Alexandra Syrokou

Proteoglycans in human malignant mesothelioma. Stimulation of their synthesis induced by epidermal, insulin and platelet-derived growth factors involves receptors with tyrosine kinase activity.

Identification of proteoglycans in two human malignant mesothelioma cell lines, one with epithelial differentiation and the other with fibroblast-like phenotype, and the effects of epidermal (EGF), insulin-like (IGF-I) and platelet-derived (PDGF-BB) growth factors on the synthesis of hyaluronan (HA) and proteoglycans (PGs) were studied. Both cell lines synthesize HA and PGs: these last were recovered both as secreted and cell-associated compounds. Chondroitin sulfate (CS) containing PGs are mainly organized as versican in the extracellular medium and as thrombomodulin and syndecan in the cell membrane. Heparan sulfate (HS) containing PGs are mainly in the form of perlecan in the culture medium, whereas cell-associated HSPGs were recovered mainly as syndecan-1, -2 and -4. Receptors for EGF, IGF-I and PDGF-BB were identified in both cell lines. In addition to cell proliferation, these growth factors stimulated the synthesis of HA and PGs, the pattern of stimulation being unique for each of them and depending on the cell phenotype. EGF increased the synthesis of HA and PGs. IGF-I showed similar stimulatory effects on the synthesis of CSPGs, whereas higher amounts were needed to influence the synthesis of HA and HSPGs, the latter only being stimulated in the epithelial cell line. PDGF-BB stimulated the synthesis of HA, HSPGs and CSPGs at low concentrations, while the stimulatory effect was abolished at higher levels. Incubation with genistein inhibited the HA and PG synthesis induced by growth factors in a mode depending on both growth factor and genistein concentrations. The results clearly suggest that the stimulatory effects of EGF, IGF-I and PDGF-BB on matrix synthesis, expressed as proteoglycan synthesis, are mediated via receptor-growth factor complexes and the protein tyrosine kinase intracellular pathway.

Effects of glycosaminoglycans on proliferation of epithelial and fibroblast human malignant mesothelioma cells: a structure–function relationship

Abstract. Proteoglycans interact with other effective macromolecules regulating a variety of cellular events via their glycosaminoglycan (GAG) chains. The effects of all known glycosaminoglycans (GAGs) produced by normal cells and tissues on the proliferation of two human malignant mesothelioma cell lines, one with fibroblast‐like morphology and the other with epithelial differentiation – both able to produce hyaluronan (HA), galactosaminoglycans (GalAGs) and heparan sulphate (HS) containing proteoglycans – have been studied. Cell proliferation was assessed by measuring [3H]thymidine incorporation and cell number. GalAGs, i.e. chondroitin sulphates (CSs) and dermatan sulphate (DS), strongly stimulate the proliferation of fibroblast‐like cells in a dose‐dependent manner (170–250% at 100 μg/ml), independently of their sulphation pattern. In epithelial cells, however, only DS stimulates cell proliferation. The effects of CSs on proliferation of epithelial cells are not depended on their sulphation pattern. Thus, CSs either with ‐[GlcA‐GalNAc‐(‐6‐O‐SO3−)]‐ or ‐[GlcA‐GalNAc‐(‐4‐O‐SO3−]‐ as the commonest unit, had no significant effect. l‐Iduronic acid (IdoA)‐rich heparin and fast‐moving HS (fm‐HS), a HS fraction with a heparin‐like structure, had significant antiproliferative effects on mesothelioma cells of both types (30–70% at 1.0 μg/ml and 85–90% at 100 μg/ml, respectively). GlcA‐rich HS, however, had no significant effects. HA inhibits only the proliferation of fibroblast‐like cells by 25% at 50 and 100 μg/ml. Keratan sulphate suppresses cell proliferation (10–30%) in both cell lines. In the view of these findings, a structure–function relationship of GAGs on cell proliferation of the two human malignant mesothelioma cell lines is discussed. Other factors, such as chain conformation and geometry, as well as interactions of growth factors with GAGs, possibly involved in the regulation of cell proliferation, are also discussed.

Identification of oligomeric domains within dermatan sulfate chains using differential enzymic treatments, derivatization with 2-aminoacridone and capillary electrophoresis.

