The effect of ascorbate on the glycosaminoglycans synthesized by normal and simian virus 40(SV40)-transformed human skin fibroblasts was examined. Cells were incubated in the presence or absence of ascorbate, and radiolabelled with [3H]glucosamine and [35S]sulphate for 48 h, 3 days after reaching confluence. Glycosaminoglycans were analysed in the medium, a collagenase extract, and in the trypsin/cell-associated fraction. Hyaluronic acid was the main 3H-labelled glycosaminoglycan in all but the collagenase extracts, and showed a large decrease in normal fibroblast cultures, but a significant increase in SV40-transformed fibroblast cultures following feeding with ascorbate. Incorporation of [3H]glucosamine into sulphated glycosaminoglycans was reduced in normal fibroblast cultures but increased slightly in SV40-transformed cultures following ascorbate supplementation. [35S]sulphate incorporation remained essentially unaltered in both cell cultures. Ascorbate stimulated the deposition of glycosaminoglycans into the insoluble matrix of normal fibroblasts while reducing the deposition in SV40-transformed fibroblast cultures. The observed changes may in part be related to ascorbate-induced deposition of collagen in normal fibroblast cultures and the inability of the transformed fibroblast cells to deposit an extensive extracellular matrix, in addition to possible changes in the specific activity of the UDP-iV-acetyl- [3H]hexosamine pool.

The extracellular matrix, in addition to its structural role, is now known to play an important role in regulating cellular properties such as growth, metabolism, migration and differentiation. Ascorbate has been shown to modulate the composition of the extracellular matrix synthesized by smooth muscle cells (Schwartz et al. 1982) and foetal skin fibroblasts (Edward & Oliver, 1983), these changes being possibly mediated by an ascorbate-stimulated increase in the synthesis and deposition of collagen (Russell et al. 1981). The presence of increased amounts of collagen in the extracellular matrix may enhance the incorporation of other matrix molecules such as glycosaminoglycans, which bind to collagen, both in their native proteoglycan form and as free polysaccharide chains (Toole, 1976). Gallagher et al. (1980) reported changes in glycosaminoglycan synthesis by human skin fibroblasts grown on collagen gels in which there was an increased incorporation of sulphated glycosaminoglycans into the insoluble matrix. Because of their relatively large polyanionic and hydrated domains, sulphated glycosaminoglycans in the extra-cellular/pericellular matrix are ideally situated to regulate interactions between the cells and their microenvironment, such interactions being of paramount importance for normal cell growth and differentiation (Hôôk et al. 1984).

Transformed fibroblasts lack the ability to assemble an extensive extracellular matrix and have a reduced ability to synthesize collagen (Kreig et al. 1980) and deposit it into the extracellular matrix (Arbogast et al. 1977). Indeed transformed fibroblasts will grow in the absence of collagen while normal fibroblasts require collagen for proliferation (Liotta et al. 1978). In addition, transformation also results in considerable changes in the synthesis, distribution and sulphation of glycosaminoglycans (Rollins & Culp, 1979; Winterbourne & Mora, 1981); such changes in these matrix macromolecules possibly contributing to the loss of growth control of the transformed cells. In view of these observations, I have examined the possibility that ascorbate may stimulate transformed fibroblasts to synthesize and assemble a more elaborate extracellular matrix.

Materials

Eagle’s minimal essential medium, heat-inactivated foetal calf serum, penicillin/streptomycin, L-glutamine, trypsin and Nunclon tissue culture flasks were obtained from Gibco Ltd, Paisley, Scotland. Chondroitinase AC lyase from Arthrobacter aurescens, chondroitinase ABC lyase from Proteus vulgaris, Pronase E from Streptomyces griseus, bovine pancreatic ribonuclease A and deoxyribonuclease I, hyaluronidase from Streptomyces hyalurolyticus and highly purified collagenase, Sigma type VII from Clostridium histolyticum, were purchased from Sigma Chemical Co., Poole, England. D-[6-3H]glucosamine (36Cimmol-1), D-[l-14C]glucosamine (58-7mCi mmol-1) and Na235SO4 (1465 Ci mmol-1) were from Amersham International pic, Amersham, England. Sephraphore III cellulose acetate strips were from Gelman International Ltd, Dublin, Ireland, and ‘Optiphase-MP’ scintillation cocktail from Fisons, Loughborough, England. All other reagents were of analytical grade.

