Glycosylation alterations ofcells in late phase apoptosis from colon carcinomas
Evgenia Rapoporta and
JacquesLe Pendub
INSERM U419, Institut de Biologie, 9 Quai Moncousu, 44035,Nantes, Cedex, France
Received on March 8, 1999. revisedon April 29, 1999; accepted on May 13, 1999.
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Abstract
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Comparisons of carbohydrate profiles between controland apoptotic colon carcinoma cells were performed by flow cytometryusing a set of lectins and anti-carbohydrate antibodies. The sixcell lines analyzed presented distinct carbohydrate profiles beforeinduction of apoptosis. PHA-L and MAA binding decreased after inductionof apoptosis by UV-treatment. In contrast an increase of PNA bindingwas observed after induction of apoptosis, except on SW-48 cellsfor which a decrease occurred. A decrease of SNA binding was observedafter induction of apoptosis from strongly positive control celllines, whereas it increased on weakly positive ones. All the bloodgroup related antigens A, H, Lewis a, Lewis x, Lewis b, and Lewisy, had their expression strongly diminished on apoptotic cells.These changes occurred irrespective of the mode of apoptosis inductionsince similar results were obtained after UV, TNF
,or anti-Fas treatment. Fucosyltransferases activities were alsodecreased after apoptosis induction, except for
1,3fucosyltransferasein anti-Fas treated HT-29 cells, where it was strongly augmented.This could be attributed to the IFN
preteatmentrequired to induce Fas expression on these cells. Fucosidase activitydecreased after induction of apoptosis suggesting that it was notresponsible for the loss of fucosylated structures. In the rat PROcell line, H blood group antigens are mainly carried by a high molecularweight variant of CD44. It could be shown that the loss of H antigenafter induction of apoptosis correlated with a loss of the carrierglycoprotein.
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Introduction
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Apoptosis, or programmed cell death, is an active mode of cell deaththat is highly conserved and regulated. It occurs physiologicallyfrom development through adult life where it participates to themaintenance of the dynamic steady state in cell turnover of manytissues. It also occurs pathologically in conditions such as cancer,autoimmune diseases, neurodegenerative diseases, or infectious diseases.Apoptosis is characterized by cell shrinkage, nuclear condensation,protease activation, and finally DNA fragmentation (12
Granvilleet al., 1998; 21
Raff,1998). Alterations in cell surface molecules and mostspecifically of glycan structures are also observed. These includea loss of sialic acid residues and inversely an increase in accessibilityof galactose, mannose, fucose, and N-acetylgalactosamineresidues (20
Morris et al.,1984; 1
Akamatsu et al.,1996; 10
Falasca et al.,1996; 23
Russel et al.,1998). Once apoptotic cells or bodies have been generated,the terminal phase of the process is their rapid elimination byneighboring cells or by cells of the macrophage type. This eliminationof apoptotic cells by phagocytes is of utmost importance since itprevents the release of unwanted molecules and the initiation ofan inflammatory response (22
Ren and Savill,1998). The engulfment of apoptotic bodies by phagocytesrequires specific recognition systems, the best characterized beingthe exposure of phosphatidylserine at the surface of the apoptoticcell and its recognition by a specific receptor located at the surfaceof the phagocyte (9
Fadok et al.,1998). It is similarly believed that some changes in glycanexposure could be key steps in the process of removal of apoptoticcells (8
Duvall et al., 1985; 7
Dini et al., 1995; 10
Falasca et al., 1996).Indeed, an unmasking of galactose residues occurs after the lossof sialic acids and phagocytosis of rat apoptotic hepatocytes byneighboring hepatocytes carrying the asialoglycoprotein receptorcould be inhibited by galactose, desialylated glycoproteins as wellas by anti-asialoglycoprotein receptor antibodies (6
Diniet al., 1992). Similarly, participationof the mannose/fucose receptor to the phagocytosis of apoptoticbodies by neighbor cells has been suggested (15
Hallet al., 1994).
