Functional changes in cellular fibronectin from late passage fibroblasts in vitro

Analysis of fibronectin synthesized by human fibroblasts, at different times during serial subcultivation, reveals functional differences. Fibronectin isolated from late passage cells is defective in promoting cell adhesion, cell spreading, and the formation of focal contacts. These changes are not the result of an inability of late passage cells to interact with fibronectin, since late passage cells become adhesive and form focal contacts in the presence of fibronectin isolated from early passage cells. Therefore, we conclude that late passage cellular fibronectin derived from late passage cells cannot support the cell substrate interactions.     

Enhanced synthesis of a Mr = 55,000 dalton peptide by senescent human fibroblasts

The secretory protein profiles of early and late passage cultures of human fibroblasts were compared using polyacrylamide gel electrophoresis. In comparison with early passage cell cultures (40-50% lifespan completed), late passage (greater than 80% of lifespan completed) cell cultures exhibited enhanced production of several peptides in the Mr range 55-60,000. One of those peptides had an apparent molecular weight of Mr = 55,000 and was constitutively present in the late passage cell conditioned medium. Late passage cell cultures synthesized the Mr = 55,000 peptide in the presence or absence of fetal bovine serum. Serum did not enhance its production by early passage cells. Further, production of the peptide was not induced in early passage cell cultures whose proliferation was arrested either by serum starvation or by contact inhibition. Pulse chase studies demonstrated that the peptide appears in the culture medium within 60 min of labeling. There was no evidence that it is derived via degradation of other proteins present either in early passage or late passage cell conditioned media. Further, the production of the 55,000 dalton peptide did not appear to be regulated by factors present in conditioned media. The peptide was detected in the conditioned media produced by late passage cultures of several different cell strains.     

The effect of a collagen tripeptide fragment (GER) on fibroblast adhesion and spreading depends on properties of an adhesive surface

The effect of collagen tripeptide fragment GER on the adhesion and spreading of mouse embryonic fibroblasts STO to different substrates (polystyrene plastic, poly-L-lysine, fibronectin, gelatin) has been studied. It was found that tripeptide GER was involved in fibroblast adhesion and spreading. The cell response depended both on the mode of tripeptide addition to culture medium and the substrate type. Coincubation of fibroblasts with tripeptide stimulated the cell attachment and spreading to untreated plastic and plastic coated with fibronectin or gelatin but did not change cell adhesion to immobilized poly-L-lysine. Preincubation of cells with tripeptide resulted in partial inhibition of fibroblast adhesion and spreading on fibronectin- and gelatin-coated substrata. It was shown that activation and inhibition of adhesive processes after tripeptide treating was higher on fibronectin than gelatin. The data obtained support the assumption about concerted action of tripeptide GER (activity was dependent both on the used concentration of the tripeptide and the mode of tripeptide addition to culture medium) and chemical characteristics of substrate (polymers of styrene and L-lysine, ECM proteins in native (fibronectin) or partly denatured (gelatin) form) on the cell adhesion and spreading. The main targets that GER peptide may affect during the formation of cell-substrate interactions are discussed.     

A GTPase controls cell-substrate adhesion in Xenopus XTC fibroblasts

Cell-substrate adhesion is crucial at various stages of development and for the maintenance of normal tissues. Little is known about the regulation of these adhesive interactions. To investigate the role of GTPases in the control of cell morphology and cell-substrate adhesion we have injected guanine nucleotide analogs into Xenopus XTC fibroblasts. Injection of GTP gamma S inhibited ruffling and increased spreading, suggesting an increase in adhesion. To further investigate this, we made use of GRGDSP, a peptide which inhibits binding of integrins to vitronectin and fibronectin. XTC fibroblasts injected with non-hydrolyzable analogs of GTP took much more time to round up than mock-injected cells in response to treatment with GRGDSP, while GDP beta S-injected cells rounded up in less time than controls. Injection with GTP gamma S did not inhibit cell rounding induced by trypsin however, showing that cell contractility is not significantly affected by the activation of GTPases. These data provide evidence for the existence of a GTPase which can control cell-substrate adhesion from the cytoplasm. Treatment of XTC fibroblasts with the phorbol ester 12-o-tetradecanoylphorbol-13-acetate reduced cell spreading and accelerated cell rounding in response to GRGDSP, which is essentially opposite to the effect exerted by non-hydrolyzable GTP analogs. These results suggest the existence of at least two distinct pathways controlling cell-substrate adhesion in XTC fibroblasts, one depending on a GTPase and another one involving protein kinase C.     