Galactosaminoglycans, i.e. dermatan sulfate (DS) and chondroitin sulfate, are linear heteropolysaccharides consisting of repeating disaccharide units of L‐iduronic acid (L‐IdoA) or D‐glucuronic acid (D‐GlcA) residues linked to N‐acetyl‐galactosamine. High‐performance capillary electrophoresis (HPCE or CE) has been successfully used for determining the disaccharide composition of glycosaminoglycans. However, only limited information is available on how to identify oligomeric domains rich in D‐GlcA or L‐IdoA. The aim of this study was therefore to develop a rapid and accurate CE procedure by which such oligosaccharides can be determined together with the variously sulfated disaccharides. Isolated dermatan sulfates of human origin were separately digested with chondroitinases ABC, AC and B and the enzymic products were derivatized with 2‐aminoacridone. CE analysis of these products was performed using a phosphate buffer, pH 3.0, and reversed polarity at 30 kV. The derivatization enabled their detection with laser‐induced fluorescence (LIF) and UV at 260 nm at much higher sensitivity than the detection of nonderivatized δ‐saccharides at 232 nm and therefore components undetectable at 232 nm were nicely detected after derivatization. Except for δ‐disaccharides, altogether five distinct oligosaccharides with differences in charge density were identified. Depending on the lyase that produced these oligomers, information on the presence of L‐IdoA‐ or D‐GlcA‐containing domains within the DS chain and the sulfation pattern of these oligomeric domains was obtained. This CE method could also be useful in studying the functional oligomeric domains in galactosaminoglycan chains.

Identity of macromolecules present in the extracellular slime layer of Staphylococcus epidermidis.

The extracellular slime layer of Staphylococcus epidermidis ATCC 35983 contains: a) two non-anionic carbohydrate containing proteins degradable with papain of molecular masses 250 and 125 kDa; b) a polydisperse but homogeneously-charged acidic population with M(r) ranging from 120,000 to 35,000 (average M(r) (80,000) containing a polysaccharide covalently bound to a small peptide; c) a papain degradable macromolecule (molecular mass 60 kDa) bearing acidic carbohydrates covalently bound to protein; and c) two acidic polysaccharides strongly retained by the anion-exchange column; one polysaccharide is sulphated and has a molecular mass of 20 kDa; the other has a higher charge density and a molecular mass of 12.5 kDa. The results obtained clearly demonstrate the presence of discrete macromolecules in the extracellular material of slime-producing S epidermidis, the majority of which contain acidic carbohydrates, whose biological role remains to be elucidated.

Microemulsion electrokinetic capillary chromatography of sulfated disaccharides derived from glycosaminoglycans.

Microemulsion electrokinetic capillary chromatography (MEEKC) is a capillary electrophoresis technique in which neutral and ionized species can be resolved according to their partitioning into moving oil droplets present in the operating buffer. In this report, we present for the first time the application of MEEKC in the analysis of glycosaminoglycans. An efficient method for the separation of the variously sulfated Δ‐disaccharides obtained following digestion of chondroitin and dermatan sulfates with chondro/dermato lyases and derivatization with 2‐aminoacridone is described. Nonsulfated, mono‐, di‐, and trisulfated Δ‐disaccharides were completely separated using the microemulsion octane/butan‐1‐ol/Sodium dodecyl sulfate (SDS) in 10 mM borate buffer, pH 9.3, at 25 kV. Agreement of the obtained disaccharide composition with literature values showed that MEEKC can be used for the analysis of glycosaminoglycans.

Effect of insulin and epidermal growth factors on the synthesis of glycosaminoglycans/proteoglycans in cultured human malignant mesothelioma cells of different phenotypic morphology

Two human malignant mesothelioma cell sublines, one with a fibroblast‐like and the other with an epithelial differentiation, were examined for their capacity to synthesize glycosaminoglycans in the presence of IGF‐I, EGF, and their combination. This synthesis depends on the morphology of the mesothelioma cells, with many‐fold higher amounts of both hyaluronan and proteoglycans being produced by the cells with epithelial morphology than by those of the fibroblast phenotype. In both cell lines this synthesis was affected in a dose‐dependent fashion by the exogenously added growth factors and exposure to IGF‐I and EGF in combination showed a synergistic effect. This effect of those factors seems to be mediated via protein tyrosin kinase‐dependent receptors and was different in the fibroblast‐like and epithelial cells. The synthetic rates of the various glycosaminoglycans formed (hyaluronan, galactosaminoglycans and heparan sulfate) were also variously affected by these factors, indicating differences in how the synthesis of the various glycosaminoglycans is regulated. The results obtained suggest a close correlation between the presence of the appropriate growth factor(s), the process of cell differentiation and the synthesis of glycosaminoglycans in these cells.