heparitin-sulfate and Cell-Transformation--Viral

In patients with diabetic nephropathy, glomerular staining for heparan sulfate proteoglycans (HSPG) side chains and for agrin is decreased. In the present study, the influence of angiotensin II (AngII) on the production of HSPG in SV40 transformed podocytes was investigated. SV40 transformed human podocytes were cultivated with or without 1 microM AngII, and HSPG production was measured by sequential DEAE-anion exchange chromatography and HPLC-DEAE separation. Expression of agrin was studied by indirect immunofluorescence and Western blot analysis using specific mono- and polyclonal antibodies. DEAE separation of total glycosaminoglycans (GAG) revealed a significant increase of GAG in the culture supernatant and decrease in the cell and matrix layer when podocytes were cultured for 72 h in the presence of AngII. This was particularly found for HS-GAG. Qualitative analysis of HSPG, using gel filtration of HNO(2)-treated fractions, showed that AngII treatment decreased N-sulfation of HS-GAG side chains. Indirect immunofluorescence staining with anti-agrin polyclonal antibody was strongly decreased after AngII stimulation. A reduction in agrin expression in cell extracts could also be detected in Western blot analysis using an mAb. No changes in agrin mRNA were found after AngII stimulation. It is concluded from this study that AngII decreases the amount of HSPG on the cell surface and in the extracellular matrix of podocytes. Because HSPG play a fundamental role in the permselectivity of the glomerular basement membrane, these results thus may explain at least partially the antiproteinuric effects of angiotensin-converting enzyme inhibition in patients with diabetic nephropathy.

Topics: Agrin; Cell Transformation, Viral; Cells, Cultured; Epithelial Cells; Glycosaminoglycans; Heparitin Sulfate; Humans; Kidney Glomerulus; Proteoglycans; Receptor, Angiotensin, Type 1; Simian virus 40; Urothelium

Topics: Animals; Carbohydrate Conformation; Cell Transformation, Viral; Fibroblasts; Heparitin Sulfate; Iduronic Acid; Molecular Structure; Spectrophotometry, Infrared

The replica filter technique has been used to isolate variants of Swiss mouse 3T3 cells which produce heparan sulfates with altered levels of sulfation. These changes in the extent of sulfation correlate with alterations in cell morphology, in the organization of cytoskeletal elements, focal contacts, and the extracellular matrix, and in the growth regulation of cells, as expressed by saturation density. An increase in the extent of heparan sulfate sulfation occurs concomitantly with a decreased saturation density and enhanced focal contact formation. In contrast, graded decreases in sulfation correlate with graded increases in saturation density and losses of cytoskeletal and extracellular matrix organization. These graded responses appear very similar to those which have been reported for the transformation of cells with fusiform mutants of Rous sarcoma virus or the adenovirus type 2 Ela transforming gene and suggest that the morphological changes observed in the transformed cells can be controlled by cellular systems.

Topics: Animals; Cell Adhesion; Cell Line; Cell Transformation, Viral; Chondroitin Sulfate Proteoglycans; Fibroblasts; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Mice; Mutation; Proteoglycans; Simian virus 40