Cultivation of mammalian cells in serum-free media]

P3 cell lines can be grown in protein- and lipid-free synthetic medium. Using the P3 cell culture, we have shown that these cells produce autocrine growth factors and cell-substrate attachment factors. Because the cultured cells produce proteinase-inhibitor, spent medium is applicable for inactivating the action of trypsin at the time of cell passage. In addition, we have tried to cultivate various types of cells in serum-free media on the market (ASF103, ASF104 and GIT). Many cell lines can grow in these media, but inoculum dependency is observed in some cell lines. Production of monoclonal antibody by a hybridoma cell line is rather enhanced in these media. These media can be preserved at 4 degrees C or -20 degrees C for a relatively long period. These media added with EGF support the growth of Syrian hamster embryo cells at an early passage. The growth of human diploid fibroblasts in GIT medium added with EGF is a little less compared in a serum-containing medium.     

Binding of soluble fibronectin and its subsequent incorporation into the extracellular matrix by early and late passage human skin fibroblasts

The specific binding of soluble 125I-labeled human plasma fibronectin (125I-HFN-P) to confluent cultures of early and late passage human skin fibroblasts was investigated. Previous studies of HFN-P bound to fibroblast cell layers indicated that HFN-P was present in the cultures in two separate pools, distinguishable on the basis of their solubility in 1% deoxycholate. Pool I contained deoxycholate-soluble fibronectin (cell-associated), whereas Pool II contained deoxycholate-insoluble fibronectin (matrix-associated). Time course studies indicated that HFN-P was initially incorporated into Pool I and then accumulated into Pool II (McKeown-Longo, P.J., and Mosher, D.F. (1983) J. Cell Biol. 97, 466-472). Examination of the kinetics of 125I-HFN-P binding to Pool I of early and late passage cultures revealed that both cultures required 2-4 h to approach steady-state conditions. Other kinetic studies showed that the rates of loss of 125I-HFN-P from either Pool I or Pool II were similar for both cultures. However, the late passage cultures bound greater than twice as much fibronectin into Pool I, per cell, than the early passage cultures. This difference was not related to a difference in the level of endogenously produced fibronectins accumulating in the medium. Late passage cultures incorporated 125I-HFN-P into the deoxycholate-insoluble Pool at an average rate 2.6 times greater than early passage cultures. The late passage cultures also chased a greater percent of their Pool I-bound fibronectin into Pool II and a lower percent into the chase medium. These results indicate that early and late passage cultures of human fibroblasts exhibit differences in the binding of soluble fibronectin and in the extent to which they incorporate soluble fibronectin into the extracellular matrix.     