Cell culture

Adult human skin fibroblasts and their simian virus 40(SV40)-transformed counterparts, which were kindly supplied by Dr P. Gallimore, Cancer Research Campaign Laboratories, Birmingham, England, were grown in Eagle’s minimal essential medium supplemented with 10% heat-inactivated foetal calf serum, L-glutamine (2nffl), penicillin (100 units ml-1) and streptomycin (100μgml-1) at 37°C in a humidified atmosphere of 5% CO2 in air. For glycosaminoglycan biosynthetic determinations, cells were seeded in 80 cm2 flasks at an initial density of 5×105 cells/flask in the presence or absence of ascorbic acid (50μml-1), and after the cells had reached confluence (6 days) were incubated with [3H]glucosamine (5/iCiml-1) and Na235SO4 (20 μCi ml-1) for 48 h. For qualitative analysis of glycosaminoglycans, cells grown in the presence of ascorbate were incubated with [14C]glucosamine (2μCiml-1), while controls were incubated with [3H]glucosamine (5μCiml-1).

Collagenase and trypsin extracts of cell cultures

After 48 h incubation with radiolabels, the medium was removed and the cell layer washed twice with 5 ml phosphate-buffered saline (Dulbecco A) at 37°C, and the washes added to the medium. The cell layer was then incubated for 2h at 37°C with 5 ml collagenase (90 units ml-1) in phosphate-buffered saline, the collagenase extract was removed, and the cell layer gently washed twice with 5 ml of phosphate-buffered saline, which was then combined with the collagenase extract. The cell layer was then treated with 5 ml of trypsin solution (0·05%, w/v in phosphate-buffered saline) for 10 min at 37 °C, and the detached cells removed with the trypsin extract. The flasks were washed twice with 5 ml phosphate-buffered saline which was combined with the trypsin/cell fraction.

Isolation and characterization of radiolabelled glycosaminoglycans

Biosynthetic studies

Radiolabelled glycosaminoglycans were isolated and estimated essentially as described (Edward & Oliver, 1983).

Qualitative analysis

The medium fraction that was labelled with [3H]glucosamine (control) was combined with the medium fraction labelled with [14C] glucosamine (ascorbate-supplemented cultures), and similarly collagenase and trypsin/cell fractions were combined, and dialysed against several changes of distilled water for 24 h and finally against Tris HCl buffer (0·05 M, pH 6’0). All fractions were digested with Pronase as described above, heat-inactivated, and dialysed against several changes of distilled water, followed by Tris * HCl buffer (0-01 M, pH 7·6 containing 0·01 M-NaCl). Glycosaminoglycans were separated from non-diffusible material by ion-exchange chromatography on columns (1 cm × 12 cm) of Whatman DEAE-cellulose DE52 equilibrated with Tris-HCl buffer (0·01 M, pH 7-6 containing 0·01 M-NaCl) and eluted at room temperature with a linear gradient of 0·01 M to 0·0M-NaCl in a total volume of 250 ml, and a flow rate of 20mlh-1. Fractions of 2-5 ml were collected, and radioactivity was determined by counting 0·5 ml samples in 5 ml Optiphase MP scintillation cocktail. Peaks corresponding to sulphated glycosaminoglycans were pooled, concentrated, and dialysed against 0·1 M-Tris HCl (pH 7-6) containing 0*25 M-NaCl. The sulphated glycosaminoglycans were then separated on a DEAE-cellulose DE52 column (lcmX8cm) equilibrated with 0·1 M-Tris·HCl, pH7*6, 0*25M-NaCl, and eluted at room temperature with a linear gradient of 0*25 M to 0*6 M-NaCl in a total volume of 250 ml at a flow rate of 20mlh-1. Fractions were collected and analysed as described above.

For cellulose acetate electrophoresis, sulphated glycosaminoglycan fractions isolated from the DEAE-cellulose columns were pooled, dialysed against distilled water, concentrated, freeze-dried, and dissolved in 0* 1 ml of distilled water. Samples (2 μl) were applied to cellulose acetate strips, and electrophoresis was carried out in 0*1 M-HCI for 1 h at 2 mA cm-1. The acetate strips were cut into 2-mrn sections and radioactivity determined in 5 ml Optiphase MP scintillant. Electrophoretic mobilities at pH 1*0 should reflect the glycosaminoglycan sulphate content (Wessler, 1971).