We have isolated a syngeneic monoclonal antibody (HepSS-1) reactive to a murine methylcholanthrene-induced fibrosarcoma, Meth-A. HepSS-1 also bound to a wide variety of established and fresh normal cells derived from not only mice but also other species such as human, monkey, rat, hamster, and chicken. Immunoprecipitation of surface iodinated Meth-A cell extract with HepSS-1, as well as Sepharose 4B gel chromatography of Meth-A cell extract and detection of antigens recognized by HepSS-1 by a sandwich-type radioimmunoassay revealed that the HepSS-1 antigens were composed of several molecular species, with one as large as approximately 10(6) daltons. The following evidence indicates that HepSS-1 specifically recognizes an epitope present in heparan sulfate glycosaminoglycan (HS-GAG). First, treatment of Meth-A cells with heparitinase or heparinase, but not with chondroitinase ABC or hyaluronidase, resulted in the loss of HepSS-1 binding. Second, HS-GAG but not seven other types of GAG (hyaluronic acid, heparin, chondroitin, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, and keratan sulfate) inhibited HepSS-1 binding to Meth-A cells. Third, HepSS-1 bound with HS-GAG but not with the seven other types of GAG. From the binding analysis of HepSS-1 to various modified HS-GAG and whale omega-heparin, it is additionally suggested that HepSS-1 recognizes an epitope closely related to O-sulfated and N-acetylated glucosamine. We found that NIH 3T3 cells expressed more HepSS-1 epitopes at a low cell density than at confluency and in G2 + M than in G1, whereas NIH 3T3 cells transformed with Kirsten-ras oncogene or SV-40 expressed high levels of HepSS-1 epitopes and ceased to show the density-dependent change in the amount of HepSS-1 epitopes. These observations were also reproduced by using NIH 3T3 cells transformed with a temperature sensitive Kirsten murine sarcoma virus maintained at permissive and non-permissive temperatures. Thus HepSS-1 is a first monoclonal antibody to HS-GAG and seems to be useful to elucidate changes in cell surface HS-GAG in normal cell growth and cell transformation.

Topics: Animals; Antibodies, Heterophile; Antibodies, Monoclonal; Antibodies, Neoplasm; Cell Cycle; Cell Transformation, Viral; Chickens; Cricetinae; Epitopes; Fibrosarcoma; Glycosaminoglycans; Heparitin Sulfate; Humans; Kirsten murine sarcoma virus; Macaca mulatta; Mice; Rats; Sarcoma, Experimental; Species Specificity

35S-Labelled heparan sulfates derived from the culture medium (extracellular), a trypsinate of the cells (pericellular) and the cell residue (intracellular) of quiescent normal, proliferating normal or SV40-transformed 3T3 cells were analyzed for charge heterogeneity, by ion exchange chromatography and for self-affinity, by chromatography on heparan sulfate-agarose gels. Quiescent normal cells retained most of their heparan sulphate intra- or pericellularly. The surface-exposed material was charge heterogeneous and had a strong affinity for heparan sulfate. In cultures of growing cells and transformed cells most of the heparan sulfate was found in the medium. The heparan sulfate retained on the surface or growing cells had a lower self-affinity than did the corresponding material from normal and transformed cells. Although cell surface heparan sulfates from transformed cells showed affinity for a matrix substituted with the total heparan sulfate pool, the affinity for one particular subtype was much less pronounced or non-existent.

Topics: Animals; Cattle; Cell Transformation, Viral; Chromatography, Affinity; Culture Media; Fibroblasts; Glycosaminoglycans; Heparitin Sulfate; Macromolecular Substances; Mice; Mice, Inbred BALB C; Simian virus 40

Changes in glycosaminoglycans (GAGs) were investigated in relation to cell density, growth and transformation of human fibroblasts. Relative amounts (percentages of the total GAGs) of heparan sulfate (HS) increased and those of hyaluronic acid (HA) decreased in growth-reduced (serum-starved, exogenous HS-treated and dense) cultures of normal (WI-38) cells. In contrast, transformed (WI-38 CT-1) cells exerted such GAG changes only in serum-starved cultures, but not in HS-treated or dense cultures. These results indicate that the changes in glucosaminoglycans (G1cAGs) (HS and HA) is coupled exclusively with cell growth.

