Heparan Sulfate Undergoes Specific Structural Changes during the Progression from Human Colon Adenoma to Carcinoma in Vitro*

Gordon C. JaysonDagger §, Malcolm LyonDagger , Christos Paraskeva, Jeremy E. TurnbullDagger , Jonathan A. DeakinDagger , and John T. GallagherDagger

From the Dagger  Cancer Research Campaign Department, Medical Oncology, University of Manchester and Christie Hospital National Health Service Trust, Withington, Manchester M20 4BX and  Cancer Research Campaign Department, Colorectal Biology, University of Bristol, Bristol, United Kingdom

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Heparan sulfate is a widespread complex linear polysaccharide that consists of alternate hexuronic acid and N-substituted glucosamine residues. The ability to bind protein effectors such as growth factors or protease inhibitors (e.g. antithrombin III; Refs. 1 and 2) is strongly influenced by the position and density of sulfate residues that occur most frequently as N-sulfates but are also present as sulfate esters at the 6-O- (or less commonly the 3-O-) position of glucosamine or the 2-O-position of iduronic acid (3). The complexity of HS is achieved through the location of sulfate groups in domains of high and low sulfation (4), the conformational flexibility of iduronate residues (5, 6), and the degree of polymorphism manifest in different tissues (7-9). The strongly anionic zones of HS,1 the S domains, consist of contiguous glucosamine N-sulfate-containing disaccharides that bear a variable number of O-sulfate moieties. Domains of less sulfated sequences called mixed sequences are believed to flank the S domains, separating them from the unsulfated domains, and these are liberated as tetrasaccharides by low pH nitrous acid, a reagent that cleaves HS at N-sulfated glucosamine-containing disaccharides (i.e. GlcNSO3-alpha 1-4-hexuronic acid).

The ability of HS to act as a growth factor activator (10-13) and as a component of focal adhesions (14) has focused attention on this molecule as a potential therapeutic target in diseases of aberrant cellular growth or migration such as cancer, diabetic retinopathy, or coronary arterial restenosis. A number of studies have investigated the structural changes in HS in animal models of malignancy with the earliest detailed analyses being performed on SV-40-transformed murine embryo cell lines (15-17). These studies showed that transformation was accompanied by a reduction in charge density that was largely due to a decrease in 6-O-sulfation in nitrous acid-resistant tetrasaccharides. Further analyses of normal and transformed mouse mammary basement membrane proteoglycans confirmed that carcinogenesis was associated with reductions in 6-O-sulfation and also of 3-O-sulfation and a reduction in the size of intact HS (18), changes that were associated with a reduced affinity for antithrombin III.

There have been no detailed studies of HS structure in human malignant tissues, but a crude comparison of normal liver and hepatoma tissue showed that there was a reduction in the charge density of HS in the neoplastic tissue (19).

The first in vitro model of the progression of human colon cancer from adenoma to carcinoma was developed recently by Paraskeva and co-workers (20). A cell line with the phenotype of an adenoma was derived from a polyp taken from a patient with familial adenomatous polyposis. Through chemical transformation, a second cell line was derived that was tumorigenic in nude mice and anchorage-independent in soft agar, in contrast to the adenoma cell line. We have used these cell lines to identify the structural changes in HS during the progression from human colon adenoma to carcinoma.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- D-[6-3H]Glucosamine hydrochloride (20-45 Ci/mmol) and Na235SO4 (carrier free; 1200-1400 Ci/mmol) were obtained from NEN Life Science Products. Heparinase I (Flavobacterium heparinum; heparin lyase, EC 4.2.2.7), heparinase II (F. heparinum; no EC number assigned), and heparinase III/heparitinase (F. heparinum; heparan-sulfate lyase, EC 4.2.2.8) were obtained from Grampian Enzymes (Aberdeen, UK). Materials for cell culture were obtained from Life Technologies, Inc. All other chemicals were from Sigma.

Cell Culture-- The adenoma and carcinoma cells were grown in conditions described by Williams et al. (20) and were used within 10 passages.

Preparation Of Heparan Sulfate-- Glycosaminoglycans were prepared from cells contained in four 175-cm2 tissue culture flasks. The adenoma and carcinoma cells were labeled at 80% confluence by incubation in normal medium containing 5 mCi/ml D-[6-3H]glucosamine and 5 mCi/ml Na235SO4.

