Synthesis of Glycosaminoglycans by Undifferentiated and Differentiated HT29 Human Colonie Cancer Cells1

[CANCER RESEARCH 47, 4478-4484, August 15, 1987] Synthesis of Glycosaminoglycans by Undifferentiated and Differentiated HT29 Human Colonie Cancer Cells1

Patricia Simon-Assmann,2 FranÃ§oiseBouziges,3 Daniele Daviaud, Katy Haffen, and MichÃ¨leKedinger

INSERM UnitÃ©61,Biologie Cellulaire et Physiopathologie Digestives, 3 avenue MoliÃ¨re,67200 Strasbourg Hautepierre, France

ABSTRACT colon carcinoma cell line established by Fogh and Trempe (11). Indeed, as discovered by Zweibaum's group (12-14), this cell Among the extracellular matrix components which have been sug gested to be involved in developmental and neoplastic changes are gly- line displays typical enterocytic differentiation characteristics cosaminoglycans (GAGs). To try to correlate their amount and nature (apical brush borders, tight junctions, presence of hydrolases) with the process of enterocytic differentiation, we studied glycosamino- when cultured in the total absence of sugar, in contrast to cells glycan synthesis of human colonie adenocarcinoma cells (HT29 cell line) grown with glucose which always remain undifferentiated (14). by [3H]glucosamine and |35S]sulfate incorporation. Enterocytic differen The differentiation process starts after the cells have reached tiation of the cells obtained in a sugar-free medium (for review, see A. confluency and continues for at least 20 days (13). Zweibaum ft al. In: Handbook of Physiology. Intestinal Transport of the In the present study, sulfated and nonsulfated GAGs from Gastrointestinal System, in press, 1987) resulted in a marked increase in undifferentiated (HT29G+) and differentiated (HT29G-) cells total incorporation of labeled precursors (20-fold for |3H)glucosamine, 4.5-fold for |35S|sulfate) as well as in uronic acid content (5-fold); most were analyzed. We report here the occurrence of changes in the of the synthesized GAGs were found associated with the cell pellet. amount, proportion, and nature of the different types of GAGs Chromatographie and electrophoretic analysis of the labeled GAGs re after differentiation of this colonie carcinoma cell line. vealed that undifferentiated cells synthesized and secreted hyaluronic acid, lit-paran sulfate, and one class of chondroitin sulfate. Differentiation of HT29 cells became associated with the synthesis of an additional class MATERIALS AND METHODS of chondroitin sulfate (CS4) concomitant to a decrease in heparan sulfate Cell Culture. The human colon adenocarcinoma cell line HT29 (11) which is no longer found secreted in the medium. Furthermore the charge given by Dr. A. Zweibaum (Paris) was used in this work. The cells were density of this latter GAG component varied as assessed by a shift of its cultured in the presence of glucose (HT29G+) or by substituting glucose affinity on ion-exchange chromatography. for inosine (HT29G-) (13). This last culture condition represents the easiest way of obtaining rapidly differentiated HT29 cells without any INTRODUCTION effect on cell viability and growth (15). The HT29G+ cells (passages 145 to 149) were grown in Dulbecco's Accumulating evidence suggests that intestinal development, modified Eagle's medium (Eurobio, Paris, France) containing glucose including epithelial cell differentiation, is dependent upon epi- (standard concentration, 25 HIM) and supplemented with 10% fetal thelial-mesenchymal interactions (for reviews see Refs. 1-3). bovine serum (Gibco Biocult, Glasgow, Scotland) and Gentalline (200 Among the mechanisms which could be involved in these proc jig/ml). The cells were subcultured by trypsinization (0.25% trypsin in esses, one can assume that extracellular matrix components 0.53 IBM EDTA; Gibco Bio-cult) and seeded at 5 x IO5 cells/28-cm2 play a