Topics: Animals; Cell Count; Cell Division; Cell Transformation, Viral; Culture Media; Fibroblasts; Glucosamine; Glycosaminoglycans; HeLa Cells; Heparitin Sulfate; Humans; Hyaluronic Acid; Mice; Mice, Inbred BALB C; Rabbits; Serum Albumin, Bovine

Both newly formed and long-term culture-generated substratum adhesion sites, generated by EGTA-mediated detachment of Balb/c SVT2 cells, were extracted with an eta-octyl-beta-D-glucopyranoside buffer containing salt and several protease inhibitors under conditions which result in maximal solubilization of the sulfate-radiolabeled proteoglycans. Because of the functional importance of heparan sulfate proteoglycans in the fibronectin-dependent cell-substratum adhesion processes of these cells, these proteoglycans were fractionated on affinity columns of octyl-Sepharose or of the heparan sulfate-binding proteins platelet factor 4 or plasma fibronectin. These affinity matrices resolved a number of both binding and nonbinding classes of heparan sulfate proteoglycan from both types of adhesion sites. In particular, the platelet factor 4 column could resolve several proteoglycans with differing binding affinities. Approximately twice as much heparan sulfate proteoglycan from newly formed sites bound to all three matrices as proteoglycan from longterm sites. The proteoglycan which bound to one matrix was then tested for binding to a second matrix; this approach resolved a number of biochemically distinct species. For example, one-half of the fibronectin-Sepharose-binding fraction from the long-term sites could also bind to platelet factor 4-Sepharose; however, over 90% of the fibronectin-binding fraction from newly formed sites could bind to platelet factor 4. A major portion of the octyl-Sepharose-binding fractions of the original extracts could bind to fibronectin-Sepharose. These studies indicate that some of these proteoglycans have overlapping affinities for fibronectin, platelet factor 4, and octyl-Sepharose and that a portion of the heparan sulfate proteoglycan from these adhesion sites cannot bind to any of these affinity matrices. These results are discussed with regard to the functional significance of these various heparan sulfate proteoglycans in mediating adhesion to extracellular matrices containing fibronectin or platelet factor 4.

Topics: Animals; Cell Adhesion; Cell Transformation, Viral; Chondroitin Sulfate Proteoglycans; Chromatography, Affinity; Fibroblasts; Fibronectins; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Mice; Mice, Inbred BALB C; Platelet Factor 4; Proteoglycans; Sepharose; Simian virus 40; Surface Properties

Chick embryo skin fibroblasts and vertebral chondroblasts were infected with a temperature-sensitive mutant of Rous sarcoma virus, LA24A, and were grown at permissive (36 degrees C) and nonpermissive (41 degrees C) temperatures. During exponential growth, infected and parallel uninfected cultures were labeled with D-[3H]glucosamine, and newly synthesized glycosaminoglycans were identified by anion exchange chromatography and by selective enzymatic and chemical degradations. Control fibroblasts synthesized low levels of hyaluronic acid (HA), and dermatan sulfate (DS), moderate levels of heparan sulfate (HS), and high levels of chondroitin sulfate (CS). In contrast, control chondroblasts synthesized very low levels of HA and DS, no HS, and very high levels of CS. Following transformation and growth at 36 degrees C, both cell types showed a dramatic increase in HA synthesis and a significant decrease in CS synthesis. In addition, transformed chondroblasts initiated the synthesis of HS and increased their synthesis of DS to levels that matched those of transformed fibroblasts. The CS chains synthesized by control chondroblasts were partially undersulfated, while those synthesized by both normal and transformed fibroblasts were fully sulfated. Upon transformation, chondroblasts grown at 36 degrees C initiated the synthesis of fully sulfated CS chains. Most of the above biosynthetic alterations were completely reversed when infected cells were grown at 41 degrees C, indicating that they were dependent on the transforming gene product of LA24A. Clearly, the profound differences that distinguish normal fibroblasts from normal chondroblasts are lost upon transformation, and these two types of terminally differentiated cells converge toward a common, though not identical, biosynthetic program for glycosaminoglycans.