The removal of extracellular fibronectin from areas of cell-substrate contact

Immunofluorescent labeling for fibronectin was largely excluded from sites of closest contact between spreading chicken gizzard fibroblasts and the substratum. This was observed by double immunofluorescent labeling of fixed cells for fibronectin and vinculin, a smooth muscle intracellular protein that is specifically associated with focal adhesion plaques, in conjunction with interference-reflection microscopy. When the cells were plated on a fibronectin-coated substratum they adhered to its surface and rapidly spread on it. The immunofluorescent labeling for fibronectin in those cultures (after fixation and triton permeabilization) was usually absent from the newly formed, vinculin-containing focal adhesion plaques. We have found, however, that the accessibility to the cell-substrate gap at the focal adhesion plaques is limited and therefore a more direct approach was adopted. We have found that cells spreading on a substrate coated with rhodamine-labeled fibronectin progressively removed the underlying protein from the substrate. The removal of fibronectin involved at least two distinct mechanisms. Part of the substrate-associated fibronectin was removed from small areas and displaced toward the cell center. The arrowhead-shaped areas from which fibronectin was removed often coincided with vinculin-rich focal contacts. We observed, however, many areas where focal contacts were found over unperturbed fibronectin carpet, as well as fibronectin-free areas with no overlapping focal contacts. The possibilities that fibronectin is actively displaced from areas of cell-substrate contact, that the focal adhesion plaques are transiently associated with these areas and their implications on the dynamics of cell spreading and locomotion are discussed. The second route of fibronectin removal from the substrate was endocytosis. The rhodamine-labeled fibronectin was found in the cells in a partial or transient association with clathrin-containing structures.     

Early and late passage C-6 glial cell growth: Similarities with primary glial cells in culture

Earlier studies in our laboratory have shown that C-6 glial cells in culture exhibit astrocytic properties with increasing cell passage. In this study, we tested the responsiveness of early and late passage C-6 glial cells to various cultures conditions: culture substrata (collagen, poly-L-lysine, plastic), or supplements for the culture medium, DMEM, [fetal calf, or heat inactivated (HI) serum, or media conditioned from mouse neuroblastoma cells (NBCM) or primary chick embryo cultured neurons (NCM)]. Glutamine synthetase (GS) and cyclic nucleotide phosphohydrolase (CNP), astrocytic and oligodendrocytic glial markers, were used. Cell numer and protein content increased exponentially with days in culture regardless of the type of the substratum or cell passage. Differences in cell morphology among the three types of substratum were also reflected on GS activity, which rose by three-fold on culture day 3 for cells grown on collagen; thereafter, GS profiles were similar for all substrata. This early rise in GS is interpreted to reflect differential cell adhesion processes on the substrata; specifically, cell adhesion on the collagen stimulated differentiation into astrocytic phenotype.Analogous to immature glia cells in primary cultures, early passage C-6 glial cells responded to neuronal factors supplied either from NCM or NBCM by expressing reduced GS activity, the astrocytic marker and enhanced CNP activity, the oligodendrocytic marker. Thus, early passage cells can be induced to express either astrocytic or oligodendrocytic phenotype. In accordance with our previous reports on primary glial cells, late passage C-6 cells exhibit their usual astrocytic behavior, responding to serum factors with GS activity. Moreover, whereas NCM or NBCM alone markedly lowered GS activity, a combination with serum restored activity. The present findings confirm our previous observations and further establish the C-6 glial cells as a reliable model to study immature glia.Special issue dedicated to Dr. Paola S. Timiras.     

Effect of low dose rate irradiation on the division potential of cells in vitro. IV. Embryonic and adult human lung fibroblast-like cells

Early and late passage human embryonic lung fibroblasts were compared with early passage adult lung fibroblasts with regards to their survival (number of population doublings), after low dose rate ionizing radiation. It was found that early passage embryonic cells are quite resistant to this type of radiation. Late passage embryonic and early passage adult fibroblasts are more sensitive to ionizing radiations. The results suggest that cell aging is accompanied by an increased sensitivity to low dose rate ionizing radiation and favor the idea that aging in vitro, expressed as a function of the fibroblast division potential, is correlated with aging in vivo.     