After 48 h, the cell culture media from the four flasks were pooled and treated with 100 µg/ml Pronase at 37 °C for 24 h. To prepare extracts of the cell layer/extracellular matrix, each flask was treated with 20 ml of phosphate-buffered saline (PBS), pH 7.4, containing 1% (v/v) of Triton X-100 detergent and 100 µg/ml Pronase for 24 h at 37 °C. Both the cell/matrix extracts and the cell culture media were pooled and frozen at -20 °C until they were analyzed.

The pooled extracts were thawed, centrifuged (1000 × g for 20 min. at 4 °C), and the supernatant was loaded under gravity onto a DEAE-Sephacel column (1.5 × 25 cm) that had been previously equilibrated with PBS. The column was washed with PBS until there was no further elution of radioactivity and then eluted with a linear gradient from 0.15 to 0.825 M NaCl in phosphate buffer, pH 7.4, at 10 ml/h.

Double-radiolabeled peaks corresponding to sulfated GAGs (HS and chondroitin sulfate/dermatan sulfate) were collected, diluted with distilled water × 3 (v/v), and then loaded under gravity onto a 1 × 4 cm DEAE-Sephacel column pre-equilibrated with PBS. The column was then washed with 30 ml of PBS, step-eluted with 5 ml of 0.3 M NaCl in phosphate buffer, pH 7.4, to remove any residual hyaluronan, then step-eluted again with 20 ml of 1.5 M NaCl, phosphate buffer, pH 7.4, to desorb the sulfated GAGs. The latter were volume-reduced to approximately 1 ml by centrifugal evaporation. They were then loaded onto a 1 × 30-cm Sephadex G-50 (superfine) column, and pre-equilibrated and eluted with 0.2 M NH4HCO3. The GAGs were mainly present in the void volume and were pooled and exhaustively lyophilized.

The GAG preparation was dissolved in 1 ml of chondroitinase buffer (50 mM Tris-HCl, 50 mM NaCl, pH 7.8-8) and treated with 20 mIU of chondroitinase ABC (E.C. 4.2.2.4) for 24 h at 37 °C. The volume was increased to 10 ml with distilled water, and the solution was reloaded onto a small DEAE-Sephacel column (1 × 4 cm) under gravity. This was washed with 20 ml of phosphate-buffered saline (0.3 M), pH 7.2, to remove degraded chondroitin sulfate fragments and then step-eluted with 1.5 M NaCl to elute the HS. This was desalted on Sephadex G-50 and lyophilized as above.

Estimation of Molecular Weight-- Core protein remnants were removed from the HS by alkaline borohydride elimination in 1 ml of 50 mM sodium hydroxide/1 M sodium borohydride incubated at 37 °C for 24 h. The pH was neutralized by careful addition of glacial acetic acid using phenol red as an indicator. The HS was then fractionated on a Sepharose CL-6B column (Pharmacia: 1.5 × 90 cm) in 0.2 M NH4HCO3 at 10 ml/h. The Vo marker was blue dextran 2000 (Mr ~ 2 × 106), and the Vt marker was sodium dichromate (Mr 294).

The relationship between the Kav and the molecular weight of the GAG chain has been previously determined (21), and the nomogram in the latter paper was used to calculate the average molecular weight of an HS chain. Alkaline borohydride was only used in the preparation of HS for the estimation of chain length and not for the other analytical techniques, to avoid the loss of any alkali-sensitive sulfate groups on the HS chain.

Heparinase I and Heparinase III (Heparitinase) Scission-- HS was dissolved in heparinase buffer (0.1 M sodium acetate, 0.1 mM calcium acetate, 0.1 mg/ml bovine serum albumin, pH 7, at 37 °C) containing 50 µg of unlabeled HS. Heparinase I or heparinase III (20 mIU/ml) were added three times over 18 h at 37 °C, and the progress of the digestion was followed by monitoring A232.

The distribution of heparinase I- and heparinase III-sensitive sites were individually determined by gel filtration chromatography through a 1 × 130-cm Bio-Gel P-10 (Bio-Rad) column pre-equilibrated with 0.2 M NH4HCO3 at 4 ml/h. The susceptibility of a sample of HS to an enzyme was calculated by the formula, percentage of disaccharides cleaved = Sigma [([3H]GlcNRn)/n] × 100/total cpm, where [3H]GlcNRn is the number of counts in the area under a peak for a particular oligosaccharide of length n disaccharides. The average molecular weight of heparinase I-resistant HS was determined by gel filtration chromatography of the digested HS on Sepharose CL-6B as described above.