major role; such an assumption is supported by the fact plastic dishes (Falcon, Los Angeles, CA). Cultures were maintained at 37Â°Cin an atmosphere of 5% COi in air. The media were changed that matrix molecules, like fibronectin, procollagen type III, laminin, nidogen, and collagen type IV, found at early devel daily. The experiments were performed 1 week after seeding when the cultures reached confluency. opment stages, exhibit changes in their distribution pattern as The HT29G- cells (passages 14 to 16), seeded at 1 x 10' cells were a function of specific morphogenetic events (4). For several cultured in Dulbecco's modified Eagle's medium specially prepared years, increasing interest focused on GAGs,4 which are long without glucose and without sodium pyruvate by Eurobio and supple polyanionic carbohydrate chains composed of repeating disac- mented with 10% dialyzed fetal bovine serum (Gibco Bio-Cult), 2.5 charide units (uronic acid and hexosamine) forming the ground HIMinosine (Sigma Chemical Co., St. Louis, MO) and Gentalline (200 substance of the extracellular matrix. These macromolecules Mg/ml). The cells were subcultured with 0.25% trypsin in 2.68 mM include hyaluronic acid, heparan sulfate, CS4, CS6, and der- EDTA. The experiments were done 4 weeks after confluency, the time matan sulfate; with the exception of hyaluronic acid (the only period necessary to obtain their optimal differentiation (13). nonsulfated GAG), GAGs in tissues are always linked cova- Cells were counted in the presence of trypan blue by using a hemo- cytometer. The final cell number attained by HT29G+ and HT29G- lently to proteins as proteoglycans. at the time of experiment was similar (22 x IO6cells/dish). Changes in the composition of GAGs have been correlated with development and differentiation in a great number of For studies of GAG synthesis as a function of cell density, two systems (for reviews see Refs. 5-7). Of interest also is the experimental approaches were utilized. In the first approach series of triplicate dishes were seeded with cells at different densities and har presence of high amounts of CS4 and CS6 in colonie tumors vested for GAG extraction at a precise culture time after plating. In the (8, 9), a phenomenon otherwise reported in the early phases of second experimental approach, series of triplicate dishes were seeded intestinal development (9, 10). with 5x10' HT29G+ cells or 1 x 10" HT29G- cells but the dishes To correlate cell differentiation and GAG pattern, an appro were harvested after different time culture periods. priate i/i vitro model system is provided by the human HT29 Isotopie Labeling of the Cells and Isolation of Extracellular, Pericel- lular, and Cellular Compartments. D-[6-3H]Glucosamine hydrochloride Received 11/5/86; revised 3/30/87; accepted 5/19/87. (Amersham; specific activity, 20-40 Ci/mmol) and Na235SO4(Amer- The costs of publication of this article were defrayed in part by the payment sham; specific activity, 100 mCi/mmol) were added to the culture of page charges. This article must therefore be hereby marked advertisement in medium at isotopie concentration of 4 /<<'Â¡/ml24 h prior to cell accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Financial support was given by INSERM, ARC (Grant 6236), and The Ligue harvesting. The former compound is a precursor of the nonsulfated Nationale FranÃ§aisecontre le Cancer. GAG hyaluronic acid and the sulfated GAGs, whereas the 3SSlabel is 2To whom requests for reprints should be addressed. incorporated into sulfated GAGs. 3Recipient of a fellowship from ARC. After the labeling period at 37Â°C,the culture medium of three dishes 4The abbreviations used are: GAG(s), glycosaminoglycan(s); CS4 and CS6, chondroitins 4- and 6-sulfate. was removed and the cell layers were washed with Tris-HCl, pH 7.0 4478