Modulation of fibronectin synthesis and fibronectin binding during transformation and differentiation of mouse AKR fibroblasts

In previous studies it was shown that transformation of AKR fibroblasts with 3-methylcholanthrene was associated with a loss of surface fibronectin and that induction of differentiation of the transformed cells with N,N-dimethylformamide (DMF) was associated with reacquisition of surface fibronectin (Chakrabarty et al., J. Cell. Physiol. 133:415, 1987). It is shown in the present study that changes in surface fibronectin reflect altered fibronectin synthesis and altered fibronectin binding. Both the nontransformed cells (AKR-2B) and their transformed counterparts (AKR-MCA) bound 125I-fibronectin in a receptor-like fashion, but the AKR-MCA cells had only 20% of the receptors found on the AKR-2B cells. Whole cell extracts prepared from the AKR-2B cells and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions were examined for 125I-fibronectin binding. Under these conditions, the majority of binding occurred to moieties with molecular weights of 180 kD, 150 kD, and 97 kD. Binding to similar moieties on the AKR-MCA cells was virtually absent but occurred rapidly after treatment with DMF. The appearance of these moieties paralleled the acquisition of 125I-fibronectin binding activity by whole cells. Antibodies to the fibronectin receptor isolated from human placenta reacted with the DMF-sensitive moieties in immunoblot assays. Both the appearance of the fibronectin binding moieties and the acquisition of 125I-fibronectin binding activity by whole cells occurred within 6 hr of DMF treatment and increased over the subsequent 4 day period. The time course of these events paralleled closely the time course for induction of fibronectin biosynthesis by DMF. These changes in fibronectin binding and fibronectin production were associated with alterations in cell-substrate adhesion. The AKR-2B cells rapidly attached and spread on bovine serum albumin-coated dishes and on fibronectin-coated dishes, whereas the AKR-MCA cells were less adhesive on both substrates. Capacity to attach and spread was regained concomitantly with the induction of fibronectin binding and fibronectin production. Adhesion on both substrates was partially inhibited by antibodies to the fibronectin receptor and by RGDS. These studies suggest that fibronectin production and fibronectin binding are coregulated in AKR fibroblasts and that they function together to bring about changes in cell-substrate adhesion.     

Effect of Conditioned Media from Chicken and Turkey Intestinal Cell Cultures on Invasion by Sporozoites of Three Species of Avian Coccidia

The effect of conditioned media from cultures of turkey and chicken intestinal cells on cellular invasion by sporozoites of avian Eimeria species was examined in vitro. Media conditioned by the growth of cells from the ceca, mid-intestine (area of the yolk stalk diverticulum), and duodenal loop were examined for their ability to enhance invasion. Conditioned medium from cultures of turkey cecal cells significantly enhanced invasion by the turkey coccidia Eimeria adenoeides, by 2.4-fold, and E. meleagrimitis , by 2.2-fold, as compared with invasion in the presence of control medium. Conditioned medium from mid-intestinal cell cultures enhanced invasion by the two coccidial species by 2.0- and 2.1-fold, respectively. The enhancement occurred with conditioned media from early (1) as well as later (11) passages of cells. This suggests that the enhancing factor was produced by fibroblast-like cells, the predominant cell type at both early and late passages, and not by epithelial-like cells that had disappeared by the first or second passage. Additionally, conditioned media from cultures of chicken cecal and duodenal loop cells significantly enhanced invasion by the turkey cecal coccidium, E. adenoeides , (1.7- and 1.6-fold, respectively). This was less enhancement than was caused by the turkey cell conditioned media. Heat treatment (56° C for 45 min) of conditioned media failed to alter the effect on invasion. Neither the turkey or chicken cecal cell media nor conditioned media from any other chicken intestinal cell cultures enhanced invasion by E. tenella , the chicken cecal coccidium. Although morphologically dissimilar when they were first plated, the gross appearance and growth of the turkey and chicken cells when conditioned media was collected was comparable.     

