heparitin-sulfate and Colonic-Neoplasms

We examined whether chondroitin sulfates (CSs) exert inhibitory effects on heparanase (Hpse), the sole endoglycosidase that cleaves heparan sulfate (HS) and heparin, which also stimulates chemokine production. Hpse-mediated degradation of HS was suppressed in the presence of glycosaminoglycans derived from a squid cartilage and mouse bone marrow-derived mast cells, including the E unit of CS. Pretreatment of the chondroitin sulfate E (CS-E) with chondroitinase ABC abolished the inhibitory effect. Recombinant proteins that mimic pro-form and mature-form Hpse bound to the immobilized CS-E. Cellular responses as a result of Hpse-mediated binding, namely, uptake of Hpse by mast cells and Hpse-induced release of chemokine CCL2 from colon carcinoma cells, were also blocked by the CS-E. CS-E may regulate endogenous Hpse-mediated cellular functions by inhibiting enzymatic activity and binding to the cell surface.

Topics: Animals; Bone Marrow Cells; Cartilage; Cell Line; Cell Line, Tumor; Cell Membrane; Chemokines; Chondroitin Sulfates; Colon; Colonic Neoplasms; Decapodiformes; Glucuronidase; Glycosaminoglycans; Heparitin Sulfate; Humans; Mast Cells; Mice; Recombinant Proteins

Glycans play a critical role in physiological and pathological processes through interaction with a variety of ligands. Altered expression and dysregulation of these molecules can cause aberrant cellular function such as malignancy. Glycomics provide information of the structure and function of glycans, glycolipids, and glycoproteins such as proteoglycans, and may help to predict cancer development and progression as biomarkers. In this report, we compared the expression of proteoglycans, the content and structure of glycosaminoglycans and glycolipids between patient-matched normal and cancer tissues obtained from colon cancer patients. Tumor-related proteoglycans, glypican-3, and syndecan-1 showed downregulation in cancer tissues compared to normal tissues. In cancer tissue, the total amount of chondroitin sulfate (CS)/dermatan sulfate and heparan sulfate were lower and, interestingly, the level of disaccharide units of both 4S6S (CS-E) and 6S (CS-C) were higher compared to normal tissue. Also, overall lipids including glycolipids, a major glycomics target, were analyzed by hydrophilic interaction liquid chromatography mass spectrometry. Increase of lyso-phosphatidylcholine (phospholipid), sphingomyelin (sphigolipid), and four types of glycolipids (glucosylceramide, lactosylceramide, monosialic acid ganglioside, and globoside 4) in cancer tissue showed the possibility as potential biomarkers in colon cancer. While requiring the need for careful interpretation, this type of broad investigation gives us a better understanding of pathophysiological roles on glycosaminoglycans and glycolipids and might be a powerful tool for colon cancer diagnosis.

Topics: Adenocarcinoma; Aged; Aged, 80 and over; Antigens, CD; Biomarkers; Carbohydrate Sequence; Case-Control Studies; Chondroitin Sulfates; Colonic Neoplasms; Dermatan Sulfate; Female; Gangliosides; Gene Expression Regulation, Neoplastic; Globosides; Glucosylceramides; Glypicans; Heparitin Sulfate; Humans; Lactosylceramides; Lysophosphatidylcholines; Male; Middle Aged; Molecular Sequence Data; Sphingomyelins; Syndecan-1

Proteoglycans (PGs), glycosaminoglycans (GAGs) and hyaluronan as a free GAG, have unique structural characteristics which enable them via specific interactions with matrix proteins and cell surface receptors to regulate key tumor cell functions and thus to affect cancer growth and progression. This article explores the many layers of interdependent signaling among transformed colon epithelial cells, neighboring stromal cells and their respective PGs / GAGs components along the insidious and often deadly route of colon cancer progression. Specifically addressed is the altered deposition of PGs / GAGs by colon cancer cells; the effects of these malignant cells on gene expression and biosynthesis of PGs / GAGs of the surrounding stromal cells and the signaling pathways involved, with the utmost goal to highlight potential therapeutic targets in the rapidly developing field of glycan-based therapy.

Topics: Animals; Antineoplastic Agents; Chondroitin Sulfates; Colon; Colonic Neoplasms; Glycosaminoglycans; Heparitin Sulfate; Humans; Hyaluronic Acid; Molecular Targeted Therapy; Proteoglycans; Signal Transduction

The cancer microenvironment and the interactions between cancer and surrounding tissue cells are thought to play a pivotal role in tumor development and progression. Glycosaminoglycans (GAGs)/proteoglycans (PGs) are major constituents of the extracellular matrix, the composition of which may affect various cellular functions. In the present study, the effects of GAGs on the proliferation of HT29, SW1116, and HCT116 human colon cancer cell lines were examined using exogenously added GAGs, an inhibitor of endogenous GAG sulfation and specific glycosidase digestions. Our results demonstrate that colon cancer cell growth was exclusively stimulated by exogenously added heparin and insensitive to endogenous GAGs/PGs production, in a sulfation pattern-related manner. Treatment of the tested cell lines with the FGF-2 neutralizing antibody showed that the stimulatory effect of heparin on the cells' growth was not FGF-2-dependent. Responsiveness of colon cancer cell lines to exogenous heparin/heparan sulfate may play a role in their growth and metastasis.