The alterations in glycosylation of apoptotic cells have previouslybeen mainly studied on hepatocytes or on subsets of leukocytes,but since a large heterogeneity in cell surface glycosylation existsbetween cell types, it was of interest to compare the changes thatwould occur on cells originating from colorectal carcinomas. Suchcells can express distinct carbohydrate structures whichhave their expression developmentally regulated and are tumor associated(14
Hakomori, 1996). These includeincreased
2,6sialylation, ß1,6GlcNAc branchingat the trimannosyl core of N-linked oligosaccharides and increasedexpression of ABH and Lewis related antigens (18
Kimand Varki, 1997). In the present study, we thus reportour investigation of some changes in glycosylation that occur at thecell surface of colon carcinoma cell lines. This was performed usinga set of lectins and of monoclonal antibodies of well defined carbohydratespecificity toward the above-mentioned developmental and tumor associatedglycan structures.
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Results
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Induction of apoptosis
Three different treatments known to induce apoptosis were testedon six colon carcinoma cell lines: UV, TNF
andanti-Fas treatments. In order to confirm that these treatments inducedapoptosis in the cell lines used, DNA fragmentation was monitored.As shown in Figure 1, anti-Fas treatmentof HT-29 cells induced DNA fragmentation characteristic of apoptosisin detached cells recovered in the supernatants 24 or 48 h afteraddition of the anti-Fas antibody. Seventy-two hours after induction,only the low molecular weight fragments were visible, the unfragmentedhigh molecular weight DNA being no longer visible (not shown). Howeverin the latter case, floating cells incorporated trypan blue andthus were not used for the study of cell surface glycans. No suchdegradation was evidenced in the untreated cells. Staining of nucleiwith Hoescht 33258 confirmed that floating treated cells presented nucleolarcondensation typical of apoptosis, no such sign being visible incontrol untreated cells (data not shown). Upon induction of apoptosis,epithelial cells are known to detach from their substrate. As alreadyreported by others (13
Günthert etal., 1996), only detached HT-29 cells showed DNAfragmentation and nucleolar condensation. Adherent treated cellsdid not present these signs of apoptosis. Similar observations were madewith the two other inducers of apoptosis, UV or TNF
treatments,on HT-29 and other cell lines (data not shown). Twenty-four hoursafter induction of apoptosis, from 10 to 20% cells werefloating. After 48 h, this proportion doubled. Therefore, comparisonsbetween floating and adherent untreated cells could be performedto compare glycosylation patterns between control and apoptoticcells.
Cytofluorimetric analysis of apoptotic cells
The binding of various lectins to the colon carcinoma cell lines wasdetermined by flow cytometry. As depicted in Figure 2A, large differences were visible among thecell lines. For example, cell lines such as PRO and SW-48 were strongly stainedby PNA, whereas the others were only weakly stained. The same 2cell lines were also strongly stained by PHA-L, but MAA stainingwas strong only on HT-29 and SW-48. After UV treatment (Figure 2B), the binding of PHA-L and of MAA was largelydecreased from all cell lines that were significantly labeled bythese two lectins before treatment. Concerning the binding of SNA,the situation was very contrasted since it increased after treatmentof cells that were previously negative (PRO and SW-48). Inversely,it decreased from cells that were previously strongly positive (LS-174T and SW-1116) and did not change on cells that were only moderatelystained before treatment. As to PNA binding, it increased on allcell lines with the exception of SW-48 from which it decreased.Figure 3 illustrates the loss of MAA bindingsites and the parallel gain of PNA binding sites on apoptotic HT-29cells. A major loss of PHA-L binding sites is also illustrated.The specificity of the increase of SNA and PNA after treatment wasassessed by inhibition of their respective binding either to PROcells with 6sialyl-lactose (Figure 4A)or to HT-29 cells with galactose (Figure 4B).