Concurrent modulation of cell surface fibronectin and adhesion to fibronectin in hepatoma cells

Rat hepatoma cells grown in vitro were poorly adhesive to plastic surfaces coated with fibronectin and lacked cell surface fibronectin matrix. They synthesized soluble fibronectin into the medium. The cell surface fibronectin matrix and the ability to attach to fibronectin-coated surface were restored in the 7777 cells upon passage as a tumor in rats and by coculturing these cells with normal liver-derived cells in vitro. Fibronectin matrix and the ability of cells to attach to fibronectin were thus modulated in a coordinated fashion, suggesting that the formation of a cell surface fibronectin matrix is dependent on the cell surface property that enables cells to interact with fibronectin.     

Culture media conditioned by wounded cells modify the fibronectin localization pattern of unwounded confluent PtK2 cells

A confluent PtK2 cell sheet was incised in a serum-free culture medium, at 15 min, 2 hr and 24 hr after wounding. The culture media were collected in the same way and used as conditioned media. Unwounded confluent cells were cultured in the conditioned medium for 24 hr. They showed a modification of fibronectin localization similar to that which we had previously observed in wounded confluent PtK2 cells: cells lost their normal fibronectin fibrils and were surrounded by fibronectin lace. This finding suggested that during wound healing, the cells released soluble chemical factors which could modify the fibronectin localization pattern of unwounded confluent cells. Subconfluent cells did not respond to conditioned media, showing that confluent cells and subconfluent cells had different susceptibilities.     

PLC-γ1 regulates fibronectin assembly and cell aggregation

Phospholipase C-γ1 (PLC-γ1) mediates cell adhesion and migration through an undefined mechanism. Here, we examine the role of PLC-γ1 in cell-matrix adhesion in a hanging drop assay of cell aggregation. Plcg1 Null (−/−) mouse embryonic fibroblasts formed aggregates that were larger and significantly more resistant to dissociation than cells in which PLC-γ1 is re-expressed (Null+ cells). Aggregate formation could be disrupted by inhibition of fibronectin interaction with integrins, indicating that fibronectin assembly may mediate aggregate formation. Fibronectin assembly was mediated by integrin α5β1 in both cell lines, while assays measuring fibronectin assembly revealed increased assembly in the Null cells. Null and Null+ cells exhibited equivalent fibronectin mRNA levels and equivalent levels of fibronectin protein in pulse-labeling experiments. However, levels of secreted fibronectin in the conditioned medium were increased in Null cells. The data implicates a negative regulatory role for PLC-γ1 in cell aggregation by controlling the secretion of fibronectin into the media and its assembly into fibrils.     

Identification of an alternatively spliced site in human plasma fibronectin that mediates cell type-specific adhesion

We have compared the molecular specificities of the adhesive interactions of melanoma and fibroblastic cells with fibronectin. Several striking differences were found in the sensitivity of the two cell types to inhibition by a series of synthetic peptides modeled on the Arg-Gly-Asp-Ser (RGDS) tetrapeptide adhesion signal. Further evidence for differences between the melanoma and fibroblastic cell adhesion systems was obtained by examining adhesion to proteolytic fragments of fibronectin. Fibroblastic BHK cells spread readily on fl3, a 75-kD fragment representing the RGDS-containing, "cell-binding" domain of fibronectin, but B16-F10 melanoma cells could not. The melanoma cells were able to spread instead on f9, a 113-kD fragment derived from the large subunit of fibronectin that contains at least part of the type III connecting segment difference region (or "V" region); f7, a fragment from the small fibronectin subunit that lacks this alternatively spliced polypeptide was inactive. Monoclonal antibody and fl3 inhibition experiments confirmed the inability of the melanoma cells to use the RGDS sequence; neither molecule affected melanoma cell spreading, but both completely abrogated fibroblast adhesion. By systematic analysis of a series of six overlapping synthetic peptides spanning the entire type III connecting segment, a novel attachment site was identified in a peptide near the COOH- terminus of this region. The tetrapeptide sequence Arg-Glu-Asp-Val (REDV), which is somewhat related to RGDS, was present in this peptide in a highly hydrophilic region of the type III connecting segment. REDV appeared to be functionally important, since this synthetic tetrapeptide was inhibitory for melanoma cell adhesion to fibronectin but was inactive for fibroblastic cell adhesion. REDV therefore represents a novel adhesive recognition signal in fibronectin that possesses cell type specificity. These results suggest that, for some cell types, regulation of the adhesion-promoting activity of fibronectin may occur by alternative mRNA splicing.     