Biosynthetic studies

Supplementation of the culture medium with ascorbate resulted in an increased maximum cell density of normal fibroblasts from 5·07×106 to 7·76×106 cells/flask, while the SV40-transformed fibroblasts showed a slight decrease in cell density from 12·2×106 to 12*0×106 cells/flask. The incorporation of [3H]glucosamine and Na235SO4 into glycosaminoglycans synthesized by ascorbate-fed and control cultures (shown in Table 1) resulted in a marked ascorbate-stimulated decrease in the incorporation of [3H]glucosamine into hyaluronate in normal fibroblast cultures, while there was a substantial increase in the incorporation of [3H]glucosamine into hyaluronate in SV40-transformed fibroblast cultures. Ascorbate also altered the incorporation of [3H]glucosamine into sulphated glycosaminoglycans, normal fibroblasts showing an overall decrease in incorporation, while SV40-transformed fibroblast cultures showed a slight increase. Total incorporation of [35S]sulphate remained essentially unaltered in both cell cultures after feeding with ascorbate. In addition to altered radiolabelled precursor incorporation, supplementation of the culture medium with ascorbate resulted in a redistribution of the glycosaminoglycans (Table 2). While ascorbate reduced the incorporation of [3H] glucosamine and increased the incorporation of [35S]sulphate into the sulphated glycosaminoglycans present in the insoluble fraction (collagenase plus trypsin/cell fractions) of normal fibroblasts, the overall percentage of total glycosaminoglycans in this fraction increased. In contrast, ascorbate feeding brought about a dramatic reduction in the percentage of glycosaminoglycans present in the insoluble fraction of SV40-transformed fibroblast cultures. Therefore, ascorbate appears to increase the percentage of glycosaminoglycans present in the insoluble fraction of normal fibroblasts, but stimulates an increased release of glycosaminoglycans into the medium of SV40-transformed fibroblast cultures.

Table 1.

Ascorbate-modulated radioisotope incorporation into glycosaminoglycans synthesized by normal and SV40-transformed fibroblasts

Ascorbate-modulated radioisotope incorporation into glycosaminoglycans synthesized by normal and SV40-transformed fibroblasts
Ascorbate-modulated radioisotope incorporation into glycosaminoglycans synthesized by normal and SV40-transformed fibroblasts
Table 2.

Distribution of glycosaminoglycans of normal and SV40-transformed fibroblasts

Distribution of glycosaminoglycans of normal and SV40-transformed fibroblasts
Distribution of glycosaminoglycans of normal and SV40-transformed fibroblasts

The composition and distribution of the individual sulphated glycosaminoglycans is shown in Fig. 1. In normal fibroblast cultures after supplementation with ascorbate, chondroitin sulphate and dermatan sulphate show slight decreases in the medium fraction, while heparan sulphate is increased substantially in the medium fraction, and shows an overall decreased synthesis. In the SV40-transformed fibroblast cultures, chondroitin sulphate is greatly increased in the medium fraction and decreased in the insoluble fractions, while dermatan sulphate shows only a slight decrease in the insoluble fractions after feeding with ascorbate. Ascorbate stimulates a large increase in SV40-transformed fibroblast medium heparan sulphate, and a significant decrease in the trypsin/cell-associated heparan sulphate.

Fig. 1.

Distribution of individual sulphated glycosaminoglycans synthesized by normal (A and B) and SV40-transformed (C and D) fibroblasts. Results here show the incorporation of [3H]glucosamine (▭) and Na235SO4 (⬚) into chondroitin sulphates A and C (CS A/C), dermatan sulphate (DS) and heparan sulphate (HS) in the medium (M), collagenase extract (C) and trypsin/cell fraction (T/C) of cells grown in the presence or absence of ascorbic acid. Glycosaminoglycans were identified by their susceptibility to chondroitinases AC and ABC, and HNO2.

Fig. 1.

Distribution of individual sulphated glycosaminoglycans synthesized by normal (A and B) and SV40-transformed (C and D) fibroblasts. Results here show the incorporation of [3H]glucosamine (▭) and Na235SO4 (⬚) into chondroitin sulphates A and C (CS A/C), dermatan sulphate (DS) and heparan sulphate (HS) in the medium (M), collagenase extract (C) and trypsin/cell fraction (T/C) of cells grown in the presence or absence of ascorbic acid. Glycosaminoglycans were identified by their susceptibility to chondroitinases AC and ABC, and HNO2.

Ascorbate, under certain conditions, is known to depolymerize hyaluronic acid, therefore the dramatic decrease in hyaluronic acid synthesized by normal fibroblasts after feeding with ascorbate may have been due to ascorbate-induced depolymerization. To check this, radiolabelled medium from normal fibroblast control cultures was incubated for 24 h at 37 °C in the presence of ascorbate (50μgml-1) and then processed as before. No loss of radioactivity was detected in the hyaluronic acid, indicating that the hyaluronate was not depolymerized by ascorbate under the conditions used here in culture.