(final concentration, 0.1 M). The media and the washings were pooled Gel Filtration. Untreated individual GAGs and their digestion prod and called the "extracellular compartment." The cells were harvested ucts were filtered on a Sepharose 4B column (0.45 x 60 cm; Pharmacia, with 0.25% trypsin in EDTA at 37'C for 10 min. Proteolysis was Uppsala, Sweden) in 0.15 M NaCI- 0.01 M Na2HPO4-NaH2PO4, pH stopped with Ham's F-10/newborn bovine serum (8/2) (Gibco Bio- 7.2. Elution was performed at 4Â°Cat a flow rate of 12 ml/h and Cult). After centrifugation (900 x g, 10 min), the supernatant was fractions of 0.9 ml were collected. Degradation was assessed by the recovered and designated as "pericellular compartment." The cell pellet disappearance or the shift of the original peak towards the total volume. was resuspended in 0.1 M Tris-HCl (pH 7.0) and designated as the Fluoroautoradiography. The isolated GAGs were subjected to cellu "cellular compartment." lose acetate electrophoresis; 1 /jl of the GAG fractions or of digested Extraction and Analysis of GAGs. The different isolated compart samples was applied to strips of Titan III (Helena Laboratories, Beau ments were digested with 0.5 mg/ml protease (from purified Strepto- mont, TX); electrophoresis was carried out in 0.2 M calcium acetate at myces caespitosus type IV; Sigma) for 24 h at 37Â°C.Then, linkage of 3 mA/strip for 2 h. The position of the GAGs was visualized by residual peptides to GAGs was cleaved by adding NaOH at a final fluoroautoradiography as follows. The electrophoretograms were dried concentration of 0.1 N and stirring overnight at 4"C. Residual undi at room temperature, impregnated with EN3HANCE (New England gested peptides were then precipitated with trichloroacetic acid (5%, Nuclear), and covered with Kodak X-ray film; an intensifying screen w/v) for 2 h at 4Â°C.After centrifugation at 2000 x g for 30 min the (Philips, The Netherlands) was interposed between the strip and the film. Development was achieved after 1 week of exposition at â€”80Â°C. supernatant was dialyzed extensively against water to remove free label and contaminating small glycopeptides (M, <2000). 3H and 35Sradio The identity of the radiolabeled GAGs was determined (a) by comparing activity of aliquots was counted by liquid scintillation spectrometry and the position of autoradiographic spots to that of the known GAG defined as total amount of GAGs synthesized in each compartment.5 standards (detected by Alcian blue staining) and (b) by monitoring the Uronic acid content was determined by the method of Bitter and Muir disappearance of the spots after enzymatic or chemical degradation. (16) using glucuronic acid as standard. The sum of either the 3H, 35S, IR Spectroscopy. Using this technique, the sulfated GAG classes can be distinguished based on their differences of spectra in the region 800- or uronic acid values found in the different compartments represents 950 cm~' (20, 21). Lyophilized samples (1-2 mg) were thoroughly the total glycosaminoglycan production. Remaining dialyzed samples were lyophilized and subjected to chromatography in order to separate ground with potassium bromide (150 mg) in an agate mortar; the the individual GAGs. In order to obtain the best separation of the mixture was then squeezed in an engineering workshop vice. Transpar different classes of GAGs, preliminary experiments were performed ent pressings were then placed in the spectrophotometer (Perkin Elmer) with different NaCI gradient ranges and different sizes of columns. The cell compartment. data led us to choose the following optimal experimental conditions. Morphological Analysis. For scanning electron microscopy, cells Samples were applied to a 9- x 1-cm column of DEAE-cellulose (DE52; were grown on plastic culture coverslips (Thermanox Lux Scientific Whatman) equilibrated with 50 mM Tris-HCl (pH 7.2), a method Corp., Newbury Park, CA). The cells were fixed in 0.2 M cacodylate- buffered 2% glutaraldehyde (pH 7.4) for 2 h at 4Â°C,dehydrated, dried adapted from that used by Gordon and Bernfield (17). The bound components were eluted by developing a I(IO-mIlinear gradient from 0 in a critical point drier, coated with gold, and examined with a Philips to 0.6 M NaCI in 50 mM Tris-HCl (pH 7.2) at