Cytoarchitecture of ras oncogene-expressing tumor cells: butyrate modulation of substrate adhesion, cytoskeletal actin content and subcellular microfilament distribution

1. The subcellular distribution of particular cytoskeletal (CSK) and cell-substrate adhesive elements was assessed during the morphologic response of cultured tumor cells to the shape modulating agent sodium butyrate (NaB). 2. NaB induced marked increases in cellular and CSK actin content and in the matrix-associated proteins fibronectin and p52. 3. Subcellular fractionation indicated disproportionate increases in the actin content of the substrate-attached cellular residue (SAM fraction) which contains the majority of cell-substrate adhesive elements. 4. Augmented cell spreading and substrate attachment characteristic of NaB-treated cells is likely due to increased elaboration of cell-to-substrate adhesive structures and reflected in an enhanced deposition of actin into the CSK and SAM compartments.     

Cell adhesion to fibronectin and tenascin: quantitative measurements of initial binding and subsequent strengthening response

Cell-substratum adhesion strengths have been quantified using fibroblasts and glioma cells binding to two extracellular matrix proteins, fibronectin and tenascin. A centrifugal force-based adhesion assay was used for the adhesive strength measurements, and the corresponding morphology of the adhesions was visualized by interference reflection microscopy. The initial adhesions as measured at 4 degrees C were on the order of 10(-5)dynes/cell and did not involve the cytoskeleton. Adhesion to fibronectin after 15 min at 37 degrees C were more than an order of magnitude stronger; the strengthening response required cytoskeletal involvement. By contrast to the marked strengthening of adhesion to FN, adhesion to TN was unchanged or weakened after 15 min at 37 degrees C. The absolute strength of adhesion achieved varied according to protein and cell type. When a mixed substratum of fibronectin and tenascin was tested, the presence of tenascin was found to reduce the level of the strengthening of cell adhesion normally observed at 37 degrees C on a substratum of fibronectin alone. Parallel analysis of corresponding interference reflection micrographs showed that differences in the area of cell surface within 10-15 nm of the substratum correlated closely with each of the changes in adhesion observed: after incubation for 15 min on fibronectin at 37 degrees C, glioma cells increased their surface area within close contact to the substrate by integral to 125- fold. Cells on tenascin did not increase their surface area of contact. The increased surface area of contact and the inhibitory activity of cytochalasin b suggest that the adhesive "strengthening" in the 15 min after initial binding brings additional adhesion molecules into the adhesive site and couples the actin cytoskeleton to the adhesion complex.     

Isolation and partial characterization of endothelial cell extracellular complexes

Human endothelial cells release components into the growth medium that stimulate cell-substratum adhesion. Several macromolecular components were isolated by ultracentrifugation of the endothelial cell conditioned medium. The components were heterogeneous, consisting of several sizes when examined by sedimentation velocity and gel filtration. When the extracellular components were evaluated by electron microscopy, structurally discrete particles were observed. The extracellular components and the complexes mediated cell-substratum adhesion to both human umbilical and arterial endothelial cells. The majority of the extracellular components that promote endothelial cell adhesion were pelleted by ultracentrifugation. Although the complexes contained fibronectin, antibodies to fibronectin did not inhibit cell adhesion to the complexes. Significant inhibition of endothelial cell adhesion was observed in the presence of heparin and heparan sulfate. The supernatant fraction following ultracentrifugation of the growth medium contained a component that suppressed endothelial cell adhesion to culture dishes coated with fibronectin, type I collagen, and endothelial cell complexes. SDS-polyacrylamide gel electrophoresis indicated that the complexes contained several components, and the majority of the large-molecular-weight components were pelleted by ultracentrifugation. The conditioned medium from human endothelial cells contains specific complexes that promote cell-substratum adhesion and components that suppress cell-substratum adhesion.     