Nitrous Acid Scission-- Freshly prepared low pH nitrous acid was made according to Shively and Conrad (22). Nitrous acid (100 µl) was added to 10 µl of sample in water. The reaction was allowed to proceed for 60 min at room temperature and then terminated by the addition of 2 M Na2CO3 with phenol red as the indicator. The digested material was mixed with 3 µl of saturated sodium dichromate solution and 100 µl of saturated bovine hemoglobin (Vo and Vt markers, respectively) and loaded onto a 1 × 130-cm Bio-Gel P-10 column pre-equilibrated with 0.2 M NH4HCO3 at a flow rate of 4 ml/h, and 1 ml fractions were collected (22).

The radioactivity elution profile for [3H]glucosamine label was used to calculate the degree of N-sulfation of HS. To analyze the nitrous acid-cleaved material further, those fractions corresponding to the disaccharides and tetrasaccharides were separately pooled and exhaustively lyophilized. The disaccharides were reduced by incubation in 2 M NaBH4, 50 mM NaOH at 37 °C for 45 min so that they could be identified by comparison with known reduced standards. Disaccharides and tetrasaccharides from nitrous acid scission were separately applied to two Propac PA1 strong anion-exchange (SAX) HPLC columns connected in series to a Dionex HPLC system. The tetrasaccharides were eluted by a continuous gradient from 0-1 M NaCl, pH 3.5, whereas the disaccharides were eluted with a biphasic gradient (0-120 mM, 0-20 min; 120-1,000 mM, 21-60 min). The latter method allowed the separation of IdceA(2S)-aManR and GlcUA(2S)-aManR disaccharides.

Enzymic Depolymerization and Analysis of Composition by SAX-HPLC-- HS was completely depolymerized by incubation with heparinases I, II, and III (20 mIU/ml each) for 24 h at 37 °C. The activity of each enzyme against HS (0.5 mg/ml) was measured separately by monitoring the increase in absorbance at A232.

The digested material was loaded onto a 1 × 100-cm Bio-Gel P-2 column (Bio-Rad) and eluted with 0.2 M NH4HCO3 at a flow rate of 5 ml/h to confirm the completion of chain scission (always 95% digestion). Fractions corresponding to the disaccharides were pooled and extensively lyophilized to remove the NH4HCO3.

The disaccharides were dissolved in 1 ml of distilled water (pH 3.5), loaded onto a 5-µm Spherisorb SAX (Technicol, Stockport, UK) column linked to a Dionex HPLC apparatus, and eluted at 1 ml/min with a linear gradient between 0 and 0.75 M NaCl, pH 3.5. Fractions of 0.5 ml were collected, and their radioactive content was determined. The column was calibrated with HS disaccharides of known composition that were detected by their A232 using the in-line UV detector in the HPLC apparatus.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Adenoma and carcinoma cells were taken at 80% confluence and metabolically radiolabeled for 48 h with [3H]glucosamine and 35SO4. GAGs were extracted from the cell layer by treatment with Pronase in 1% (v/v) Triton X-100; the culture medium was also digested with Pronase, and the medium and cell layer extracts were then pooled and clarified by centrifugation (see "Experimental Procedures"). The GAGs were fractionated by anion-exchange chromatography, and the HS (identified by its degradation by nitrous acid) eluted in a double-labeled peak at ~0.55 M NaCl (not shown). There was no difference in the NaCl concentration required to desorb the HS produced by adenoma and carcinoma cells. The 3H/35S-labeled HS was cleared of any contaminating chondroitin sulfate and hyaluronan by treatment with chondroitinase ABC. The Sepharose CL-6B elution profiles of intact adenoma and carcinoma HS species were symmetrical, and the position of the peak of elution of each sample corresponded to a chain of average molecular mass 20 kDa, equivalent to 45-50 disaccharides (Fig. 1).


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Fig. 1.   Sepharose CL-6B gel filtration chromatograph showing the position of elution of intact and heparinase I-resistant fragments of adenoma HS (a) and carcinoma HS (b). Solid line, intact material; dotted line, heparinase I-resistant material.

