heparitin-sulfate and Chondrosarcoma

Multiple osteochondromas (MO) is a hereditary disorder associated with benign cartilaginous tumors, known to be characterized by absence or highly reduced amount of heparan sulfate (HS) in the extracellular matrix of growth plate cartilage, which alters proper signaling networks leading to improper bone growth. Although recent studies demonstrated accumulation of HS in the cytoplasm of MO chondrocytes, nothing is known on the structural alterations which prevent HS from undergoing its physiologic pathway. In this work, osteochondroma (OC), peripheral chondrosarcoma, and healthy cartilaginous human samples were processed following a procedure previously set up to structurally characterize and compare HS from pathologic and physiologic conditions, and to examine the phenotypic differences that arise in the presence of either exostosin 1 or 2 (

Topics: Adolescent; Adult; Bone Neoplasms; Cartilage; Child; Child, Preschool; Chondrosarcoma; Chromatography, High Pressure Liquid; Female; Heparitin Sulfate; Humans; Magnetic Resonance Imaging; Mass Spectrometry; Mutation; N-Acetylglucosaminyltransferases; Osteochondroma

Heparan sulfate proteoglycans (HSPGs), strategically located at the cell-tissue-organ interface, regulate major biological processes, including cell proliferation, migration, and adhesion. These vital functions are compromised in tumors, due, in part, to alterations in heparan sulfate (HS) expression and structure. How these modifications occur is largely unknown. Here, we investigated whether epigenetic abnormalities involving aberrant DNA methylation affect HS biosynthetic enzymes in cancer cells. Analysis of the methylation status of glycosyltransferase and sulfotransferase genes in H-HEMC-SS chondrosarcoma cells showed a typical hypermethylation profile of 3-OST sulfotransferase genes. Exposure of chondrosarcoma cells to 5-aza-2'-deoxycytidine (5-Aza-dc), a DNA-methyltransferase inhibitor, up-regulated expression of 3-OST1, 3-OST2, and 3-OST3A mRNAs, indicating that aberrant methylation affects transcription of these genes. Furthermore, HS expression was restored on 5-Aza-dc treatment or reintroduction of 3-OST expression, as shown by indirect immunofluorescence microscopy and/or analysis of HS chains by anion-exchange and gel-filtration chromatography. Notably, 5-Aza-dc treatment of HEMC cells or expression of 3-OST3A cDNA reduced their proliferative and invading properties and augmented adhesion of chondrosarcoma cells. These results provide the first evidence for specific epigenetic regulation of 3-OST genes resulting in altered HSPG sulfation and point to a defect of HS-3-O-sulfation as a factor in cancer progression.

Topics: Azacitidine; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chondrosarcoma; CpG Islands; Decitabine; DNA Methylation; Enzyme Repression; HeLa Cells; Heparan Sulfate Proteoglycans; Heparitin Sulfate; HL-60 Cells; Humans; Neoplasm Invasiveness; Sulfotransferases; Up-Regulation

On October 29, 2009, researchers and physicians gathered at the Sheraton Four Points Hotel in Boston for 4 days to discuss a disease called multiple hereditary exostoses (MHE). MHE is an autosomal dominant disease that is associated with mutations in two enzymes that are required for heparan sulfate (HS) synthesis. Children with the disease form numerous benign bone tumors (osteochondromas) and have >2% chance of developing chondrosarcoma. The aim of the meeting was to generate new ideas for the diagnoses, treatment, and cure of this disease. Discussions ranged from orthopedic surgical treatment and patients' personal experiences to fundamental questions in skeletal biology and the precise molecular role that HS plays in developmental signaling pathways.

Topics: Bone and Bones; Bone Neoplasms; Boston; Carbohydrates; Child; Chondrosarcoma; Exostoses, Multiple Hereditary; Heparitin Sulfate; Humans; Mutation; Osteochondroma

Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type serine proteinase inhibitor that plays a central role in the extrinsic pathway of blood coagulation and is mainly expressed by endothelial cells. In this study we examined the in vitro effects of heparin and other glycosaminoglycans on TFPI mRNA-expression in cultivated human endothelial (Ea.hy 926) and in chondrosarcoma (SW 1353) cells.. We used a LightCycler-based method for relative quantification of the TFPI-mRNA expression before and after stimulation. The cells were stimulated with different concentrations of heparin (with and without addition of protamin), heparan sulfate (HS) and chondroitin-6-sulfate (CS). Cells were harvested after incubation times of 4, 8 and 24h, total RNA was isolated, and cDNA was synthesized and quantified relatively to a constantly expressed housekeeping gene.. Stimulation of Ea.hy 926 cells with unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH) caused a time- and dose-dependent upregulation of TFPI-mRNA expression with LMWH showing the stronger effect. In contrast to this, HS led to a strongly and CS to a slightly decreased TFPI-mRNA expression. SW 1353 cells which were stimulated with LMWH/UFH and HS/CS did not show a significant up- or downregulative effect.. Our results show that we have developed a versatile method for the relative quantification of TFPI-mRNA expression. As a conclusion, the determined heparin-induced upregulation of TFPI-mRNA expression can be considered a major component of the modulation of the anticoagulant properties of the endothelium.