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Fig. 2. Cytofluorimetric analysis ofcell surface glycosylation using fluorescent lectins before andafter UV treatment. (A) After culture in standardconditions, untreated cells, PRO, LS-174 T, SW-707, SW-1116, HT-29,or SW-48 were detached and stained with the four FITC labeled lectinsPHA-L, MAA, SNA, and PNA. Intensities of fluorescence in arbitraryunits are given from a representative experiment. At least two experimentswere performed for each lectin on each cell line. (B)Percentages of fluorescence intensity from floating UV treated cells(24 h after treatment) relative to that of adherent control untreatedcells. Dashed lines indicate the level of the 100% values.n.a., Not applicable since either a decrease after UV treatmentwas observed from low control values no more than three times abovebackground, making the significance of the decrease uncertain, orthat an increase in MAA binding, not inhibitable by 3'sialyllactose,therefore nonspecific, was observed.
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Fig. 3. Examples of changes in cytofluorimetricprofiles after induction of apoptosis by UV. The logs of fluorescenceintensities are plotted against cell numbers. Adherent untreatedHT-29 cells (filled histograms) or floating UV treated cells (unfilledhistograms) were stained with the labeled lectins MAA, PNA, PHA-L,the anti-Lewis x antibody 3E1 or the control irrelevant antibodyE4 (controls).
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Fig. 4. Inhibition of the binding ofSNA and PNA lectins to apoptotic cells by free ligands. (A)UV-treated floating PRO cells were stained with FITC-labeled SNA aloneor in the presence of 420 µM 6'sialyl-lactoseand analyzed by flow cytometry. Fluorescence intensities are givenin arbitrary units. (B) anti-Fas-treated HT-29cells were stained with FITC-labeled PNA alone or in the presenceof 0.2 M galactose. Adherent untreated cells were also stained withthe lectins (control) showing the low binding of SNA to standardPRO cells and of PNA to standard HT-29 cells.
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The fate of fucosylated structures was next analyzed using theUEA-I lectin. It showed an increased binding to all cell lines afterinduction of apoptosis. However, while the binding to spontaneouslypositive cells such as PRO was readily inhibited by 0.2 M fucose,the binding to apoptotic cells could not be inhibited at all, indicatingthat it was nonspecific (data not shown). A panel of antibodiesspecific for some of the fucosylated antigens was therefore used.A control irrelevant antibody showed only a small background onboth untreated and treated cells. As shown on Figure 5A, each specific antibody presented a uniquebinding pattern on the six cell lines. Only two of them SW-707 andHT-29 were blood group A positive; three cell lines expressed Htype 3/4 antigens (PRO, SW-1116, and SW-48). Similarly,the Lewis type antigens were unevenly distributed among cell lines.After UV induction of apoptosis, the binding of the antibodies toall cell lines largely decreased (Figures 3, 5B). Different inducers of apoptosis were used: TNF
on PRO and SW-48 and anti-Fas on HT-29.The same results were obtained in each case. The expression of fucosylatedantigens and of
2,3 linked sialic acidsdecreased after apoptosis induced by each of the three treatments(Figure 6). In the case of HT-29, inductionof apoptosis by TNF
and anti-Fas requiredan IFN
pretreatment. This pretreatmentitself did not induce any detectable apoptosis. However, it strongly enhancedthe binding of the anti-Lewis y and Lewis x antibodies on adherentcells.

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Fig. 5. Cytofluorimetric analysis ofcell surface glycosylation before and after UV treatment, usingantibodies toward fucosylated structures. (A) Afterculture in standard conditions, untreated cells were detached andstained with MAbs against Lewis x, Lewis a, H types 3/4,Lewis y, A blood group antigen, or H/Lewis b. Intensityof fluorescence in arbitrary units is given from a representativeexperiment. Non specific binding given by the irrelevant antibodyE4 were not deduced. Mean fluorescence intensities for these negativecontrols were comprised between 10 and 20. At least two experimentswere performed for each antibody on each cell line. (B)Percentages of fluorescence intensity from floating UV treated cells(24 h after treatment) relative to that of adherent control untreatedcells. Dashed lines indicate the level of the 100% values.n.a., Not applicable since a decrease after UV treatment was observedfrom control values no more than three times above background.