Qualitative analysis

Sulphated glycosaminoglycans in both normal and SV40-transformed fibroblast cultures were separated from hyaluronic acid and glycopeptides by chromatography on DEAE-cellulose by elution with a 0·01M to 0·6M-NaCl gradient. The peaks corresponding to sulphated glycosaminoglycans were pooled, and possible differences in the charge density of the glycosaminoglycans from control ([3H]glu-cosamine-labelled) and ascorbate-fed ([14C]glucosamine-labelled) cultures examined on a DEAE-cellulose column eluted with a 0·25 M to 0·6M-NaCl gradient (Fig. 2). No significant difference was detected in the elution positions of the sulphated glycosaminoglycans from control and ascorbate-supplemented cultures. However, it was found that when the sulphated glycosaminoglycans were pooled from the SV40-transformed fibroblast extracts, and re-run on DEAE-cellulose, a significant amount of hyaluronic acid was carried over. This appeared to suggest that the hyaluronic acid from the transformed fibroblast cultures was binding more tightly to the DEAE-cellulose. To examine this further, normal fibroblast cultures were labelled with [3H] glucosamine and SV40-transformed fibroblasts with [14C]glucosamine, and the glycosaminoglycans extracted and prepared for ion-exchange chromatography as previously described. The hyaluronate was isolated following separation from other glycoconjugates on a DEAE-cellulose column eluted with a 0·01 M to 0·6M-NaCl gradient, and rerun on a DEAE-cellulose column to which a 0·01 M to 0·3 M-NaCl gradient was applied. It was found that the hyaluronate from the SV40-transformed fibroblast cultures eluted at a higher salt concentration than that from the normal fibroblast cultures (Fig. 3). These observed differences in the elution profiles of the hyaluronate may be due to the SV40-transformed fibroblasts synthesizing hyaluronate with a higher charge density or molecular weight.

Fig. 2.

Anion exchange profiles of glycosaminoglycans produced by normal and SV40-transformed fibroblasts in the presence or absence of ascorbate. Sulphated glycosaminoglycans were isolated from the medium, collagenase extract and trypsin/cell fraction by DEAE-cellulose chromatography using a 0·01 M to 0·6 M-NaCl gradient, followed by dialysis against 0·01 M-Tris - HCl, 0·25 M-NaCl, pH 7·6, applied to a DEAE-cellulose column, and eluted with a 0·25 M to 0·6 M-NaCl gradient.

Fig. 2.

Anion exchange profiles of glycosaminoglycans produced by normal and SV40-transformed fibroblasts in the presence or absence of ascorbate. Sulphated glycosaminoglycans were isolated from the medium, collagenase extract and trypsin/cell fraction by DEAE-cellulose chromatography using a 0·01 M to 0·6 M-NaCl gradient, followed by dialysis against 0·01 M-Tris - HCl, 0·25 M-NaCl, pH 7·6, applied to a DEAE-cellulose column, and eluted with a 0·25 M to 0·6 M-NaCl gradient.

Fig. 3.

Anion exchange profile of hyaluronic acid produced by normal and SV40-transformed fibroblasts. Cells were radiolabelled with [3H]glucosamine (normal fibroblasts) and [14C]glucosamine (SV40-transformed fibroblasts), and hyaluronic acid separated from other glycoconjugates by DEAE-cellulose chromatography. Fractions corresponding to hyaluronic acid were pooled, dialysed against 0’01 M-Tris HCl, 0·01 M-NaCl, pH 7·6, and applied to a DEAE-cellulose column that was eluted with a 0·01 M-0·3 M-NaCl gradient, and fractions counted for radioactivity.

Fig. 3.

Anion exchange profile of hyaluronic acid produced by normal and SV40-transformed fibroblasts. Cells were radiolabelled with [3H]glucosamine (normal fibroblasts) and [14C]glucosamine (SV40-transformed fibroblasts), and hyaluronic acid separated from other glycoconjugates by DEAE-cellulose chromatography. Fractions corresponding to hyaluronic acid were pooled, dialysed against 0’01 M-Tris HCl, 0·01 M-NaCl, pH 7·6, and applied to a DEAE-cellulose column that was eluted with a 0·01 M-0·3 M-NaCl gradient, and fractions counted for radioactivity.

Any possible change in overall sulphation of the glycosaminoglycans as a result of ascorbate treatment was determined by cellulose acetate electrophoresis at pH 1-0; however, there was no apparent difference.