Topics: Blotting, Western; Cell Proliferation; Chondroitin Sulfates; Colonic Neoplasms; Dermatan Sulfate; Fibrinolytic Agents; Fibroblast Growth Factor 2; Heparin; Heparitin Sulfate; Humans; Hyaluronic Acid; Receptors, Fibroblast Growth Factor; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sulfates; Tumor Cells, Cultured

We describe here a new organ culture system for the evaluation of viral tropism to colon carcinomas and normal colon tissues. Organ cultures of mouse and human colon retained viability for several days and thus facilitated studies of viral tropism. Two adenoviral vectors (AD) were compared in the study: AD5, that utilizes the CAR receptor, demonstrated poor infectivity to both normal and carcinoma tissues, while a capsid-modified-AD, recognizing haparan-sulfate receptor, demonstrated efficient infectivity of both tissues. Immunohistochemistry analysis demonstrated different viral tropism; while AD5 infected only the colon epithelia, the capsid-modified-adeno infected both the epithelia and mesothelial layers. To investigate other determinants in the tissue that influence viral tropism, human cancer tissues were pretreated with collagenase and infected with the AD viruses. Increased infectivity and altered tropism were noted in the treated tumor tissue. Taken together, this ex vivo system indicated that receptor utilization and extracellular-matrix components influence AD viral tropism in solid tissues.

Topics: Adenoviridae; Adenovirus E1 Proteins; Animals; Capsid; Collagenases; Colon; Colonic Neoplasms; Coxsackie and Adenovirus Receptor-Like Membrane Protein; Epithelium; Gene Deletion; Genetic Vectors; Heparitin Sulfate; Humans; Mice; Mice, Inbred BALB C; Neoplasms, Experimental; Organ Culture Techniques; Receptors, Virus; Species Specificity; Virus Replication

The tyrosine kinase receptor c-Met and its ligand HGF/SF, ezrin, and splice variants of CD44 have independently been identified as tumor metastasis-associated proteins. We now show that these proteins cooperate. A CD44 isoform containing variant exon v6 sequences is strictly required for c-Met activation by HGF/SF in rat and human carcinoma cells, in established cell lines as well as in primary keratinocytes. CD44v6-deficient tumor cells were unable to activate c-Met unless they were transfected with a CD44v6-bearing isoform. Antibodies to two v6-encoded epitopes inhibited autophosphorylation of c-Met by interfering with the formation of a complex formed by c-Met, CD44v6, and HGF/SF. In addition, signal transduction from activated c-Met to MEK and Erk required the presence of the cytoplasmic tail of CD44 including a binding motif for ERM proteins. This suggests a role for ERM proteins and possibly their link to the cortical actin cytoskeleton in signal transfer.

Topics: Adenocarcinoma; Cell Line; Colonic Neoplasms; Heparitin Sulfate; Hepatocyte Growth Factor; Humans; Hyaluronan Receptors; Keratinocytes; Protein Isoforms; Proto-Oncogene Proteins c-met; Signal Transduction

CD44 variant exon (CD44v) 3 is a heparan sulfate-binding isoform of CD44. The role of CD44v3 in invasion and metastasis associated with heparan sulfate in colon cancer cell lines and cases of colon cancer was examined. Expression of CD44v3 mRNA and protein was observed in five of six human colorectal cancer cell lines. Colo320 and WiDr cells expressed CD44v3 at high levels. Heparan sulfate treatment increased the invasive activity of Colo320 and WiDr cells to rates 14.3 and 12.6 times higher, respectively, than that of untreated cells. However, heparan sulfate treatment did not affect cell growth. Repression of CD44v3 protein production by antisense S-oligodeoxynucleotide treatment reduced the binding affinities and capacities for heparan sulfate by Colo320 and WiDr cells in comparison with that of control cells, and it also reduced the invasiveness of both cell lines to one-fifth that of control cells. In heparan sulfate-treated Colo320 cells, the levels of CD44v3 protein in the Triton X-100-insoluble fraction and moesin-precipitated fraction were increased, suggesting that heparan sulfate treatment facilitates association of CD44 molecules with the cytoskeleton. Immunohistochemical analysis showed CD44v3 to be expressed in 21 of 37 (57%) colorectal cancer cases. Positive CD44v3 expression was associated with more advanced pathological stage and poorer prognosis than negative CD44v3 expression. These data support a role for CD44v3 in invasion and metastasis by colorectal carcinoma cells.

Topics: Antigens, CD; Base Sequence; Cell Division; Colonic Neoplasms; Colorectal Neoplasms; Cytoskeleton; Exons; Gene Expression Regulation, Neoplastic; Genetic Variation; Heparitin Sulfate; Humans; Hyaluronan Receptors; Kinetics; Neoplasm Invasiveness; Oligodeoxyribonucleotides, Antisense; Protein Isoforms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Survival Rate; Thionucleotides; Time Factors; Tumor Cells, Cultured

Basic fibroblast growth factor (bFGF) is dependent on heparan sulphate for its ability to activate the cell surface signal transducing receptor. We have investigated the FGF dual receptor mechanism in a novel model of the transformation from human colon adenoma to carcinoma in vitro. Reverse transcription-polymerase chain reaction showed that mRNA for FGF receptors 1 and 2 were expressed in both the adenoma and carcinoma cells whereas immunocytochemistry showed that the expression of the FGF R1 was reduced significantly in the carcinoma cells. We have reported previously that the composition and sequence of human colon adenoma and carcinoma heparan sulphate (HS) differ in a defined and specific manner. The functional significance of these changes was assessed by affinity co-electrophoresis, which showed that the affinity of adenoma HS for bFGF was 10-fold greater than that of the carcinoma HS (Kd 220 nM vs. 2493 nM, respectively). In addition, Northern studies of the expression of syndecan 1 and 4 mRNA showed that proteoglycan core protein expression was reduced significantly in the carcinoma cells. These findings were associated with a reduced biological response to bFGF in the carcinoma cells that could be partially reversed by the addition of exogenous heparin, suggesting that both the proteoglycan and signal transducing receptor control the cells' response to bFGF.