a flow rate of 13 ml/h, 501B scanning electron microscope. followed by the passage of 20 ml of 2 M NaCI. Fractions of 2 ml were Immunocytochemical Localization of Sucrase. The expression of a collected and aliquots (0.1 ml) were counted using Biofluor scintillation digestive enzyme, sucrase, at the microvillar level has been revealed by fluid (New England Nuclear, Boston, MA). The different fractions immunofluorescent staining with monoclonal antibodies specific for sucrase-isomaltase (HBB 2/614/88), kindly provided by Dr. H. P. corresponding to individual radioactive peaks were pooled, dialyzed, and lyophilized for subsequent determination of uronic acid content Hauri (Biocenter, Basel, Switzerland) (22). Fixed cryosectioned cell sheets were prepared and stained according to the methods described and GAG identification. Enzymatic and Chemical Degradation of the GAGs. To identify GAG previously (23). components, isotopically labeled peaks from DEAE-cellulose chroma tography were subjected to enzymatic or chemical degradation, followed by gel filtration or fluoroautoradiography. In order to obtain enough RESULTS radiolabeled material, the individual peaks obtained from several ex periments and which have been eluted at the same concentration of Criteria of HT29 Differentiation. Differentiation of HT29 NaCI were pooled. cells resulting from the replacement of glucose by inosine in Aliquots of the different peaks were enzymatically digested with the the culture medium, already demonstrated by Zweibaum's following enzymes: 2.5 units of Streptomyces hyaluronidase (Sigma) in group (13), has been assessed in this study morphologically and 0.1 M sodium acetate buffer-0.13 M NaCI (pH 5.0) for 16 h at 37Â°C; enzymatically. Under the scanning electron microscope, the 0.25 unit of chondroitinase AC or ABC (Sigma) in 0.15 M Tris-NaCl- sodium acetate (pH 8.0) for l h at 37Â°C.The aliquots were placed in a differentiated cells (Fig. \d) exhibited clearly defined contours when compared to undifferentiated ones (Fig. la). In addition boiling water bath for 1 min. Samples of known standard substrates hyaluronic acid, chondroitins 4- and 6-sulfate, and dermatan sulfate the former cells exhibited typical elongating apical brush bor (Sigma) were digested with the above mentioned enzyme preparations. ders as illustrated at a higher magnification (Fig. le), the surface Chemical degradation of GAGs with 11\( ): was carried out by a method of the HT29 G+ cells being at the opposite covered by irregular (18) derived from that described by Lindahl et al. (19). Aliquots (50 M!) membrane processes (Fig. Ib). The enzymatic differentiation of were treated with 50 MlNaNO2 (5%) and 50 Â¿ilCH3COOH (33%) for the cells was proved by a positive fluorescence with the mono 80 min at room temperature; the degradation products were discarded clonal against sucrase-isomaltase at the brush border level of by successive washings in ethanol saturated with NaCI and centrifuga- groups of cells (Fig. If versus Fig. le). tions. Glycosaminoglycan Production as a Function of Cell Density. Under these conditions Streptomyces hyaluronidase and nitrous acid To investigate growth-related changes in GAGs, we measured degraded exclusively hyaluronic acid and heparan sulfate, respectively, [3H]glucosamine and [35S]sulfate incorporations over 24 h and whereas chrondroitinase ABC degraded hyaluronic acid, chrondroitins the overall uronic acid in confluent HT29G+ cells 7 days after 4- and 6-sulfate, and dermatan sulfate; this latter component is not seeding at different cell densities (2 to 12 x 10* cells/dish). At degraded by chondroitinase AC. the end of the experiment, the number of cells varied between s We could show that, respectively, 83 and 94% of the postdialyzed 3H- and 120 and 250 x IO5cells/dish (Fig. 2A). As the cells progressed "S-labeled material were degraded by hyaluronidase, chondroitinase ABC, and from low to high densities, [3H]glucosamine and [35S)sulfate nitrous acid treatments in both HT29G+ and HT29G- cells. Furthermore, approximately 90% of the radioactivity was recovered in the individual GAGs incorporation as well as uronic acid content decreased. The after DEAE chromatography. percentage of distribution of GAGs present in the cellular, 4479