Topics: Animals; Avian Sarcoma Viruses; Cartilage; Cell Transformation, Viral; Chick Embryo; Chondroitin Sulfates; Dermatan Sulfate; Fibroblasts; Glycosaminoglycans; Heparitin Sulfate; Hyaluronic Acid; Sarcoma, Avian; Skin

We used antibodies raised against both a heparan sulfate proteoglycan purified from a mouse sarcoma and a chondroitin sulfate proteoglycan purified from a rat yolk sac carcinoma to study the appearance and distribution of proteoglycans in cultured cells. Normal rat kidney cells displayed a fibrillar network of immunoreactive material at the cell surface when stained with antibodies to heparan sulfate proteoglycan, while virally transformed rat kidney cells lacked such a surface network. Antibodies to chondroitin sulfate proteoglycan revealed a punctate pattern on the surface of both cell types. The distribution of these two proteoglycans was compared to that of fibronectin by double-labeling immunofluorescent staining. The heparan sulfate proteoglycan was found to codistribute with fibronectin, and fibronectin and laminin gave coincidental stainings. The distribution of chondroitin sulfate proteoglycan was not coincidental with that of fibronectin. Distinct fibers containing fibronectin but lacking chondroitin sulfate proteoglycan were observed. When the transformed cells were cultured in the presence of sodium butyrate, their morphology changed, and fibronectin, laminin, and heparan sulfate proteoglycan appeared at the cell surface in a pattern resembling that of normal cells. These results suggest that fibronectin, laminin, and heparan sulfate proteoglycan may be complexed at the cell surface. The proteoglycan may play a central role in assembly of such complexes since heparan sulfate has been shown to interact with both fibronectin and laminin.

Topics: Animals; Butyrates; Cell Membrane; Cell Transformation, Viral; Cells, Cultured; Chondroitin Sulfate Proteoglycans; Extracellular Space; Fibronectins; Fluorescent Antibody Technique; Glycoproteins; Glycosaminoglycans; Heparitin Sulfate; Laminin; Proteoglycans; Rats; Surface Properties

Topics: Animals; Avian Sarcoma Viruses; Cell Transformation, Viral; Cells, Cultured; Culture Media; Fibronectins; Fluorescent Antibody Technique; Glycoproteins; Glycosaminoglycans; Heparitin Sulfate; Laminin; Oncogene Protein pp60(v-src); Peptides; Rats; Temperature; Viral Proteins

1. Heparan sulphates from normal 3T3 fibroblasts are association-prone as indicated by their affinity for agarose gels substituted with cognate heparan sulphate species. Heparan sulphates from SV40-transformed or polyoma-virus-transformed cells have no affinity for the same gels. 2. Heparan sulphates from the medium, the pericellular and intracellular pools of normal, SV40-transformed and polyoma-transformed 3T3 cells were separated into four subfractions (HS1-HS4) by ion-exchange chromatography. In general, HS1-HS3 were found in cell-derived heparan sulphates, whereas HS3-HS4 were present in the medium. The heparan sulphates from transformed cells were more heterogeneous and of lower charge density than those from the normal counterpart. 3. Degradations via periodate oxidation/alkaline elimination yielded the oligomers glucosamine-(hexuronate-glucosamine)(n)-R with n=1-5 and a large proportion of N-sulphate groups. There was a large contribution of fragments n=4-5 from heparan sulphates of normal cells. These fragments were less common in low-sulphated heparan sulphates of transformed cells. In the case of medium-drived heparan sulphates all species had a low content of fragments n=4-5. 4. The size distribution of (glucuronate-N-acetylglucosamine)(n) regions was assessed after deaminative cleavage. It was broad and ranged from n=1-10 for all heparan sulphate species. In the case of medium-derived heparan sulphates there were distinct differences between normal and transformed cells. In the latter chains the N-acetyl-rich segments were both shorter and longer than in the normal case. The shape of the disaccharide peak was consistent with a lower content of O-sulphate in the heparan sulphates from transformed cells. 5. It was concluded that heparan sulphates from medium or transformed cells exhibit the greatest structural deviation from the normal case. The finding of lower proportions of extended, iduronate/glucuronate-bearing, N-sulphate-rich segments in heparan sulphates of transformed cells was particularly interesting in view of the fact that these elements have been associated with ability to self-interact.