Topics: Adenoma; Carcinoma; Colonic Neoplasms; Disease Progression; Down-Regulation; Fibroblast Growth Factor 2; Fibroblast Growth Factors; Gene Expression Regulation, Neoplastic; Heparin; Heparitin Sulfate; Humans; Neoplasm Proteins; Proteoglycans; Receptor Protein-Tyrosine Kinases; Receptor, Fibroblast Growth Factor, Type 1; Receptor, Fibroblast Growth Factor, Type 2; Receptors, Fibroblast Growth Factor; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Signal Transduction; Tumor Cells, Cultured

We report a detailed analysis of heparan sulfate (HS) structure using a model of human colon carcinogenesis. Metabolically radiolabeled HS was isolated from adenoma and carcinoma cells. The chain length of HS was the same in both cell populations (Mr 20,000; 45-50 disaccharides), and the chains contained on average of two sulfated domains (S domains), identified by heparinase I scission. This enzyme produced fragments of approximate size 7 kDa, suggesting that the S domains were evenly spaced in the intact HS chain. The degree of polymer sulfation and the patterns of sulfation were strikingly different between the two HS species. When compared with adenoma HS, the iduronic acid 2-O-sulfate content of the carcinoma-derived material was reduced by 33%, and the overall level of N-sulfation was reduced by 20%. However, the level of 6-O-sulfation was increased by 24%, and this was almost entirely attributable to an enhanced level of N-sulfated glucosamine 6-O-sulfate, a species whose data implied was mainly located in the mixed sequences of alternating N-sulfated and N-acetylated disaccharides. The results indicate that in the transition to malignancy in human colon adenoma cells, the overall molecular organization of HS is preserved, but there are distinct modifications in both the S domains and their flanking mixed domains that may contribute to the aberrant behavior of the cancer cell.

Topics: Adenoma; Carcinoma; Chromatography, Gel; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Colonic Neoplasms; Heparitin Sulfate; Humans; Molecular Structure; Molecular Weight; Nitrous Acid; Oligosaccharides; Tumor Cells, Cultured

Colon carcinoma cells provide a useful model to study the biochemical processes associated with cell differentiation. Undifferentiated HT29, differentiated HT29MTX(-3) and HT29MTX(-6), and Caco2 human colon carcinoma cells have been used to study the production of proteoglycans and to characterize the glycosaminoglycan structure of the heparan sulphate chains. All the cell lines produce mainly a heparan sulphate proteoglycan that is found partly in the extracellular medium and associated to the cell membrane. The heparan sulphate proteoglycans from the media were purified by ion-exchange chromatography and subjected to structural analysis. The heparan sulphate proteoglycan from differentiated cells is larger and more homogeneous in size than the heparan sulphate proteoglycan from undifferentiated HT29 cells. No differences in protein core structure were observed when cells were labeled with [35S]methionine and the protein cores visualized by gel electrophoresis. Nevertheless, differences in glycosaminoglycan composition were found correlated with the degree of differentiation. The heparan sulphate chains from differentiated HT29MTX(-3) and HT29MTX(-6) cells have a higher sulphation degree than those from undifferentiated HT29 cells. The heparan sulphate from Caco2 cells is the most highly sulphated species. The differences are mainly attributed to O-sulphate groups. The increase in O-sulphation was more pronounced for D-glucosamine 6-O-sulphate than for L-iduronic acid 2-O-sulphate groups.

Topics: Carbohydrate Sequence; Cell Differentiation; Colonic Neoplasms; Disaccharides; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Models, Biological; Molecular Structure; Proteoglycans; Sulfates; Tumor Cells, Cultured

Heparan sulfate species expressed by different cell and tissue types differ in their structural and functional properties. Limited information is available on differences in regulation of heparan sulfate biosynthesis within a single tissue or cell population under different conditions. We have approached this question by studying the effect of cell differentiation on the biosynthesis and function of heparan sulfate in human colon carcinoma cells (CaCo-2). These cells undergo spontaneous differentiation in culture when grown on semipermeable supports; the differentiated cells show phenotypic similarity to small intestine enterocytes. Metabolically labeled heparan sulfate was isolated from the apical and basolateral media from cultures of differentiated and undifferentiated cells. Compositional analysis of disaccharides, derived from the contiguous N-sulfated regions of heparan sulfate, indicated a greater proportion of 2-O-sulfated iduronic acid units and a smaller amount of 6-O-sulfated glucosamine units in differentiated than in undifferentiated cells. By contrast, the overall degree of sulfation, the chain length and the size distribution of the N-acetylated regions were similar regardless the differentiation status of the cells. The structural changes were found to affect the binding of heparan sulfate to the long isoform of platelet-derived growth factor A chain but not to fibroblast growth factor 2. These findings show that heparan sulfate structures change during cell differentiation and that heparan sulfate-growth factor interactions may be affected by such changes.

Topics: Acetylation; Caco-2 Cells; Carbohydrate Conformation; Cell Differentiation; Colonic Neoplasms; Growth Substances; Heparitin Sulfate; Humans; Protein Binding

Perlecan, a ubiquitous heparan sulfate proteoglycan, possesses angiogenic and growth-promoting attributes primarily by acting as a coreceptor for basic fibroblast growth factor (FGF-2). In this report we blocked perlecan expression by using either constitutive CMV-driven or doxycycline- inducible antisense constructs. Growth of colon carcinoma cells was markedly attenuated upon obliteration of perlecan gene expression and these effects correlated with reduced responsiveness to and affinity for mitogenic keratinocyte growth factor (FGF-7). Exogenous perlecan effectively reconstituted the activity of FGF-7 in the perlecan-deficient cells. Moreover, soluble FGF-7 specifically bound immobilized perlecan in a heparan sulfate-independent manner. In both tumor xenografts induced by human colon carcinoma cells and tumor allografts induced by highly invasive mouse melanoma cells, perlecan suppression caused substantial inhibition of tumor growth and neovascularization. Thus, perlecan is a potent inducer of tumor growth and angiogenesis in vivo and therapeutic interventions targeting this key modulator of tumor progression may improve cancer treatment.