Low pH Nitrous Acid Scission of HS-- The adenoma and carcinoma HS chains were cleaved with nitrous acid, which breaks HS at the glycosidic bond between N-sulfated glucosamine and uronic acids. The products of scission were eluted by Bio-Gel P-10 gel filtration, and the chromatographs (Fig. 2, a and b) show that the major low molecular mass products were either disaccharides derived from the S domains (contiguous N-sulfated disaccharides) or tetrasaccharides of structure hexuronic acid-GlcNAc-GlcUA-aManR, where the aManR (anhydromannitol) is derived from GlcNS in the HS chains. The 35S in these peaks represents O-sulfated sugars in the tetrasaccharides, and O-sulfated sugars plus inorganic 35S, released from the N-sulfate groups, in the disaccharide peak.


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Fig. 2.   Bio-Gel P-10 gel filtration chromatographic analysis of adenoma HS (a, c, and e) and carcinoma HS (b, d, and f) after scission with nitrous acid (a and b), heparinase III (c and d), and heparinase I (e and f). The insets in e and f represent enlargements of the 3H radiolabel profiles in each graph. Solid line, 3H label; dotted line, 35S label; dp, degree of polymerization; dp2, disaccharides, dp4, tetrasaccharides; dp6, hexasaccharides.

The nitrous acid profiles (Fig. 2, a and b) can be used to calculate the degree of N-sulfation of HS (3, 25). The adenoma HS was more susceptible to nitrous acid scission (37% disaccharides) than the carcinoma HS (32% scission), and 16% of the adenoma HS chain and 13% of the carcinoma HS were present as contiguous N-sulfated sequences (Table I), as revealed by the level of 3H radioactivity in the disaccharide peaks in Fig. 2, a and b). In general the data also suggest that the adenoma and carcinoma HS chains maintain a close clustering of O- and N-sulfate groups and that the general distribution of N-sulfated residues is similar in both chains.

                              
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Table I
Summary of the comparative sensitivities of the adenoma and carcinoma HS species to specific reagents

Heparinase III Scission-- Heparinase III (heparitinase) largely cleaves HS at hexosaminidic linkages to glucuronic acid residues (23-25), although the enzyme also shows activity against some glucosaminidic linkages to iduronic acid (26) but with reduced efficiency. However, under the conditions of this investigation, resistant fragments are likely to contain mainly nonsulfated and 2-O-sulfated iduronic acid residues (see "Discussion").

The samples of digested adenoma and carcinoma HS gave broadly similar elution profiles, and it was calculated that 69% of the adenoma HS was sensitive to heparinase III scission, whereas 74% of the carcinoma HS was cleaved by the enzyme (Fig. 2, c and d; Table I). One notable difference, however, was that the adenoma HS yielded more hexa- than octasaccharide, but the converse was true in the carcinoma (Fig. 2, c and d). The heparinase III profiles demonstrate that in both HS species, the majority of the GlcUA-bearing disaccharides occur in contiguous sequences, as revealed by the large disaccharide peaks (Fig. 2, c and d). The fragments that are resistant to heparinase III digestion are the sulfated domains (S domains).

Heparinase I Scission-- To gain further information on the structure and location of the S domains, the enzyme heparinase I was used. This enzyme cleaves HS essentially where GlcNS(±6S)-IdceA(2S) residues occur (23, 24), although the enzyme is active against glucuronate-2-O-sulfate (28), a rare constituent in HS (29, 30). Scission of the intact adenoma and carcinoma chains with heparinase I and elution of the products by Sepharose CL-6B gel filtration (Fig. 1) showed that the Kav of the main peak of eluted material was 0.7, suggesting that the average molecular weight of heparinase I-resistant fragments was 7,000 (21).

Smaller fragments were also produced by heparinase I, and these were examined by chromatography on Bio-Gel P-10. The results showed that the adenoma HS gave a significantly higher yield of low molecular weight products (disaccharides and tetrasaccharides) than the carcinoma HS (Fig. 2, e and f). Although 13% of the hexosaminidic linkages in the adenoma HS were susceptible to heparinase I, only 7% of such linkages were cleaved in the carcinoma HS. In addition, the chromatographs suggest that approximately 4% of the adenoma HS disaccharides were present as contiguous heparinase I-sensitive disaccharides, whereas the corresponding figure for the carcinoma HS was about 2% (Fig. 2, e and f; Table I). This was a consistently observed difference and was noted in three separate experiments.