Topics: Animals; Biopolymers; Cell Line; Cell Transformation, Viral; Chromatography, Gel; Chromatography, Ion Exchange; Fibroblasts; Glycosaminoglycans; Heparitin Sulfate; Mice; Mice, Inbred BALB C; Polyomavirus; Simian virus 40

Topics: Animals; Cell Adhesion; Cell Transformation, Viral; Cells, Cultured; Chondroitin Sulfates; Fibroblasts; Glycoproteins; Guanidine; Guanidines; Heparitin Sulfate; Hyaluronic Acid; Hyaluronoglucosaminidase; Mice; Mice, Inbred BALB C; Simian virus 40; Solubility

Fibronectin, previously also termed LETS-protein, is a high-molecular-weight protein (mol. w. ca. 450,000) present in the form of thin fibrils in the pericellular space of fibroblasts and other adherent cells, as well as in distinct areas of the connective tissue. A soluble form, immunologically identical and chemically at least very similar to the cell-attached protein, is found in plasma in a concentration of about 300 micrograms/ml. It is also denominated cold-insoluble globulin. The protein has affinity both to cell surfaces and to various matrix substances such as fibrin and collagen and, therefore, is capable of mediating cell attachment to these substrates. In addition, it serves as an opsonin for the phagocytosis of gelatin-containing compounds and probably is essential for the removal of soluble fibrin from the circulating blood by the reticulo-endothelial system. Bacterial cell walls are also recognized by fibronectin. A conversion of soluble fibronectin to fibrils is achieved by heparin which also enhances the binding of soluble fibronectin to cells. Heparin or, as suggested, the related heparan sulfate present on the surface of various cells, appears to function as a cofactor in the formation of pericellular fibrils. The fibronectin fibrils precipitated with heparin, compared to soluble fibronectin, show a considerably improved affinity to native collagen, especially to type III. Hyaluronic acid has an antagonistic function which, at higher concentrations, prevents the fibronectin fibrils from interacting with collagen and cell surfaces. Masking of fibronectin fibrils was also achieved by sulfated proteoglycans of cartilage. Virus-transformed fibroblasts produce less fibronectin and are less capable of maintaining surface pericellular fibrils. A reasonable explanation is that they have an elevated secretion of hyaluronic acid. The transformed cells attach only weakly to a surface and exhibit a rounded shape in contrast to healthy ones. This phenotype can be corrected to a great extent with fibronectin. It is suggested that fibronectin also influences the formation of connective tissue by accumulating collagen precursors on the surface of fibroblasts and facilitating fibrillogenesis.

Topics: Cell Adhesion; Cell Communication; Cell Transformation, Viral; Chemical Phenomena; Chemistry; Collagen; Connective Tissue; Fibroblasts; Fibronectins; Heparin; Heparitin Sulfate; Humans; Isoelectric Point; Membrane Proteins; Microscopy, Electron; Molecular Weight; Receptors, Cell Surface; Receptors, Fibronectin; Spermine

A cloned embryonic mouse cell line contained specific cell-surface receptors for heparin and both the number and affinity appeared to be unchanged in a simian-virus-40-transformed subclone. In competitive binding assays heparan sulphate from the control clone was bound preferentially compared to that from the transformed subclone, indicating that the altered sulphation of heparan sulphate from transformed cells results in a lowered affinity for cell-surface receptors. Evidence was obtained suggesting that endogenous proteoglycans were not held at the cell surface by binding to these receptors alone. However the possibility that proteoglycans embedded in the plasma membrane may interact with the receptor has not been ruled out.

