecular-weight chondroitin sulfates with different degrees of sulfation; a minor component has been characterized as heparan sulfate.

Topics: Animals; Bacteria; Cell Line; Cell Nucleus; Cetylpyridinium; Chemical Precipitation; Chondroitin; Chromatography; Glycosaminoglycans; Hyaluronoglucosaminidase; Lyases; Male; Melanoma; Mice; Pronase; Sulfatases; Testis

B16 melanoma cells were treated in culture with 5-bromo-2-deoxyuridine. The cell-associated and released proteoglycans and sialoglycopeptides were compared to those of control cultures treated with thymidine. The 5-bromo-2-deoxyuridine-treated cultures showed a marked reduction in the proportion of cell-associated proteoglycans and sialoglycopeptides, an increase in the synthesis of hyaluronic acid, the absence of high-molecular-weight chondroitin sulfate, and the presence of increased amounts of heparan sulfate in the media. In addition, the 5-bromo-2-deoxyuridine-treated cells had a higher DNA content and were larger than controls.

Topics: Animals; Bromodeoxyuridine; Cells, Cultured; Chondroitin; Depression, Chemical; DNA; Glucosamine; Glycopeptides; Heparitin Sulfate; Hyaluronic Acid; Melanoma; Mice; Neoplasms, Experimental; Polysaccharides; Proteoglycans; Sialic Acids; Thymidine

Topics: Animals; Cell Line; Chondroitin; Clone Cells; Culture Media; Galactosamine; Glucosamine; Glycoproteins; Heparitin Sulfate; Hyaluronic Acid; Iris; Melanocytes; Melanoma; Mice; Neoplasms, Experimental; Neuraminic Acids; Neuraminidase; Osmolar Concentration; Polysaccharides; Sodium Chloride; Sulfates; Sulfur Radioisotopes; Sulfuric Acids; Tritium