Fig. 1. Morphological and enzymatic char acteristics of undifferentiated (HT29G+; a-c) and differentiated (HT29G-;

extracellular, and pericellular compartments did not vary as HT29 cells in both undifferentiated and differentiated states cell number increased (not illustrated). were able to synthesize GAGs. As depicted in Fig. 3, the In the second experimental approach, the HT29G+ cells amounts of [3H]glucosamine and [35S]sulfate incorporated into seeded at 5 x IO5 cells/dish were harvested for GAG determi GAGs over a 24-h labeling period were, respectively, 20 and nation at different periods varying between 3 and 37 days. As 4.5 times increased in the differentiated cells (HT29Gâ€”) when exemplified by [3H]glucosamine incorporation, an increase in compared to the undifferentiated ones (HT29G+). Analysis of cell number due in this experiment to a longer culture period uronic acid also revealed that HT29Gâ€” contained amounts 5 led to a reduction of 3H-synthesized GAGs (Fig. 2B). From 3 times higher than those present in HT29G+ cells. weeks of culture onwards, no major changes could be observed. Fig. 4 shows the relative contents of the 3H- and 35S-labeled In the HT29Gâ€” cells a similar correlation between cell den GAGs among the cellular, pericellular, and extracellular com sity and GAG production could be observed during the expo partments of HT29 cells. In HT29G+ cells, analysis of GAGs nential phase of growth (not illustrated). However, from 10 synthesis over 24 h revealed that almost one-half (46%) of the days of culture onwards when the HT29Gâ€” cells have reached 35S-labeled GAGs were found in the extracellular compartment, confluency, they behaved differently; indeed, 3H incorporation whereas in HT29Gâ€”cells, they were predominant in the cellular at 10 days (41,397 cpm/106 cells) increased up to 98,907 cpm/ compartment (62%). Concerning the 3H-labeled GAGs, they IO6cells at 4 weeks for the same final cell number. were also found predominantly in the cellular compartment of Total Amounts of Glycosaminoglycans in Undifferentiated and HT29 cells whatever their differentiation state. Differentiated HT29 Cells. Owing to the importance of cell Nature of the Synthesized GAGs. Ion-exchange chromatog- density on GAG synthesis (see preceding section), the latter has raphy (DEAE-cellulose) of GAGs produced by undifferentiated been analyzed in the HT29Gâ€” cells when the differentiated HT29G+ cells during 24 h (Fig. 5A) revealed three distinct state had been attained at 4 weeks in culture and in their radioactive peaks: a singly labeled 3H peak (I) eluting at 0.1 M- undifferentiated HT29G+ counterpart at 7 days, at which time NaCl; a 35Speak (II) eluting at 0.2 M NaCl; and a second 35S- a similar final cell number has been obtained. labeled peak (III) emerging as a shoulder at 0.43 M NaCl. The 4480

8,000

10 20 30 2 5 8 12 Iroction number IO5 < / DISH AT PLATING 2M 12 (I) HT29G- (2MI â€¢Hâ€”

200 05 I i 0-1 Q I01M) 05 Â§ Q < 02 o: 01 358 IS 2ÃŽ 31 37 DAYS OF CULTURE 10 20 30 "10 50 Fig. 2. Changes in glycosaminoglycan production as a function of cell density. (faction nurnbei In .I, cells (t) were seeded at different concentrations and were harvested 1 week Fig. S. Representative isotopically labeled GAG pattern obtained by ion- after plating. In B, cells seeded at S x 10' cells/dish were harvested after different exchange chromatography on DEAE-cellulose of (A) undifferentiated HT29G+ time culture periods. Each column corresponds to a mean of six values. The and (B) differentiated HT29G- cells (pericellular compartments). Elution was values presented herein are the sum of those found in the cellular, extracellular, achieved with a linear gradient of 0-0.6 M NaCl in Tris buffer and then by a and pericellular compartments. further passage of 2 M NaCl; 2-ml fractions were collected. Similar patterns were obtained in at least 4 separate experiments. 3H-GLUCOSAMINE No, URONIC ACID L1râ„¢-I'â€¢i; IOOÃ›OO^I50XXÂ»*Pâ€” In differentiated HT29Gâ€” cells, changes in the elution pro files of GAGs were obvious (Fig. SB). The striking difference is the appearance of an important 'H labeled peak coeluting 1rXsonr=T with small amounts of 3SS radioactivity after passage of 2 M NaCl (peak IV). When this peak was rechromatographed on the same column and eluted in a 0.4-1 M NaCl linear gradient,