Total Disaccharide Composition-- HS from adenoma and carcinoma cells was completely depolymerized with heparinases I, II, and III. The disaccharides were separated by strong anion-exchange HPLC, and the results are shown graphically (Fig. 3, a and b) and numerically (Table II). The major disaccharide in both HS species was Delta UA-GlcNAc, which comprised just over half the total disaccharide units; the Delta UA-GlcNSO3 was also a prominent constituent (Table II). However, a number of reproducible differences were present in the O-sulfated disaccharides. In particular, progression to carcinoma was associated with an increase in Delta UA-GlcNAc(6S) but a reduction in Delta UA(2S)-GlcNS. The latter disaccharide is most likely to contain IdceA(2S) in the HS chain and to be located in the S domains, where it will be cleaved by heparinase I. The findings on the content of IdceA(2S) in the two HS species are compatible with the reduced heparinase I sensitivity in the carcinoma-derived material noted earlier (Fig. 2, e and f).


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Fig. 3.   Strong anion-exchange HPLC analysis of disaccharides and tetrasaccharides derived from adenoma HS (a, c, and e) and carcinoma HS (b, d, and f). Panels a and b show the separation of disaccharides released by complete depolymerization of HS using heparinases I, II, and III (peak 1, Delta UA-GlcNAc; peak 2, Delta UA-GlcNS; peak 3, Delta UA-GlcNAc(6S); peak 4, Delta UA(2S)-GlcNAc; peak 5, Delta UA-GlcNS(6S); peak 6, Delta UA(2S)-GlcNS; peak 8, Delta UA(2S)-GlcNS(6S)). Panels c and d show the separation of nitrous acid-liberated disaccharides (peak 1, IdceA/GlcUA-aManR; peak 2, IdceA(2S)-aManR; peak 3, GlcUA-aManR(6S); peak 4, IdceA-aManR(6S); peak 5, IdceA(2S)-aManR(6S); panels e and f show the analysis of nitrous acid-resistant tetrasaccharides (peak 1, nonsulfated; peak 2, monosulfated; peak 3, disulfated). Solid line, 3H label; dotted line, 35S label.

                              
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Table II
Comparative disaccharide compositions of the adenoma and carcinoma HS species
HS samples were degraded by combined heparinase I, II, and III digestion, and the resulting disaccharides were analyzed by SAX-HPLC. The results represent the mean of values obtained from three determinations, with the S.E. values in all cases being <= 1.5%.

The data also show that the average number of sulfate groups per 100 disaccharides in the adenoma and carcinoma HS was 73 and 61, respectively. The lower sulfation in the carcinoma HS was due to a 20% reduction in N-sulfation and a 33% reduction in 2-O-sulfation, the observed 24% increase in 6-O-sulfation only partially offsetting the fall in 2-O- and N-sulfation.

Analysis of Disaccharides Liberated by Nitrous acid; Comparison with Heparinase-released Disaccharides-- The foregoing analyses indicated that transformation was associated with a reduction in 2-O-sulfation and an increase in 6-O-sulfation. To establish the principal locations of these changes, disaccharides and tetrasaccharides released by nitrous acid scission were examined further by SAX-HPLC. Disaccharides released by nitrous acid provide an indication of the composition of the S domains.

The relative proportions of each disaccharide were largely equivalent in the adenoma and carcinoma material except for a striking decrease in the IdceA(2S)-aManR in the carcinoma HS (Fig. 3, c and d). This component corresponds to the Delta hexuronic acid(2S)-GlcNS unit in the heparinase-released disaccharides (Table II). Disaccharides of structure GlcUA(2S)-aManR(6S) that elute after IdceA(2S)-aManR(6S) (peak 5) on SAX-HPLC were not detected. The data indicate that the reduction in 2-O-sulfation on transformation occurs chiefly in the S domains. The major 6-O-sulfated unit in the nitrous acid-released disaccharides was IdceA(2S)-aManR(6S), and only very small amounts of the mono-6-O-sulfated units GlcUA/IdceA-aManR (6S), were present (Fig. 3, c and d). This indicates that the majority of the Delta UA-GlcNS (6S) detected in the heparinase digests (Table II) must be present in the tetrasaccharides released by nitrous acid, which correspond to the mixed sequences. Because of the close coupling of N- and O-sulfation in HS, the Delta UA-GlcNAc (6S), which represents 12% of adenoma and 16% of carcinoma HS disaccharides, will also be present mainly in the mixed sequences, which thus contain significantly more 6-O-sulfates than the contiguous N-sulfated regions. Combining the data in Table II and Fig. 3, c and d, we can calculate that approximately 70 and 80% of the total 6-O-sulfates are present in the mixed sequences in the adenoma and carcinoma HS, respectively.