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Changes in fucosyltransferases activities uponinduction of apoptosis
There exist various fucosyltransferases which can participate tothe synthesis of the antigens revealed by the panel of antibodiesthat was tested as described above (5
Costacheet al., 1997). We previously showed thatPRO cells contain a significant
1,2fucosyltransferaseactivity, in accordance with their expression of H antigens. AfterUV and TNF
treatments, PRO apoptoticcells presented a largely decreased enzymatic activity (Figure 7). HT-29 present a strong
1,3fucosyltransferaseactivity detected using the H type 2 trisaccharide as acceptor.This enzyme activity was largely diminished after induction of apoptosisby UV irradiation. Inversely, after induction by anti-Fas, the activitywas increased over 2.5-fold. Yet, this increase was not due to theapoptotic process itself, since it was already visible in cell extractsfrom IFN
-treated cells, which do notshow any sign of apoptosis (Figure 7). The synthesisof Lewis x and Lewis y antigens requires an
1,3fucosyltransferase.Thus, the increased expression of these antigens after IFN
treatmentcan be explained by an induction of the enzyme activity. Nevertheless,this does not correlate with the antigenic expression which decreasesfrom apoptotic cells, irrespective of the IFN
pretreatment. Synthesisof the Lewis a and Lewis b antigens requires an
1,4fucosyltransferasewhich was assayed using the biotinylated H type 1 trisaccharideas acceptor. This enzyme activity was unchanged after IFN
treatment,but was decreased in cell extracts from apoptotic cells irrespectiveof the inducer (data not shown).
Changes in fucosidase activity upon induction ofapoptosis
Since the loss of fucosylated antigens observed after induction ofapoptosis could not always be correlated to a decrease of the correspondingfucosyltransferase activity, we tested whether there was an increaseof fucosidase activity that could be responsible for the degradationof the antigens. After UV treatment, both HT-29 and SW-1116 showeda diminished fucosidase activity (Figure 8).The other cell lines presented a lower fucosidase activity thatwas unchanged after UV treatment (data not shown). Therefore, thechanges in this enzymatic activity could not explain the drop offucosylated antigens.

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Fig. 8. Fucosidase activity in HT-29and SW-1116 cells in control adherent cells and floating apoptoticUV-treated cells. Fucosidase activities were determined from cellextracts using 4-methylumbelliferyl- -L-fucose as substrate. Fluorescence of releasedfree methylumbelliferone is shown in arbitrary units±SDfrom triplicates.
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Loss of the carrier protein from apoptotic cells
Various proteases are known to be involved in the process of apoptosis.Such enzymes could potentially release the glycoproteins that carrysome of the glycan lost after induction of apoptosis. We had shownearlier by immunoprecipitation experiments, that most of the H antigenpresent at the surface of PRO cells was born by a high molecularweight variant of CD44 carrying the product of exon v6 (19
Labarrière et al., 1994; 16
Hallouin et al., 1999).We therefore tested the expression of the CD44v6 peptide on PROcells, using a specific antibody, together with that of the H antigen,before and after induction of apoptosis. After both UV and TNF
treatments, the expression of H antigenstrongly decreased (Figure 9). A parallel decreasewas noted for the CD44v6 epitope, indicating that the loss of thefucosylated antigen was due to a loss of the carrier protein itself.