Topics: Animals; Binding Sites; Cell Transformation, Viral; Cells, Cultured; Glycosaminoglycans; Heparin; Heparitin Sulfate; Kinetics; Mice; Simian virus 40; Sulfur Radioisotopes

Cell lines, selected from two independent clones of an established mouse embryo cell line by their ability to grow as solid tumors in immunocompetent syngeneic hosts, were found to have the same alteration in anion exchange properties as was previously reported for simian virus 40 (SV40)-transformed subclones. One tumor cell line (219CT) and one SV40-transformed subclone (215CSC) were selected for further detailed comparison with their common parent clone (210C). Cellulose acetate electrophoresis at pH 1.0 showed that 215CSC heparan sulfate had a slight overall decrease in sulfation compared with heparan sulfate from 210C; however, no gross difference in sulfation could be detected between heparan sulfate from 219CT and 210C. Analysis of the products of deaminative cleavage of heparan sulfate by nitrous acid under conditions where cleavage occurs quantitatively at N-sulfated glucosamine residues showed that, although heparan sulfate from the three cell lines gave similar yields of O-sulfated disaccharides, both 215CSC and 219CT had only about half as many O-sulfate residues in higher molecular weight oligosaccharides compared to heparan sulfate from 210C. Enzymatic degradation of heparan sulfate with a mixture of enzymes from Flavobacterium heparinum showed that this common alteration in heparan sulfate from both 215CSC and 219CT resulted from a 30% decrease in glucosamine residues bearing 6-O-sulfate groups. As this decrease in 6-O-sulfate glucosamine residues occurs in regions of the chain containing relatively few sulfate groups, it is clear that certain sequences of charged groups present in heparan sulfate frm 210C will be found only rarely in heparan sulfate from 215CSC and 219CT. It is suggested that this will result in alterations of the interaction of heparan sulfate with other molecules in the microenvironment at the cell surface which may be important in the control of such phenomena as cell growth and adhesion.

Topics: Animals; Cell Line; Cell Transformation, Viral; Clone Cells; Embryo, Mammalian; Glycosaminoglycans; Heparitin Sulfate; Kinetics; Mice; Oligosaccharides; Simian virus 40; Sulfates; Sulfur Radioisotopes; Trypsin

Topics: Animals; Cell Line; Cell Transformation, Viral; Cells, Cultured; Fibroblasts; Glycosaminoglycans; Heparitin Sulfate; Humans; Lung; Mice; Polyomavirus; Sepharose; Simian virus 40

A difference in the extent of sulfation between the heparan sulfate isolated from Swiss 3T3 mouse cells and that from Swiss 3T3 cells transformed by the DNA virus SV40 has been reported previously. This variance is manifested by different chromatographic and electrophoretic properties. Heparan sulfates from the two cell types were treated with nitrous acid under conditions that gave selective deaminative cleavage of glucosaminyl residues with sulfated amino groups in order to define the nature of the difference in sulfation further. The O-sulfate containing fragments from the heparan sulfates were compared by gel filtration and ion-exchange chromatography. The results showed that the 3T3 heparan sulfate contains 8% more O-sulfate than does the SV3T3 heparan sulfate. Analysis of uronic acids revealed that both types of heparan sulfates contain 45% L-iduronic acid and 55% D-glucuronic acid. These and other observations indicate that the primary difference in sulfation between the 3T3 and SV3T3 heparan sulfates lies in the extent of O-sulfation.

Topics: Animals; Carbon Radioisotopes; Cell Line; Cell Transformation, Viral; Chromatography, Ion Exchange; Glycosaminoglycans; Heparitin Sulfate; Isotope Labeling; Mice; Simian virus 40; Sulfur Radioisotopes; Sulfuric Acids; Tritium

Topics: Adult; Animals; Cell Division; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Chemical Phenomena; Chemistry; Chondroitin Sulfates; Dogs; Glycosaminoglycans; Guinea Pigs; Heparitin Sulfate; Humans; Liver; Mice; Rabbits; Regeneration; Swine; Tissue Distribution