;.:oEQ.uIOOOO-â€¢W"on it was recovered from the 0.48 M NaCl fraction (not illustrated). Peak IV could be detected in all three compartments with only G+G- G+G- G+G- small variations in labeling intensity. All over the 0-0.6 M NaCl gradient, no significant "H-labeled peak but a broad shoulder Fig. 3. [3H]Glucosamine and [3SS]sulfateincorporations into GAGs and uronic acid content in undifferentiated O and differentiated (D) HT29 cells. Columns, could be detected; it should be noted that only one 3SS-labeled mean of, respectively, 8 and 3 experiments; ears, SE; ', P < 0.005; **,/>< 0.001. peak was observed, eluting at 0.25 M NaCl (instead of 0.2 M C, cells. NaCl with HT29G+ cells). The presence of uronic acid was confirmed in peaks II and IV as well as in the pooled fractions incorporation os o % of tolal GAGs of regions corresponding to peaks I and III determined in the O 50 100 HT29G+ cells. In all compartments, the 3H radioactivity elu "S-SULFATE tion profiles were very similar. However, in the extracellular compartment, no [35S]sulfate material could be detected be tween 0 and 0.6 M NaCl. Examination of the GAGs profiles obtained in the different experiments further indicated that the 'H-GLUCOSAMINE appearance of peak IV occurred to the detriment of peak II. To identify the Chromatographie peaks of both cell types, isotopically labeled material was treated with Streptomyces hyaluronidase, chondroitinase ABC, chrondroitinase AC, or Fig. 4. Relative distribution of [3H]glucosamine and ("Sjsulfate-labeled GAGs nitrous acid. Peak I, which was devoid of 35S label, contained among cellular (Â¡71).pericellular (D). and extracellular (D) compartments in HT29G+ and HT29G- cells; 100% represents the sum of the counts from the uronic acid suggesting its correspondence to a hyaluronate-like different compartments. material. Indeed it was degraded by Streptomyces hyaluronidase as revealed by the disappearance of the radioactive spot on DEAE elution profiles were identical in the three compartments cellular acetate electrophoresis (Fig. 6). Almost the entire peak although variations in the amounts of radiolabeled material II was sensitive to nitrous acid treatment (Fig. 7) and resistant occurred (not illustrated). The GAG nature of each individual to digestion with either chondroitinase ABC and AC or hyalu peak was confirmed by the presence of uronic acid. ronidase (not illustrated), suggesting that the major constituent 4481 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1987 American Association for Cancer Research. GAGS AND COLONIC CANCER CELL DIFFERENTIATION

(A) II Ve: 13.5 ml i.ooo Ve: 38ml I 3.000

2,000 I

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Fig. 6. Auloradiogram obtained after cellulose acetate electrophoresis of peak mi200MI100"LJ50VVe: 38 I before (a) and after treatment with Streptomyces hyaluronidase (b). Degradation was assessed by the disappearance of the radioactive spot in h. I x Ve: 35ml m 1000

20 30 40. 50 fraction number IVO IVI Fig. 8. Sepharose 4B chromatograms of untreated peak IV from HT29G- cells (.-()and after treatment with chondroitinase ABC (H) or AC (C). Degradation was assessed by the shift of the peak towards the total volume. Arrows, void (Ko) and total (Vt) volumes of the gel column.