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Discussion
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In this study, the previously suggested loss of cell surfacesialic acid residues on apoptotic cells was confirmed on some cell linessince the binding of the
2,3 linkedneuraminic acid specific lectin MAA to apoptotic HT-29 and SW-48cells was much lower than on their untreated counterparts. Similarlya decrease in
2,6 linked neuramic acidcould be monitored on apoptotic bodies from LS-174 T and SW-1116using the SNA lectin. It has been previously suggested that theappearance of galactosyl residues at the surface of apoptotic cellswould stem from a loss of sialic acid residues (20
Morriset al., 1984; 6
Diniet al., 1992; 10
Falascaet al., 1996). This was indeed the casefor HT-29 and SW-1116. However, this phenomenon cannot be consideredas universal since it was not observed in the case of SW-48. Anincrease in PNA reactive terminal ß galactosides wasnevertheless observed on apoptotic bodies from the five other celllines. In the case of PRO and SW-707, it could not be accountedfor by a decrease of sialic acid residues since these cells werehardly stained by SNA or MAA before induction of apoptosis. Theuse of SNA yielded interesting results since the two cell lines(SW-48 and PRO) that primarily expressed very little SNA bindingsites became significantly stained after induction of apoptosis,whereas the opposite was true for those cell lines that expressedhigh levels of SNA binding sites prior induction of apoptosis (SW-1116and LS-174T), no change being visible for the two cell lines thatexpressed intermediate levels of SNA binding sites (SW-707 and HT-29).The increase in SNA binding to apoptotic PRO cells could not be inhibitedwhen apoptosis was induced in the presence of either benzyl-N-acetylgalactosamineor of deoxymannojirimycin which are inhibitors of O-linked and N-linkedglycosylation processes, respectively (data not shown). This wouldindicate that the appearance of
2,6linked neuramic acid residues detected by SNA after induction ofapoptosis results from an unmasking of previously undetectable moleculesand not from a de novo synthesis. This unmaskingcould result from the loss of larger molecules such as polylactosaminessynthesized from the ß1,6GlcNAc branchat the trimannosyl core of N-linked oligosaccharides, as assessedfrom the major decrease of PHA-L reactivity from all cell linesafter induction of apoptosis. It could also result from a loss ofglycoproteins such as high molecular weight variants of CD44, aswe observed in the case of PRO cells and that others previouslyobserved in the case of HT-29 (13
Günthertet al., 1996). After the loss of largeglycans the access of the SNA lectin to shorter structures, closeto the membrane, could become possible.
Concerning the fucosylated developmentally and tumor associatedantigens, their pattern of expression was quite variable from acell line to the other. Yet, all of them were lost after inductionof apoptosis irrespective of the mode of induction. The loss ofsuch structures from apoptotic cells could complement the loss ofsialic acids and of ß1,6GlcNAc branchingat the trimannosyl core of N-linked oligosaccharides to allow an unmaskingof underlying galactose residues or of SNA binding sites. Two fucosyltransferaseactivities involved in the synthesis of these antigens were alsostrongly decreased when apoptosis was induced by UV irradiationor TNF
alone. However, fucosidase activitywas also diminished in apoptotic cells, making it unlikely thatthe loss of fucosylated structures merely resulted from a degradationby fucosidases. Instead, it could result from a degradation of polylactosaminestructures on which they are often carried, or from the releaseof the entire carrier glycoprotein, as exemplified in the case ofPRO cells that mainly carry H antigens on a high molecular weight variantof CD44 which expression dropped to the same extent as that of theH antigenic sites after induction of apoptosis.
In contradiction with the results presented above, earlier reportsproposed that fucosylated structures increase rather than decreaseon apoptotic cells. 23
Russel etal. (1998) recently observed an increased bindingof the lectin from Tetragonolobus purpurea (LTA)on apoptotic thymocytes and on the mouse mastocytoma cell line P815.It could be that the use of different cell types explains the discrepancybetween the two studies. Alternatively, the increase of LTA bindingto these apoptotic cells could have been nonspecific since it was notshown that this increase corresponded to a higher fucosyltransferaseactivity or that it could be inhibited by fucose. We observed anincrease in UEA-I binding to all tested cell lines that could notbe inhibited by fucose (data not shown). Similarly an increase inMAA binding to some cell lines that could not be inhibited by 3sialyllactosewas observed after induction of apoptosis. In addition, the bindingof an irrelevant antibody to apoptotic bodies, although quite low,was always higher than on native cells (see controls in Figure 3). It is therefore quite likely that the apoptoticcell surface is prone to bind proteins nonspecifically. The lossof glycan structures as observed in the present study could be inpart responsible for this effect.