Transformed fibroblasts are commonly distinguished from their normal counterparts by the expression of certain phenotypic characteristics in vitro, such as anchorage-independent growth, reduced synthesis of matrix components, and increased production of proteolytic enzymes. In the present study, ascorbate modulated the growth and deposition of glycosaminoglycans into the extracellular matrix of normal fibroblast cultures, but failed to increase the deposition of glycosaminoglycans into the matrix, or alter the growth of SV40-transformed fibroblasts. The maximum cell density of normal fibroblasts was increased by exposure to ascorbate, which may have been facilitated by an enhanced synthesis and deposition of a collagen-rich extracellular matrix, while the SV40-transformed fibroblasts maintained their reduced ability to assemble such a matrix (Vaheri et al. 1978). The slightly increased incorporation of sulphated glycosaminoglycans into the collagenase-extractable fraction of normal fibroblast cultures after ascorbate supplementation may reflect an increase in the number of binding sites, and affinity for the extensively deposited collagen, such interactions undoubtedly contributing to the structural organization of the extracellular matrix (Comper & Laurent, 1978). Indeed glycosaminoglycans occur in close association with other matrix components such as fibronectin (Hedman et al. 1982; Hayman et al. 1982), and promote fibronectin-collagen interactions (Ruoslahti & Engvall, 1980), resulting in a fibrillar network (Hedman et al. 1979). The incorporation of sulphated glycosaminoglycans into the extracellular matrix of the normal fibroblast cultures following feeding with ascorbate may have been more pronounced had the cultures been incubated longer (Edward & Oliver, 1983), as the synthesis and deposition of collagen is cooperative (Russell et al. 1981). However, unlike normal fibroblasts, prolonged incubation of the transformed cultures results in the detachment of many cells.

The inability of the transformed cells to synthesize and deposit a matrix rich in collagen and fibronectin (Chen et al. 1984) probably contributes to the greatly reduced amounts of sulphated glycosaminoglycans incorporated into the matrix, in addition to structural changes in glycosaminoglycans resulting in a reduced affinity for other matrix components. Ascorbate failed to promote the assembly of a more elaborate matrix in the transformed cultures, but actually induced the release of glycosaminoglycans from the extracellular matrix into the medium after ascorbate supplementation. From Table 2, it can be seen that the transformed fibroblasts incorporate a fairly large percentage of their hyaluronate (32%) into the insoluble matrix compared to normal fibroblast cultures, but most of this hyaluronate is shed into the medium, although the amount of [35S]sulphate incorporated into SV40-transformed cultures is very much less than that from normal fibroblasts. While the presence of a collagenous matrix in normal fibroblast cultures may reduce the degradation of proteoglycans and glycosaminoglycans (David & Bernfield, 1981), transformed cells synthesize and secrete increased amounts of proteases (Mahdavi & Hynes, 1979), which may result in the degradation of the protein core of proteoglycans thereby releasing the free glycosaminoglycan chains, or degrade the matrix proteins to which glycosaminoglycans bind. Ascorbate may therefore promote the release of glycosaminoglycans from the extracellular matrix of transformed cells by enhancing the synthesis or secretion of proteases.

Analysis of the individual glycosaminoglycans reveals that in normal fibroblasts, there is a substantial increase in the incorporation of heparan sulphate into the insoluble fraction compared to that present in the medium following ascorbate supplementation. This probably reflects the high affinity heparan sulphate has for the other matrix components. All glycosaminoglycan species in the transformed cells show a decreased incorporation into the extracellular matrix, following exposure to ascorbate. Of particular interest is the ascorbate-mediated decrease in the incorporation of [3H] glucosamine into glycosaminoglycans synthesized by normal fibroblasts, and an increased incorporation into SV40-transformed cultures. These observed differences may be brought about by modulation of the extracellular matrix, which could evoke changes in radiolabel-uptake rates or nucleotide sugar concentrations, thereby altering the specific activities of the UDP-iV-acetyl- [3H]hexosamine precursor pools, or ascorbate may have a more direct effect. Structural alterations in the extracellular/pericellular matrix glycosaminoglycans of normal and transformed cells may affect their interactions with other matrix macromolecules; however, no apparent differences were detected in their charge density or sulphation following feeding with ascorbate.

Whatever the mechanism by which ascorbate acts, these data suggest significant differences in the synthesis and assembly of the extracellular matrix of normal and transformed cells, which appear to be more pronounced following exposure to ascorbate.

I gratefully acknowledge financial assistance from the Medical Research Council and the Cancer Research Campaign.

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