Analysis of Nitrous Acid-derived Tetrasaccharides-- The structure of the tetrasaccharide products of nitrous acid scission were analyzed by SAX-HPLC and shown to contain non-, mono-, and disulfated species (Fig. 3, e and f). 35S radiolabel in these peaks is O-sulfate that is mainly present at C-6 of GlcNAc and C-6 of aManR (see "Discussion"). The sulfation of each peak was based on the ratio of 3H/35S. The results showed that the adenoma-derived nitrous acid-resistant tetrasaccharides consisted mainly of non- and mono-O-sulfated structures (Fig. 3e), whereas the carcinoma contained a significantly higher proportion of monosulfated structures, with some di-O-sulfated material also present (Fig. 3f). These differences were reproduced in three experiments. The results confirm that variation in O-sulfation of the mixed sequences is an important distinguishing feature between adenoma and carcinoma HS.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

This paper describes a detailed analysis of the changes in structure of HS in a model of human colon carcinogenesis in vitro (20). The study shows that HS from cultured adenoma and carcinoma cells share a common molecular organization in the form of similar chain lengths and spacing of S domains (Fig. 1). When HS was degraded with either low pH nitrous acid or heparinase III (Fig. 2), the results showed that many of the linkages susceptible to these reagents were contiguous especially in the case of heparinase III (Fig. 2). The data indicate that the HS from both samples conform to a domain structure that is characteristic of the HS family (4, 31). The adenoma and carcinoma HS were approximately 45-50 disaccharides in length (20 kDa), and the fragmentation by heparinase I to yield a major peak of resistant material with a molecular mass of 7 kDa (Fig. 1) implied that the HS chains contained two evenly spaced S domains.

Progression to malignancy was associated with specific structural changes, including a reduction in 2-O-sulfation in the S domains and an increase in 6-O-sulfation in the mixed sequences, which consist of alternate N-sulfated and N-acetylated disaccharides. Previous studies have suggested that O-sulfates in these sequences are mainly present on C-6 of the amino sugars (32), and we assume that this is the case in the HS studied here. Structural changes in the mixed sequences have been reported before in murine models of transformation. These studies reported that tumor cells synthesized HS with a reduced content of 6-O-sulfate (15-17) and 3-O-sulfate (18) in the mixed sequences. Our results confirm that these regions are a major target of structural change in carcinogenesis. However, the data show that malignant transformation of human adenomas is accompanied by an increase in mixed sequence 6-O-sulfation mainly associated with GlcNAc residues (Table II). This increase is also revealed by the high content of mono-O-sulfated and di-O-sulfated tetrasaccharides in the nitrous acid scission products (Fig. 3, e and f).

The biosynthesis of HS is assumed to follow the heparin pathway of polymer modification in which the addition of 2-O-sulfates to iduronic acid residues occurs before 6-O-sulfation of GlcNS/GlcNAc (1, 33, 34). The composition of the S domains is compatible with this sequence of events, in that most of the 6-O-sulfate in these domains occur in disaccharides bearing 2-O-sulfate moieties (Fig. 3, c and d). However, in the adenoma HS and particularly in the carcinoma HS, there are more 6-O-sulfate residues in the flanking sequences than in the S domains. Taken in conjunction with the reduction in 2-O-sulfation in the carcinoma HS, the data suggest that the 6-O-sulfotransferases that act on the mixed sequences operate independent of the presence of 2-O-sulfate groups, in accord with findings on the structure of HS produced by a Chinese hamster ovary cell mutant defective in 2-O-sulfotransferase (35). It is interesting that in other species of HS the content of 6-O-sulfates is higher in the mixed sequences than in the S domains (28).

The data also indicate that there is a reduction in 2-O-sulfation after malignant transformation (Table II; Fig. 2, e and f). Since the 2-O-sulfotransferase has a similar amount of iduronate (Table I, heparinase III-resistant fraction) to act on in both HS species, the implication is that the enzyme is acting more efficiently in the adenoma than the carcinoma.