Topics: Animals; Carcinoma; Colonic Neoplasms; DNA, Antisense; Fibroblast Growth Factor 10; Fibroblast Growth Factor 2; Fibroblast Growth Factor 7; Fibroblast Growth Factors; Gene Expression; Growth Substances; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Melanoma, Experimental; Mice; Neoplasm Transplantation; Neoplasms, Experimental; Neovascularization, Pathologic; Protein Binding; Proteoglycans

Neisseria gonorrhoeae (GC) is a human pathogen that adheres to and invades genital surfaces. Although pili are required for the initial adherence, the interaction of GC with epithelial cells is also promoted by a family of outer membrane proteins, the opacity (Opa) proteins such as OpaA protein from strain MS11. Studies have demonstrated that the interaction of the OpaA GC with epithelial cells involves binding to heparan sulfate attached to syndecan receptors. However, other Opa proteins interact with CEA gene family member 1 (CGM1) or biliary glycoprotein (BGP), members of the CD66 antigen family. In this study, we demonstrate that, in addition, the 180-kD carcinoembryonic antigen (CEA) is a receptor for Opa proteins. This conclusion was based on the following observations. First, transfected HeLa cells expressing CEA (HeLa-CEA) and the CEA-expressing colon cancer cell line (LS 174T) bound and subsequently engulfed the Opa+ bacteria. These interactions were inhibited by anti-CEA antibody, but could not be inhibited by addition of heparin. Furthermore, OpaI E. coli directly bound purified CEA. We also compared the adherence and invasion by Opa+ bacteria of CD66 transfected HeLa cells: HeLa-BGPa, HeLa-CGM6, HeLa-NCA, HeLa-CGM1a, HeLa-CEA, and HeLa-Neo serving as negative control. Using OpaI as the prototype, the relative ability of the transfected HeLa cell lines to support adherence was (CEA = BGPa >CGM1a >NCA >>CGM6 = Neo). The ability to mediate invasion of the transfectant cells was (CGM1a >CEA >BGPa >NCA >CGM6 = Neo). Among the Opa proteins tested, OpaC proved to be bifunctional, able to mediate adherence to both syndecan receptors and to CD66 antigens.

Topics: Animals; Antigens, Bacterial; Antigens, CD; Bacterial Adhesion; Carcinoembryonic Antigen; CHO Cells; Colonic Neoplasms; Cricetinae; HeLa Cells; Heparitin Sulfate; Humans; Microscopy, Electron; Neisseria gonorrhoeae; Tumor Cells, Cultured

Undifferentiated HT29 and differentiated HT29G-human colon carcinoma cells have been used to study the changes in proteoglycan production and structure associated with enterocytic cell differentiation. Differentiated cells incorporate twice as much sulfate than undifferentiated cells when labeled with [35S]sulfate. Both cell lines produce a heparan sulfate proteoglycan which was purified by ion-exchange. The heparan sulfate proteoglycan from differentiated HT29G- cells is larger and more homogeneous in size than that produced by undifferentiated HT29 cells. No differences in the core protein structure were observed. The detailed structural analysis of the heparan sulfate chains revealed that the structure of these chains follows the standard rules for these glycosaminoglycans with N-sulfated domains and N-acetylated domains. The main finding was that differentiated HT29G- cells have a degree of higher sulfation than HT29 cells. These differences were found to affect primarily 6-O-sulfated positions.

Topics: Carcinoma; Cell Differentiation; Chromatography, Gel; Chromatography, Ion Exchange; Colonic Neoplasms; Heparan Sulfate Proteoglycans; Heparitin Sulfate; HT29 Cells; Humans; Proteoglycans; Sulfates

Several processes that occur in the luminal compartments of the tissues are modulated by heparin-like polysaccharides. To identify proteins responsible for the expression of heparan sulfate at the apex of polarized cells, we investigated the polarity of the expression of the cell surface heparan sulfate proteoglycans in CaCo-2 cells. Domain-specific biotinylation of the apical and basolateral membranes of these cells identified glypican, a GPI-linked heparan sulfate proteoglycan, as the major source of apical heparan sulfate. Yet, most of this proteoglycan was expressed at the basolateral surface, an unexpected finding for a glypiated protein. Metabolic labeling and chase experiments indicated that sorting mechanisms, rather than differential turnover, accounted for this bipolar expression of glypican. Chlorate treatment did not affect the polarity of the expression of glypican in CaCo-2 cells, and transfectant MDCK cells expressed wild-type glypican and a syndecan-4/glypican chimera also in an essentially unpolarized fashion. Yet, complete removal of the heparan sulfate glycanation sites from the glypican core protein resulted in the nearly exclusive apical targeting of glypican in the transfectants, whereas two- and one-chain mutant forms had intermediate distributions. These results indicate that glypican accounts for the expression of apical heparan sulfate, but that glycanation of the core protein antagonizes the activity of the apical sorting signal conveyed by the GPI anchor of this proteoglycan. A possible implication of these findings is that heparan sulfate glycanation may be a determinant of the subcellular expression of glypican. Alternatively, inverse glycanation-apical sorting relationships in glypican may insure near constant deliveries of HS to the apical compartment, or "active" GPI-mediated entry of heparan sulfate into apical membrane compartments may require the overriding of this antagonizing effect of the heparan sulfate chains.