Glycosaminoglycan production in cultures of early and late passage human endothelial cells: the influence of an anionic endothelial cell growth factor and the extracellular matrix

An endothelial cell (EC) growth factor isolated from bovine brain stimulates in vitro growth of human umbilical vein endothelial cells, and permits long term serial propagation. In the presence of increasing concentrations of EC growth factor, confluent cultures of early (CPDL less than or equal to 20) and late (CPDL greater than 20) passage human endothelial cells exhibit an increased incorporation of 3H-glucosamine and Na235SO4 into the glycosaminoglycans (GAG), hyaluronic acid, chondroitin, chondroitin-4-sulfate, dermatan-4-sulfate, and chondroitin-6-sulfate. An increase in both labelled sulfated and nonsulfated GAG was observed in the cytosol, membrane, secreted and extracellular matrix fractions. In contrast, endothelial cells grown in the presence of EC growth factor contained decreased amounts of labelled heparan sulfate than cells grown without EC growth factor. Confluent cultures of early passage cells had significantly more labelled GAG but significantly less heparan sulfate than cultures of late passage cells on a per cell basis. Extracellular matrix from early passage cells contained about two- to seven-fold more labelled GAG than extracellular matrix from late passage cells, but only about half as much labelled heparan sulfate. Cell adhesion was enhanced when cells were grown in the presence of EC growth factor as compared to adhesion of cells grown without EC growth factor. Conversely, trypsin-mediated detachment of cells grown in the presence of growth factor was inhibited as compared to detachment of cells grown in medium without EC growth factor. The composition of the extracellular matrix influenced incorporation of labelled GAG into extracellular matrix. Early passage cells grown to confluence on a matrix from late passage cells incorporated significantly less labelled GAG into extracellular matrix than when grown to confluence on matrix from early passage cells. Incorporation of labelled GAG into extracellular matrix was significantly higher when late passage cells were grown on a matrix from early passage endothelial cells than when grown on matrix from late passage cells. We conclude that EC growth factor selectively stimulates incorporation of isotopic precursors into GAG in cultures of early and late passage endothelial cells but inhibits incorporation of radiolabel into heparan sulfate; early passage cells contain more GAG but less heparan sulfate than late passage cells, extracellular matrix controls the amount of GAG and heparan sulfate incorporated into matrix.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of sodium butyrate on the membrane glycoconjugates of murine sarcoma virus-transformed rat cells

The temporal relationship between butyrate-induced cellular flattening of murine sarcoma virus-transformed rat cells (MSV-NRK) and alterations in certain surface-associated biochemical markers of transformation, e.g., surface glycopeptides, glycolipids, fibronectin, hexose uptake, and cell-substrate adhesion was examined. The induction of elevated levels of the ganglioside GM3 and of a GDla-like ganglioside were observed to precede or to parallel cellular flattening. Likewise, enhanced incorporation of radioisotopically labeled fucose into a novel fucose-containing component, i.e., glucopyranosyl (1 leads to 3) fucopyranosyl-threonine, was also observed to occur at an early stage of cellular flattening. In contrast, a shift in the molecular weight distribution of trypsin-sensitive, surface fucopeptides was observed to occur at a late stage of cellular flattening. Moreover, surface fibronectin was not detectable in the butyrate-flattened MSV-NRK cells despite the fact that the cells manifested significantly enhanced cell- substrate adhesion. Thus, butyrate appears to be a useful tool for understanding the sequential changes associated with expression of the transformed phenotype of MSV-NRK cells.