peak IV

Fig. 7. Sepharose 4B chromatograms of untreated peak II (A) and after treatment with nitrous acid (B). Degradation was assessed by the disappearance of the majority of the radioactive material. Arrows, void (Vo) and total (Vt) volumes of the gel column. in this peak is heparan sulfate. Peaks III and IV were both degraded by chondroitinases ABC and AC (illustrated for peak IV in Fig. 8), unchanged by either hyaluronidase or nitrous acid treatments (not illustrated), and thus correspond to chondroitin sulfates. The following arguments allowed to identify peak IV as a chondroitin 4 sulfate: (a) it is eluted at the same NaCl molarity as a chondroitin 4 standard whatever the NaCl gra dient used; (b) the IR absorption bands of this peak corre sponded to those of the chondroitin 4 standard (Fig. 9). 1000 Wavenumber DISCUSSION ( cm - 1 ) Fig. 9. IR absorption spectra in the region 1000-800 cm'1 of peak IV as In the present study, marked quantitative and qualitative compared to that of chondroitins (Cs) A and C standards. Peak IV presents two absorption bands like the chondroitin A standard while chondroitin C reveals differences in glycosaminoglycan biosynthesis have been found only one. The wave numbers of the standards, 855 and 925 cm"' for chondroitin between differentiated and undifferentiated HT29 human co- A and 822 cm"1 for chondroitin C, are those described in Ref. 21. Note a slight Ionic adenocarcinoma cells. The most prominent findings re shift in the wave numbers of peak IV as compared to the chondroitin A standard lated to the shift from undifferentiated to differentiated cells due to the isotopie sulfate group. are: (a) an enhancement in the overall GAG synthesis; (h) the appearance of a new class of chondroitin, CS4 sulfate, to the mÃ©rous(for reviews see Refs. 5-7). However, GAG studies detriment of heparan sulfate; (e) a modification in the charge during cellular differentiation are still fragmentary and conflict density of the heparan sulfate molecule. ing, thus suggesting that patterns of GAG production are Actually attempts of correlation between GAG production characteristic of individual tissues. Nevertheless, the relative and morphogenetic events during development are quite nu- increase in chondroitin sulfate (partly due to the appearance of 4482