Topics: Animals; Antibodies, Monoclonal; Cell Line; Cell Membrane; Colonic Neoplasms; Dogs; Epithelium; Gene Expression; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Kidney; Kinetics; Liposomes; Membrane Glycoproteins; Phosphatidylinositol Diacylglycerol-Lyase; Phosphoric Diester Hydrolases; Proteoglycans; Recombinant Proteins; Syndecan-4; Syndecans; Time Factors; Transfection; Tumor Cells, Cultured

Two human colon carcinoma cell lines, HT29 and Caco-2 were co-cultured with fetal rat or human skin fibroblasts. Their morphological features, ultra-structural characteristics at the heterologous cell interface, and the deposition of basement-membrane molecules [laminin, type-IV collagen, heparan sulfate proteoglycan (HSPG)] at the epithelial-stromal junction were analyzed. The 2 cell lines behaved differently. HT29 cells did not spread on the fibroblasts and grew as clusters, while Caco-2 cells formed a monolayer over the fibroblastic feeder layer. Only the latter carcinoma cells exhibited cytoplasmic processes towards the fibroblasts and, after 5 days in co-cultures, a structured basement membrane (BM). The immunocytochemical analysis of the BM constituents revealed the absence of the molecules studied at the sites of heterologous contacts in the case of HT29 cells. In contrast, in the co-cultures comprising Caco-2 cells, laminin and type-IV collagen were progressively deposited in a polar fashion at the epithelial-fibroblastic interface which, however, remained devoid of HSPG molecules. Together with earlier data indicating a dual origin of the BM molecules located at the epithelial-fibroblastic interface in normal intestine, the present study shows that the cancer cells as well as the fibroblastic ones under the influence of carcinoma cells display an altered capacity to synthesize and/or secrete BM molecules. The extent of such abnormalities correlates with the differentiation of the cells. Finally, these modifications occur concomitantly with alterations in cell interactions which vary among cell lines.

Topics: Adenocarcinoma; Animals; Basement Membrane; Cell Adhesion; Cell Communication; Cell Differentiation; Cell Line; Cells, Cultured; Chondroitin Sulfate Proteoglycans; Collagen; Colonic Neoplasms; DNA Replication; Epithelium; Fetus; Fibroblasts; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Intercellular Junctions; Laminin; Microscopy, Electron; Rats; Skin; Skin Physiological Phenomena

Transforming growth factor beta (TGF-beta) stimulated the synthesis of heparan sulfate proteoglycan in cultured human colon carcinoma cells without affecting its rates of intracellular degradation or secretion. The overall hydrodynamic size, electrophoretic mobility, and degree of sulfation of the TGF-beta-induced proteoglycan was indistinguishable from that of untreated cells. The synthesis of the protein core was significantly stimulated by TGF-beta, although total cellular protein was unaltered. The stimulatory effects of TGF-beta were prevented by the inhibitors of protein synthesis and DNA transcription, cycloheximide, or actinomycin D, respectively. Analysis of protein core mRNA levels using a murine cDNA encoding a basement membrane protein core, revealed a marked elevation of the steady state levels of mRNA for this gene product. In contrast, the mRNA levels for glyceraldehyde-3-phosphate dehydrogenase or beta-actin genes were not significantly affected by TGF-beta. Finally, nuclear run-off experiments showed increases in neither protein core-specific transcription nor in general transcriptional activity. Taken together, these results indicate that TGF-beta is a potent modulator of heparan sulfate proteoglycan expression in human colon carcinoma cells and that its effect is mediated primarily through an increase in mRNA levels encoding the protein core, perhaps a result of enhanced RNA stability. The TGF-beta-induced elevation of heparan sulfate proteoglycan may contribute to the control of stromal cell proliferation and matrix production by human colon carcinoma cells.

Topics: Cell Line; Cell Nucleus; Chondroitin Sulfate Proteoglycans; Colonic Neoplasms; Cycloheximide; Cysteine; Dactinomycin; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Kinetics; Molecular Weight; Poly A; RNA; RNA, Messenger; RNA, Neoplasm; Transcription, Genetic; Transforming Growth Factor beta

We have recently shown that the heparan sulfate proteoglycan of human colon carcinoma cells is acylated with both myristate and palmitate, two long-chain saturated fatty acids. In this study we show that cycloheximide did not significantly inhibit the incorporation of myristic acid into either proteoglycan or total protein pool. This lack of inhibition occurred under a condition in which protein synthesis was inhibited greater than 90%. Cycloheximide, on the other hand, did not affect the incorporation of [3H]myristic acid into fatty acid nor the intracellular interconversion of myristate to palmitate. Characterization of fatty acyl moiety in the proteoglycan and protein by reverse-phase HPLC revealed that approximately 60% of the covalently bound fatty acids was myristate and the remaining 40% was palmitate. These results indicate that in human colon carcinoma cells myristoylation of heparan sulfate proteoglycan and proteins occurs post-translationally, presumably in the Golgi complex.

Topics: Acylation; Chondroitin Sulfate Proteoglycans; Colonic Neoplasms; Cycloheximide; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Kinetics; Myristic Acid; Myristic Acids; Neoplasm Proteins; Palmitic Acid; Palmitic Acids; Protein Processing, Post-Translational

Tumor-producing phorbol esters [e.g., 12-O-tetradecanoylphorbol-13-acetate (TPA)] induce changes in a human colon cancer cell line, VACO 10MS, that mimic terminal differentiation: a rapid blockade of DNA replication and cell division, a marked increase in cell adhesion properties with striking changes in morphology, and the acquisition of ion-transporting activities. The present report shows that the triggering of this terminal differentiation sequence by TPA is associated with a rapid release of heparan sulfate proteoglycans from the cell surface that is soon followed by an acceleration of proteoglycan synthesis. The activation of the release mechanism is independent of ongoing protein synthesis, whereas the resynthesis of the proteoglycans requires the production of new proteins. A persistent high rate of proteoglycan synthesis and release appears correlated with the progression of the colon cell into the terminal differentiation state. Bryostatin 1, an agent which has been shown previously to block the TPA-induced terminal differentiation of this cell line, also largely prevents the TPA effects on proteoglycan metabolism. Since both TPA and bryostatin 1 produce their effects through the activation of members of the protein kinase C class of enzymes, it is proposed that the differentiation state of these colon cancer cells may be regulated by a differential activation of isozymes or a ligand-directed phosphorylation of proteins that are involved in proteoglycan metabolism.