Three major pools of heparan sulfate have been isolated from cultures of Swiss mouse 3T3 and SV40-transformed 3T3 cells: cell-surface, medium, and intracellular heparan sulfates. The cell-surface heparan sulfate is a high molecular weight proteoglycan which is partially degraded by pronase. Before pronase treatment, it has a peak molecular weight (as estimated by gel filtration) of approx. 7.2 . 10(5) in contrast to only 2.4 . 10(5) after pronase treatment. The medium heparan sulfate appears to be similar in structure to the cell-surface heparan sulfate, since they coelute on Bio-Gel A-15m and DEAE-cellulose, and are both proteoglycans. In contrast, the intracellular heparan sulfate has a low molecular weight (6.0 . 10(3)) and has little if any attached protein. Both the medium and intracellular heparan sulfate exhibit the transformation-associated change in structure reported earlier for cell-surface heparan sulfate (Underhill, C.B. and Keller, J.M. )1975) Biochem. Biophys. Res. Commun. 63, 448--454). This transformation-associated change, detected by DEAE-cellulose chromatography is not the result of changes in either molecular weight or protein core. Cellulose acetate electrophoresis of the cell-surface heparan sulfate at pH 1 suggests that the transformation-associated change in structure is due to a difference in sulfate content. Both types of heparan sulfate are produced in mixed cultures of 3T3 and SV3T3 cells, indicating that neither serum factors in the culture medium nor secreted cell products are responsible for the transformation-associated change in heparan sulfate structure. The presented data are discussed with respect to the postulated role of heparan sulfate in cell social behavior.

Topics: Animals; Cell Line; Cell Membrane; Cell Transformation, Viral; Culture Media; Fibroblasts; Glycosaminoglycans; Heparitin Sulfate; Mice; Molecular Weight; Pronase; Simian virus 40

The medium and cell surface heparan sulfates isolated from SV40-transformed Swiss mouse 3T3 cells were examined in the presence and absence of 1.0 mM p-nitrophenyl-beta-D-xyloside. Incubation of the SV3T3 cells with this beta-xyloside resulted in: (a) a 4- to 5-fold reduction in the molecular weight distribution of medium heparan sulfate, (b) a 10-fold increase in the total synthesis of medium heparan sulfate, and (c) a small reduction in cell growth. There was little, if any, change in either the total level of synthesis or the molecular weight distribution of cell surface heparan sulfate. The covalent association of the beta-xyloside to the medium heparan sulfate was demonstrated by an analysis of the medium heparan sulfate produced by cells grown in the presence of [35S]sulfate and the fluorogenic beta-xyloside, 4-methylumbelliferyl-beta-D-xyloside. Treatment of the purified radiolabeled and fluorogenic heparan sulfate with either nitrous acid or heparitinase resulted in a decrease in the molecular weight of both radiolabeled and fluorogenic material. The data presented in this paper are discussed with respect to both the structure of heparan sulfate and the putative role of heparan sulfate in cell social behavior.

Topics: Animals; Cell Line; Cell Membrane; Cell Transformation, Viral; Glycosaminoglycans; Glycosides; Heparitin Sulfate; Mice; Simian virus 40; Xylose