CS4) in differentiated HT29 cells parallels observations re HT29G- cells exhibited a higher rate of GAG synthesis as ported in other systems. For example, differentiation of the culture time proceeded and (b) chondroitin 4-sulfate, absent at avian cornea has been shown to be accompanied with an overall the beginning of confluency (not shown), was produced only increased level of chondroitins (24). An other example is pro when the cells underwent sufficient differentiative features. It vided by the observation of an enhanced synthesis of chondroi- remains, however, impossible to conclude that the changes in tin 4-sulfate in an in vitro system in which somites undergo GAG pattern reported in the differentiated HT29 cells repre chondrogenic differentiation when stimulated by notochord or sent an additional marker of differentiation or rather that they spinal cord (25). Finally, the formation of an oversulfated act as a triggering event of differentiation via modifications in galactosaminoglycan (corresponding to the class of chondroitin the cellular metabolism. sulfates) during differentiation of monocytes has been reported Finally, whether HT29 differentiation corresponds to the (26). state found in primary tumors from patients or mimics that of Another point of special interest in the present study is the normal absorptive cells remains unclear in view of the following decrease in heparan sulfate. Furthermore the facts that heparan observations. On one hand, tumors developed in nude mice sulfate has not been found secreted by differentiated HT29 cells from cultured HT29G+ cells exhibit morphological and enzy in the culture medium and differed in its charge density as matic characteristics similar to those present in in vitro differ visualized by ion-exchange chromatography argue in favor of entiated HT29G- cells (40, 41). On the other hand, although modifications in its attachment to the cell surface and in its similarities in surface membrane components between colon biosynthesis. From a general point of view, this molecule has tumor HT29 cells and normal small intestinal epithelial cells been demonstrated in various organs to be preferentially located (rather than colonie ones) exist (15, 42), differentiated HT29 in the cell or in close contact with its surface and to be released cells exhibit the closest analogies with human fetal colon (40, into the culture medium at a very low rate (27-30). In the 43-45). intestine, the presence of heparan sulfate possessing a high In conclusion, the results reported here of changes in the metabolic activity has been shown in normal epithelial cells glycosaminoglycan pattern in a human colonie adenocarcinoma (31). More recently, the biosynthesis of heparan sulfate has cell line which is capable of enterocytic differentiation in a been investigated in detail by lozzo (32) demonstrating the sugar-free medium raise the question of whether such a corre existence in human colon carcinoma cells (WiDr) of a large lation between differentiation degree and GAG content can be secretory pool with high turnover localized at the cell surface established by screening primary colorectal tumors from pa and of a nonsecretory intracellular pool with low turnover. tients. Finally heparan sulfate alterations have been found to exhibit reduced degree of sulfatation in virus-transformed cell lines (33) ACKNOWLEDGMENTS and in human (34) or rat (35) hepatoma as compared to normal cells. We wish to thank Maymouna N'Dir (recipient of Senegal govern The occurrence of GAGs in colonie tumors and cultured ment support) for help with the setting up of some techniques. We are malignant cells has been reported previously (8, 9, 32, 36, 37). very grateful to Drs. G. Normand, P. Levy, and J. F. Launay for helpful lozzo et al. (8) found elevated amounts of chondroitin 4- and comments and discussions. We would like to thank Dr. H. P. Hauri 6-sulfate in human colon carcinoma when compared to normal (Biocenter, Basel, Switzerland) for kindly providing sucrase antibody colon; histochemically, the connective tissue cells surrounding and Dr. A. Pousse (Institut de Chimie, Strasbourg, France) for the IR the tumors have been demonstrated to be the major sites of absorption analyses. We are also obliged to Dr. A. Zweibaum for his interest in this work and for critically reading the manuscript. biosynthesis of these sulfated components. Furthermore, it has been shown ;'// vitro that colonie malignant cells elicited an increase of GAG production by fibroblasts (37, 38). REFERENCES In this study, the major advantage of the HT29 cell model developed by Zweibaum et al. (12-14) is that it allows the direct 1. Multai, K., Kedinger, M., and Lacroix, B. CytodifTerentiation of the intestinal villus epithelium. In: P. Desnuelle, H. SjÃ¶strÃ¶m,and O. Noren (eds.), comparison among the same cell line of the phenotypic expres Molecular and Cellular Basis of Digestion, pp. 303-314. Amsterdam: Elsev- sion of the cells at two states of differentiation. Indeed as ier Science Publishers B.V., 1986. emphasized by the authors (15), "HT29 cells, because their 2. Kedinger, Mâ€žHaffen, K., and Simon-Assmann, P. Control mechanisms in the ontogenesis of villus cells. In: P. Desnuelle, H. SjÃ¶strÃ¶m,andO. Noren differentiation can be modulated, are particularly appropriate (eds.), Molecular and Cellular Basis of Digestion, pp. 315-326. Amsterdam: to look at the onset of structural and molecular events leading Elsevier Science Publishers B.V., 1986. to cell polarity." Although these cells are malignant, crucial 3. Haffen, K., Kedinger, M., and Simon-Assmann, P. Mesenchyme-dependent differentiation of epithelial progenitor cells in the gut. J. Pediatr. Gastroen- morphological changes accompanying their differentiation (ac terol. Nutr., 6: 14-23, 1987. quirement of specialized membrane domains such as brush 4. Simon-Assmann, P., Kedinger, M., and Haffen, K. Immunocytochemical borders) closely parallel those which occur during normal in localization of extracellular matrix proteins in relation to rat intestinal testinal ontogenesis and epithelial crypt-vi 11us migration (1,2). morphogenesis. Differentiation, 32: 59-66, 1986. 5. ToÃ³le,B. P. Glycosaminoglycans in morphogenesis. In: E. D. Hay (ed.), Cell However, starvation of sugar leading to the differentiation of Biology of Extracellular Matrix, pp. 259-294. New York: Plenum Publishing HT29 cells has been shown to be accompanied by modifications Corp., 1981. 6. Bissel, M. 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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1987 American Association for Cancer Research. Synthesis of Glycosaminoglycans by Undifferentiated and Differentiated HT29 Human Colonic Cancer Cells

Patricia Simon-Assmann, Françoise Bouziges, Danièle Daviaud, et al.

Cancer Res 1987;47:4478-4484.

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