Topics: Bryostatins; Cell Differentiation; Cell Division; Colonic Neoplasms; Cycloheximide; Diglycerides; DNA Replication; Enzyme Activation; Heparitin Sulfate; Humans; Isoenzymes; Lactones; Macrolides; Membrane Proteins; Neoplasm Proteins; Phosphorylation; Protein Kinase C; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

We provide direct evidence for the presence of unsulfated, but fully elongated heparan glycosaminoglycans covalently linked to the protein core of a heparan sulfate proteoglycan synthesized by human colon carcinoma cells. Chemical and enzymatic studies revealed that a significant proportion of these chains contained glucuronic acid and N-acetylated glucosamine moieties, consistent with N-acetylheparosan, an established precursor of heparin and heparan sulfate. The presence of unsulfated chains was not dependent upon the exogenous supply of sulfate since their synthesis, structure, or relative amount did not vary with low exogenous sulfate concentrations. Culture in sulfate-free medium also failed to generate undersulfated heparan sulfate-proteoglycan, but revealed an endogenous source of sulfate which was primarily derived from the catabolism of the sulfur-containing amino acids methionine and cysteine. Furthermore, the presence of unsulfated chains was not due to a defect in the sulfation process because pulse-chase experiments showed that they could be converted into the fully sulfated chains. However, their formation was inhibited by limiting the endogenous supply of hexosamine. The results also indicated the coexistence of the unsulfated and sulfated chains on the same protein core and further suggested that the sulfation of heparan sulfate may occur as an all or nothing phenomenon. Taken together, the results support the current biosynthetic model developed for the heparin proteoglycan in which unsulfated glycosaminoglycans are first elongated on the protein core, and subsequently modified and sulfated. These data provide the first evidence for the presence of such an unsulfated precursor in an intact cellular system.

Topics: Chondroitin Sulfate Proteoglycans; Chromatography, Gel; Chromatography, Ion Exchange; Colonic Neoplasms; Glucosamine; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Proteoglycans; Sulfates; Sulfur Radioisotopes; Tritium; Tumor Cells, Cultured

Human colon carcinoma cells synthesize a high-molecular-weight heparan sulfate proteoglycan which is localized at the cell surface. In this study we have performed a series of immunoprecipitation and pulse-chase experiments associated with various pharmacological agents that interfere with the synthesis and post-translational modification of the proteoglycan. We demonstrate that colon carcinoma cells synthesize the heparan sulfate proteoglycan from a 400-kDa precursor protein that is immunologically related to the Engelbreth-Holm-Swarm (EHS) tumor cell proteoglycan. The cells contain a large pool of precursor protein with a half-life of about 75 min. Most of the precursor protein receives heparan sulfate side chains and is then transported to the cell surface and released into the medium. A portion of the precursor pool, however, does not receive heparan sulfate chains but is secreted into the medium. The glycosylation and subsequent secretion of the 400-kDa precursor protein was inhibited by NH4Cl and even more by monensin, indicating that the transit of precursor from the rough endoplasmic reticulum to the cell surface occurred through the Golgi complex and acidic compartments. The existence of a sizable pool of precursor protein was confirmed by additional experiments using cycloheximide and xyloside. These experiments showed that the half-life of the precursor protein was also 75 min and that stimulation of heparan sulfate synthesis by xyloside was greatly enhanced (about 12-fold) after new protein core synthesis was blocked by cycloheximide. Although the structural models proposed for the EHS and colon carcinoma heparan sulfate proteoglycans differ, the observation that they are derived from a precursor protein with dimensional and immunological similarities suggests that they may be genetically related.

Topics: Ammonium Chloride; Blotting, Western; Carcinoma; Cell Line; Chondroitin Sulfate Proteoglycans; Colonic Neoplasms; Cycloheximide; Glycosaminoglycans; Glycosides; Heparitin Sulfate; Humans; Monensin; Polysaccharide-Lyases; Precipitin Tests; Protein Precursors; Protein Processing, Post-Translational; Proteoglycans; Trypsin

The human colon cancer cell line Caco-2 cultured in vitro displayed morphological differentiation which was shown to be a growth-related event. We have investigated this phenomenon further in relation to the cell surface glycosaminoglycans produced by growing (5-day, i.e., prior to differentiation) and confluent (9-day, i.e., after morphological and functional differentiation) cultures. Neosynthesized [35S]glycosaminoglycans were purified on DEAE-cellulose; at confluency, they were bound more strongly to the column than the corresponding fractions from the growing cells. Analysis of Kav values of heparan sulfate and chondroitin sulfates from growing and confluent cells indicated an increase in chain length of both glycosaminoglycans in morphologically differentiated cells. Heparan sulfate was the main 35S-labeled glycosaminoglycan of the cell surface of both 5-day and 9-day cultures. Paper chromatography of the unsaturated disaccharides obtained by chondroitinase digestion showed that chondroitin sulfate chains were primarily 6-sulfated in the 2 studied extracts. Heparan sulfate chains were isolated as chondroitinase-resistant material and treated with nitrous acid. Analysis of N- and O-sulfate group-related radioactivity showed an increase in the amount of 35S-label in the form of N-sulfate groups and an increase in the O-35S-sulfation pattern in heparan sulfate from morphologically differentiated cells. Thus, the structural features of both chondroitin sulfates and heparan sulfate were significantly different when the growing cells became morphologically differentiated.