Glycoconjugates have been analyzed from a family of closely related mouse cells: a parent clone and three daughter subclones, two of which expressed the simian virus 40 (SV40) T-antigen. The experimental procedure involved the simultaneous comparison by DEAE-cellulose chromatography of papain-digested macromolecules from the parent, labeled with [3H]glucosamine, and one of the daughter subclones, labeled with [14C]-glucosamine. Three cultures compartments (the medium, the cell surface trypsinate, and the cells) from the paired cell lines were combined at the earliest time during the harvesting of the cells. Heparan sulfate on the surface of cells and secreted into the medium from T-antigen-positive subclones was eluted at lower salt concentrations from the anion exchange column than that from the parent clone. In the viable trypsinized cells a marked reduction of heparan sulfate was detected in the T-antigen-positive subclones. These changes were highly reproducible, were observed during both logarithmic and stationary phase of growth, and neither change was observed in the T-antigen-negative sister subclone. The elution point of heparan sulfate from Sepharose 6B was unaltered. Ratios of 35S to 3H for heparan sulfate obtained from cells doubly labeled with [35S]sulfate and [3H]glucosamine were lower in the T-antigen-positive subclones. Similar changes for the 35S to 3H ratio of chondroitin sulfate were associated with only small alterations in elution from anion exchange columns. Kinetic experiments suggested a reduced rate of incorporation of [35S]sulfate with no change in turnover rate. A substantial portion of the labeled heparan sulfate was associated with the cell surface; in contrast most of the hyaluronic acid and a large proportion of the chondroitin sulfate was apparently secreted. Quantitative changes in hyaluronic acid labeling did not correlate with expression of T-antigen. Glycosaminoglycans left on the dish after detaching cells with ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid were nearly completely released by subsequent trypsinization. Cell detachment by trypsinization left an insignificant amount of labeled glycosaminoglycan on the dish surface. The alterations in heparan sulfate metabolism correlated with the expression of T-antigen and with the cells' ability to grow to high densities in monolayer culture, but not with growth in suspension in viscous medium. Tumorigenicity of the subclones was essentially the same a

Topics: Antigens, Viral; Cell Line; Cell Transformation, Viral; Glucosamine; Glycosaminoglycans; Heparitin Sulfate; Kinetics; Simian virus 40; Sulfates

Footpad adhesion sites pinch off from the rest of the cell surface during EGTA-mediated detachment of normal or virus-transformed murine cells from their tissue culture substrates. In these studies, highly purified trypsin and testicullar hyaluronidase were used to investigate the selective destruction or solubilization of proteins and polysaccharides in this substrate-attached material (SAM). Trypsin-mediated detachment of cells or trypsinization of SAM after EGTA-mediated detachment of cells resulted in the following changes in SAM composition: (a) solubilization of 50-70% of the glycosaminoglycan polysaccharide with loss of only a small fraction of the protein, (b) selective loss of one species of glycosaminoglycan-associated protein in longterm radiolabeled preparations, (c) no selective loss of the LETS glycoprotein or cytoskeletal proteins in longterm radiolabeled preparations, and (d) selective loss of one species of glycosaminoglycan-associated protein, a protion of the LETS glycoprotein, and proteins Cd (mol wt 47,000 and Ce' (mol wt 39,000) in short term radiolabeled preparations. Digestion of SAM with testicular hyaluronidase resulted in: (a) almost complete solubilization of the hyaluronate and chondroitin sulfate moieties from long term radiolabeled SAM with minimal loss of heparan sulfate, (b) solubilization of a small portion of the LETS glycoprotein and the cytoskeletal proteins from longterm radiolabeled SAM, (c) resistance to solubilization of protein and polysaccharide in reattaching cell SAM which contains principally heparan sulfate, and (d) complete solubilization of the LETS glycoprotein in short term radiolabeled preparations with no loss of cytoskeletal proteins. Thus, there appear to be two distinct pools of LETS in SAM, one associated in some unknown fashion with hyaluronate-chondroitin sulfate complexes, and a second associated with some other component in SAM, perhaps heparan sulfate. These data, together with other results, suggest that the cell-substrate adhesion process may be mediated principally by a heparan sulfate--LETS complex and that hyaluronate-chondroitin sulfate complexes may be important in the detachability of cells from the serum-coated substrate by destabilizing LETS matrices at posterior footpad adhesion sites.

Topics: Animals; Cell Adhesion; Cell Line; Cell Separation; Cell Transformation, Neoplastic; Cell Transformation, Viral; Chondroitin Sulfates; Glycoproteins; Glycosaminoglycans; Heparitin Sulfate; Hyaluronic Acid; Mice; Neoplasm Proteins; Solubility