Approximately 70% of both the adenoma and carcinoma HS species were sensitive to heparinase III (Fig. 2, c and d), which acts mainly on GlcUA-containing disaccharides (23, 24). IdceA-containing disaccharides are relatively poor substrates by comparison. Although activity has been demonstrated against IdceA-containing disaccharides (25, 26), studies with desulfated heparins indicate that in polymeric structures, cleavage of such units is substantially incomplete (27). Our data show that only 13% of the adenoma HS and 7% of the carcinoma HS contained IdceA(2S)-bearing disaccharides (Table II). If heparinase III cleaved all disaccharides containing nonsulfated uronates, then 87% of the adenoma HS and 93% of the carcinoma HS would be depolymerized by the enzyme, levels well above the ~70% values actually observed (Table I; Fig. 2, c and d). This suggests that the enzyme is predominantly acting on the preferred substrate species, i.e.. GlcNAc/GlcNSO3-GlcUA (23, 24), although there may be some limited scission at IdceA residues.

The data presented above allow the construction of a simplified composite model of the structure of HS produced by the adenoma and carcinoma cells (Fig. 4). The chains are shown to contain two evenly spaced S domains with internal sites of cleavage for heparinase I. The variations in 2-O-sulfation and 6-O-sulfation in the S domains and mixed sequences of the two HS species are also illustrated. The proportions of material contained in the di- and tetrasaccharide peaks of heparinase I-released fragments (Fig. 2, e and f) were used to calculate the number of heparinase I scission sites in the S domains. These data suggested that, on average, heparinase I cleavage of an S domain in the adenoma HS would liberate one disaccharide and one tetrasaccharide, whereas the same treatment of a carcinoma HS S domain would release either a disaccharide (as shown in Fig. 4) or a tetrasaccharide.


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Fig. 4.   Domain structures of adenoma and carcinoma HS species. HS from both adenoma and carcinoma cells is depicted with two S domains (open rectangles) evenly spaced along the chain from the core protein (left) to the nonreducing terminus (right). These S domains will contain the sites of cleavage for heparinase I. Distinct O-sulfation patterns are present in the S domains and the adjacent mixed sequences. In the carcinoma HS, the mixed sequences are relatively enriched in 6-O-sulfate, but a lower concentration of 2-O-sulfate occurs in the S domain. As a consequence of this reduction in 2-O-sulfate, the carcinoma HS has fewer sites of cleavage for heparinase I than the adenoma HS. Circles, GlcUA-GlcNAc; squares, GlcUA/IdceA-GlcNSO3; numbers 2 and 6 refer to 2-O-sulfates on IdceA residues and 6-O-sulfates on GlcNAc or GlcNSO3, respectively; thin arrows, linkages susceptible to nitrous acid scission; thick arrows, linkages susceptible to heparinase I.

In conclusion, the findings from the present study indicate that the overall molecular architecture of HS is preserved in colon adenoma and carcinoma cells, although distinct changes are imposed on this structure in the malignant cell through changes in the content and pattern of sulfation, particularly the O-sulfates. The data also imply that 2-O-sulfation and 6-O-sulfation may be differentially regulated. The significance of these changes for the malignant phenotype is unclear, but in view of the role of HS in controlling cell growth and adhesion, it seems reasonable to assume that the structural modifications in the carcinoma HS may promote a more proliferative or invasive type of behavior.

    ACKNOWLEDGEMENTS

The authors are very grateful to N. Gasiunas, E. Dignan, J. Niland, and G. Lebens for their technical assistance. We thank the Cancer Research Campaign and the Association for International Cancer Research for their financial support.

    FOOTNOTES

* This work was supported by the Cancer Research Campaign (United Kingdom) and the Association for International Cancer Research (United Kingdom).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Cancer Research Campaign, Dept. of Medical Oncology, Christie Hospital NHS Trust, Wilmslow Rd., Withington, Manchester M20 4BX, UK. Fax: ++44 161 446 3299.

1 The abbreviations used are: HS, heparan sulfate; aManR, anhydromannitol; GAG, glycosaminoglycans; GlcUA, glucuronic acid; GlcNAc: N-acetylated glucosamine; GlcNS: N-sulfated glucosamine; GlcNS(6S), N-sulfated glucosamine 6-O-sulfate; HPLC, high performance liquid chromatography; IdceA, iduronic acid; IdceA(2S), IdceA 2-O-sulfate; PBS, phosphate-buffered saline; SAX, strong anion exchange; UA, uronic acid.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
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