Topics: Cell Differentiation; Chromatography, Gel; Chromatography, Ion Exchange; Colonic Neoplasms; Glycosaminoglycans; Heparitin Sulfate; Humans; Tumor Cells, Cultured

The metabolism of heparan sulfate proteoglycan, a major product of human colon carcinoma cells, was investigated in a series of pulse-chase experiments using a combination of quantitative biochemistry and electron microscope autoradiography. This was possible primarily because these cells incorporate [35S]sulfate exclusively into heparan sulfate proteoglycan, thus allowing the possibility of correlating the two sets of information. The results showed a progressive movement of the newly synthesized proteoglycan from the Golgi to the cell surface, where it became closely associated with the plasma membrane and was labeled ultrastructurally by both ruthenium red and radiosulfate. Subsequently, about 55% was released into the medium (t1/2 approximately 2.5 h) where it resided as intact macromolecule and was neither endocytosed nor degraded further. The remaining 45% was internalized and converted into smaller species through a series of degradative steps. Initially (Step 1) there was proteolytic cleavage of the protein core and partial endoglycosidic cleavage of the heparan sulfate chains (t1/2 approximately 6 h), with generation of larger glycosaminoglycan-peptide intermediates with chains of Mr approximately 10,000, about one-third their original size. These components were subsequently converted (Step 2) to yet smaller, limiting fragments of Mr approximately 5,000, which were finally depolymerized (Step 3) with quantitative release of free sulfate. The intracellular degradation of the proteoglycan, particularly Steps 2 and 3, was markedly inhibited by choloroquine, implicating the involvement of acidic compartments in the catabolism of these macromolecules. This was corroborated by the autoradiographic studies which showed the close association of 35S-labeled products with secondary lysosomes. However, the initial degradation of the proteoglycan might have occurred in a prelysosomal compartment since Step 1 was not totally blocked by chloroquine. The combined results indicate that the intracellular degradation of heparan sulfate follows structural as well as functional compartmentalization and provide a model that may be shared by other cell systems.

Topics: Autoradiography; Cell Line; Chondroitin Sulfate Proteoglycans; Colonic Neoplasms; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Kinetics; Microscopy, Electron; Models, Biological; Molecular Weight; Peptide Fragments; Proteoglycans

We have investigated the biosynthesis of heparan sulfate proteoglycan in human colon carcinoma cells cultured in either the absence of L-glutamine or the presence of 6-diazo-5-oxo-L-norleucine (DON), a glutamine analogue. Following a 24-h incubation with 100 micrograms/ml DON (0.58 mM) or without L-glutamine, the incorporation of [35S]sulfate was maximally inhibited to about 50%, whereas the incorporation of [3H]leucine or [3H]serine and their specific activity were not significantly affected. Several lines of evidence indicate that the inhibition of [35S]sulfate incorporation was mediated by a reduction in the intracellular pool of UDP-N-acetylhexosamine: the intracellular hexosamine levels were reduced by about 50%; taking into account the changes in specific activity, the incorporation of [3H]glucosamine was also significantly inhibited; and exogenous D-glucosamine (2.3 mM) was capable of substantially reversing the inhibitory effects of DON. This decrease in endogenous sugar supply resulted in the generation of an underglycosylated, lower buoyant density proteoglycan which contained significantly fewer heparan sulfate chains of otherwise normal size and sulfation and even fewer O-linked oligosaccharides. These biochemical changes were corroborated ultrastructurally by the appearance of smaller ruthenium red-stained proteoglycans on the surface of the cells. DON also caused a marked inhibition of cell proliferation and profound morphological changes, both of which were reversible upon culturing in DON-free medium. These results demonstrate that perturbations in glutamine metabolism have profound effects on the structure of heparan sulfate proteoglycan and on the phenotype of human colon carcinoma cells and indicate that DON treatment could be useful for studying post-translational modifications of proteoglycans in various cell systems.

Topics: Azo Compounds; Cell Division; Cells, Cultured; Centrifugation, Density Gradient; Chemical Phenomena; Chemistry; Chondroitin Sulfate Proteoglycans; Colonic Neoplasms; Diazooxonorleucine; Electrophoresis, Polyacrylamide Gel; Glucosamine; Glutamine; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Hexosamines; Microscopy, Electron; Oligosaccharides; Protein Biosynthesis; Proteoglycans; Sulfates

We demonstrate that the cell surface heparan sulfate proteoglycan of human colon carcinoma cells has an affinity for a hydrophobic matrix. This property is mediated by sequences in the core protein, since papain-or alkaline borohydride-released heparan sulfate chains do not bind to the matrix. Trypsin releases a [3H]leucine-rich, unsulfated, hydrophobic peptide, with Mr approximately 5000. This domain is present in neither the proteoglycan released into the medium nor in the intracellular degradation products. It is proposed that this peptide may represent the portion of the core protein intercalated into the plasma membrane.

Topics: Borohydrides; Cells, Cultured; Chondroitin Sulfate Proteoglycans; Colonic Neoplasms; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Humans; Leucine; Papain; Peptide Fragments; Proteoglycans; Trypsin