Topics: Cell Line; Cell Proliferation; Chondroitin Sulfates; Chondrosarcoma; Endothelial Cells; Glycosaminoglycans; Heparin; Heparitin Sulfate; Humans; Lipoproteins; RNA, Messenger; Up-Regulation

Chondrosarcomas are malignant cartilage-forming tumors arising centrally in bone (central chondrosarcoma) or within the cartilaginous cap of osteochondroma (peripheral chondrosarcoma). For hereditary multiple osteochondromas, two responsible genes, EXT1 and EXT2, have been cloned. Their recently elucidated role in heparan sulfate biosynthesis and Hedgehog diffusion leads to the hypothesis that EXT inactivation affects fibroblast growth factor (FGF) and Indian Hedgehog (IHh)/parathyroid hormone-related peptide (PTHrP) signaling, two important pathways in chondrocyte proliferation and differentiation. The expression of PTHrP, PTHrP-receptor, Bcl-2, FGF2, FGFR1, FGFR3, and p21 is investigated by immunohistochemistry in osteochondromas (n = 24) and peripheral (n = 29) and central (n = 20) chondrosarcomas. IHh/PTHrP and FGF signaling molecules are mostly absent in osteochondromas. Although no somatic EXT mutations were found in sporadic osteochondromas, the putative EXT downstream targets are affected similarly in sporadic and hereditary tumors. In chondrosarcomas, re-expression of FGF2, FGFR1, PTHrP, Bcl-2, and p21 is found. Expression levels increase with increasing histological grade. Up-regulation of PTHrP and Bcl-2 characterizes malignant transformation of osteochondroma because PTHrP and Bcl-2 expression is significantly higher in borderline and grade I peripheral chondrosarcomas compared with osteochondromas. In contrast, up-regulation of PTHrP and Bcl-2 seems to be a late event in central cartilaginous tumorigenesis because expression is mainly restricted to high-grade central tumors.

Topics: Adolescent; Adult; Aged; Bone Neoplasms; Child; Child, Preschool; Chondrosarcoma; Cloning, Molecular; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Disease Progression; Female; Fibroblast Growth Factor 2; Gene Expression Regulation, Neoplastic; Genes, bcl-2; Heparitin Sulfate; Humans; Male; Middle Aged; N-Acetylglucosaminyltransferases; Osteochondroma; Parathyroid Hormone-Related Protein; Proteins; Proto-Oncogene Proteins c-bcl-2; Receptor Protein-Tyrosine Kinases; Receptor, Fibroblast Growth Factor, Type 1; Receptors, Fibroblast Growth Factor

Aggrecan, a chondroitin/keratan sulfate-containing proteoglycan, is a major component of cartilaginous tissues. Immunolocalization studies, using antibodies directed to perlecan, a heparan sulfate proteoglycan first detected in basement membranes, and laminin (another major component of basement membranes), indicate that perlecan and laminin are also present in the matrices of hyaline cartilage in the nasal septum, the articular surface of the bone and the growth plate of the developing bone. Consequently, we used antibodies to both aggrecan and perlecan to characterize their synthesis and secretion by primary cultures of chondrocytes derived from the rat chondrosarcoma. Chondrocytes were pulsed for 20 minutes with [35S]methionine and then chased for up to six hours. The radiolabeled perlecan and aggrecan were immunoprecipitated and analyzed by SDS-PAGE. The results show that chondrocytes synthesize precursor proteins to both proteoglycans, but that only the aggrecan precursor protein is secreted as a proteoglycan. Perlecan was also secreted but with less posttranslational modifications than aggrecan. Northern blot analyses of the RNAs from immortalized rat chondrocytes indicated that the major mRNA encoding for perlecan was approximately 13 kb in length, similar in size to that expressed by other cell types, which synthesize 400 kDa core protein perlecan. Analyses of the proteoglycan fractions from the extracts of bovine articular surface indicated that perlecan in this tissue contains both chondroitin and heparan sulfate side-chains. Purified perlecan and laminin were found to promote attachment of immortalized rat chondrocytes in vitro. These studies indicated that perlecan, once thought to be a unique component of the basement membranes, is more widely distributed and is an important component of the cartilage matrix, where it may provide for cell adhesion to the matrix.

Topics: Animals; Autoradiography; Blotting, Western; Cartilage; Cartilage, Articular; Cattle; Cell Adhesion; Cells, Cultured; Chondrosarcoma; Extracellular Matrix; Fluorescent Antibody Technique, Indirect; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Kinetics; Laminin; Methionine; Protein Processing, Post-Translational; Proteoglycans; Rats; RNA, Messenger; Sulfur Radioisotopes; Time Factors; 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

When treated with a retinoic acid analog (Ro 11-1430), the Swarm rat chondrosarcoma regressed (t 1/2 = 11-12 days) with a rapid removal of tumor proteoglycan, histologic evidence of mineralization, and cartilage proteoglycan synthesis was suppressed down to a value of 1% of the control. During the first 3 weeks of treatment, the newly synthesized proteoglycan was similar both in aggregation and size to the proteoglycan present in the control. However, after 5 weeks of treatment synthesis shifted to a small nonaggregating proteoglycan with longer glycosaminoglycan chains now containing dermatan sulfate, possibly representing a switch in proteoglycan synthesized. Heparan sulfate was also detected. Unlabeled proteoglycan released from the tissue during Ro 11-1430 treatment was large (Kav = 0.25 on Sepharose CL-2B) but incapable of aggregation, suggesting the initial proteolytic cleavage was in or near the hyaluronic acid-binding region of the proteoglycan. Degradative enzyme activity varied during the period of treatment. Since other tissues remained histologically normal during the treatment with Ro 11-1430, this drug may have possible therapeutic value.

Topics: Animals; Chondrosarcoma; Glycosaminoglycans; Heparitin Sulfate; Male; Proteoglycans; Rats; Sarcoma, Experimental; Tretinoin