After 24 h of continuous labeling with radioactive precursors, a high molecular weight heparan sulfate proteoglycan (HS-PG) was isolated from both the medium and cell layer of human colon carcinoma cells (WiDr) in culture. The medium HS-PG eluted from a diethylaminoethyl anion exchange column with 0.45-0.50 M NaCl, had an average density of 1.46-1.49 g/ml on dissociative CsCl density-gradient ultracentrifugation, and eluted from Sepharose CL-2B with a Kav = 0.57. This proteoglycan had an estimated Mr of congruent to 8.5 X 10(5), with glycosaminoglycan chains of Mr = 3 X 10(4) which were all susceptible to HNO2 deaminative cleavage. Deglycosylation of the HS-PG with polyhydrogen fluoride resulted in a 3H-core protein with Mr congruent to 2.4 X 10(5). The cell layer contained a population of HS-PG with characteristics almost identical to that released into the medium but with a larger Mr = 9.5 X 10(5). Furthermore, an intracellular pool contained smaller heparan sulfate chains (Mr congruent to 1 X 10(4)) which were mostly devoid of protein core. In pulse chase experiments, only the large cell-associated HS-PG was released (approximately 58%) into the medium as intact proteoglycan and/or internalized and degraded (approximately 42%), with a t1/2 = 6 h. However, the small intracellular component was never released into the medium and was degraded at a much slower rate. When the cells were subjected to mild proteolytic treatment, only the large cell-associated HS-PG, but none of the small component, was displaced. Addition of exogenous heparin did not displace any HS-PG into the medium. Both light and electron microscopic immunocytochemistry revealed that the cell surface reacted with antibody against an HS-PG isolated from a basement membrane-producing tumor. Electron microscopic histochemistry using ruthenium red and/or cuprolinic blue revealed numerous 10-50-nm diam granules and 70-220-nm-long electron-dense filaments, respectively, on the surface of the tumor cells. The results indicate that colon carcinoma cells synthesize HS-PGs with distinct structural and metabolic characteristics: a large secretory pool with high turnover, which appears to be synthesized as an integral membrane component and localized primarily at the cell surface, and a small nonsecretory pool with low turnover localized predominantly within the cell interior. This culture system offers an opportunity to investigate in detail the mechanisms involved in the regulation of proteoglycan

Topics: Amino Acids; Cell Line; Cell Membrane; Chondroitin Sulfate Proteoglycans; Colonic Neoplasms; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparin; Heparitin Sulfate; Humans; Kinetics; Microscopy, Electron; Molecular Weight; Proteoglycans; Trypsin

A transplantable colorectal adenocarcinoma and the normal colonic mucosa derived from rats of ACI/N strain were digested with pronase, and the glycosaminoglycan fractions were obtained by fractionation with cetylpyridinium chloride. The glycosaminoglycan fraction derived from the adenocarcinoma contained substantial amounts of chondroitin sulfate A/C, dermatan sulfate, heparan sulfate, and hyaluronic acid, whereas chondroitin sulfate A/C and dermatan sulfate were undetectable in that derived from the normal colonic mucosa. An increment in the heparan sulfate content was also apparent in the adenocarcinoma, while the level of hyaluronic acid appeared to be unchanged. Analyses of the extract of the tumor tissue with 5mM ethylenediaminetetraacetate (pH 7.0) indicated that heparan sulfate was present largely, if not completely, in the form of proteoglycan.

Topics: Adenocarcinoma; Animals; Chemical Fractionation; Chondroitin Sulfates; Colon; Colonic Neoplasms; Dermatan Sulfate; Glycosaminoglycans; Heparitin Sulfate; Hyaluronic Acid; Mucous Membrane; Neoplasms, Experimental; Rats; Rats, Inbred ACI; Rectal Neoplasms

The glycosaminoglycans from normal colonic mucosa and colons with a variety of inflammatory diseases, as well as benign and malignant neoplasms were analyzed. Normal colonic mucosa contains predominantly chondroitin sulfates and dermatan sulfate. Increases in the levels of hyaluronic acid and heparan sulfate, as well as substantial increases in the amount of total glycosaminoglycans were characteristic of invasive colonic adenocarcinoma. Lesser elevations in the amount of total glycosaminoglycans and hyaluronic acid and heparan sulfate were present in neonatal colonic mucosa, villous adenoma, ulcerative colitis, and mucosa adjacent to carcinoma. The degree of elevation was proportional to the dysplastic potential. Since dysplastic lesions have scant connective tissue, the epithelial component of colonic neoplasms may contribute to these neoplasm-related alterations in glycosaminoglycan composition.

Topics: Adenocarcinoma; Adult; Aged; Chondroitin Sulfates; Colitis; Colon; Colonic Neoplasms; Dermatan Sulfate; Female; Glycosaminoglycans; Heparitin Sulfate; Humans; Hyaluronic Acid; Intestinal Mucosa; Male; Middle Aged

Normal, transitional, and carcinoma areas of five colons resected for carcinoma were examined morphologically, histochemically, and biochemically. The transitional area contained a larger amount of non-sulphated acid mucin than the normal mucosa as verified histochemically. Normal mucosa contained mainly sulphated mucin. The hexosamine-containing macromolecules present in different areas were isolated and characterized. They were divided into the following groups: 1) acid glycosaminoglycans, 2) high-molecular-weight glycopeptides, and 3) low-molecular weight glycopeptides. The concentration of the total hexosamine-containing material was in the carcinoma area twice as high as in normal areas. Acid glycosaminoglycans were identified as hyaluronate, heparan sulphate, dermatan sulphate, and chondroitin 4-(6)-sulphate. Their concentraitons were found to increase from normal to transitional and from transitional to carcinoma areas. The high-molecular-weight glycopeptide was composed of fucose, galactose, glucosamine, galactosamine, sialic acid, and variable amounts of sulphate. The sulphation degree of the glycopeptide was higher in normal mucosa than in transitional or carcinoma areas: The low-molecular-weight glycopeptides consisted of about a half of the total hexosamine-containing substances. The concentration of saline-insoluble fraction of the low-molecular-weight glycopeptides was in transitional areas about two times, and in carcinoma areas about four times, higher than in normal mucosa.

Topics: Adenocarcinoma; Chondroitin Sulfates; Colonic Neoplasms; Dermatan Sulfate; Fucose; Galactosamine; Galactose; Glucosamine; Glycopeptides; Glycosaminoglycans; Heparitin Sulfate; Hexosamines; Histocytochemistry; Humans; Hyaluronic Acid; Intestinal Mucosa; Macromolecular Substances; Molecular Weight; Mucins; Sialic Acids; Sulfates