Akamatsu and colleagues (1996) also reported that Fas-inducedapoptosis in HT-29 was accompanied by an increase in the expressionof the Lewis x antigen and of the FUT IV
1,3fucosyltransferaseactivity necessary for its synthesis. This is in clear contradictionwith our results since using the same cell line, we observed a majordecrease of the Lewis x antigen, as well as of the other fucosylatedantigens that we tested. In our hands, this decrease occurred whencells were induced in apoptosis by UV irradiation, but also by TNF
or anti-Fas treatments. Since inductionof apoptosis by TNF
or anti-Fas inHT-29 required a pretreatment with IFN
,we tested the effect of IFN
alone.It induced a strong increase in both Lewis x and Lewis y antigenicexpressions as well as in
1,3fucosyltransferaseactivity on adherent cells. The enzymatic activity was still highafter induction of apoptosis. Since Akamatsu and colleagues (1996)did not provide a control experiment with IFN
treatmentalone, it is quite possible that the increase in FUT IV transcriptionand enzyme activity that they reported was due to an induction byIFN
and not to the apoptotic process.The reported corresponding increase in Lewis x antigen on apoptoticcells could be due to a contamination of the apoptotic cells bynon apoptotic cells since in their analysis the authors mixed upfloating and adherent cells. In our hands and those of others (13
Günthert et al., 1996),HT-29 IFN
+ anti-Fas treatedadherent cells show no sign of apoptosis. Yet, such cells couldbe at an early stage of apoptosis.
Hiraishi and colleagues (1993) showed earlier by histologicalmethods that expression of the Lewis y antigen in normal and tumoraltissues correlated with apoptosis. This might also appear in contradictionwith the results presented herein since we observed a loss of thatantigen from apoptotic cell surfaces. However, we analyzed a ratherlate phase of the apoptotic process since DNA fragmentation waswell advanced. The nick-end labeling technique used by Hiraishiand colleagues (1993) should detect earlier steps of DNA fragmentation.In addition, the expression of Lewis y antigen that they observed extendedaround the apoptotic areas to nonapoptotic cells. We recently showedan increase in resistance to apoptosis of rat colon carcinoma cellsexpressing
1,2fucosylated structures aftertransfection of cDNAs coding for either human or rat
1,2fucosyltransferases(Goupille et al., unpublished observations). Basedon these results and those reported by 17
Hiraishiet al. (1993), we suggest that before inductionof apoptosis, an overexpression of such fucosylated antigens occursthat will slow down the apoptotic process, these fucosylated antigens beingremoved in late phases of the process, as observed in the presentstudy. The slow down of the apoptotic process would be necessaryto prevent the appearance of a massive amount of apoptotic bodiesthat could not be cleared up by neighboring phagocytes. Inversely,once apoptotic bodies are generated, the removal of large glycanstructures and the uncapping of subjacent galactoses or mannoseswould facilitate the engulfment of apoptotic bodies by providinga better access to the membrane for receptors present on phagocytes,such as the phosphatidylserine receptor, the asialoglycoproteinreceptor, or the mannose/fucose receptor. Since we studieda late stage of the apoptotic process, the observed changes in glycanpatterns could have appeared at an irreversible stage of apoptosis,when DNA fragmentation already began. If so, they would only accompanythe apoptotic process and not directly participate to the deathprogram.
In conclusion, we have shown in the present study that colon carcinomaapoptotic cells have lost ß1,6 branchedN-glycans, as well as most sialic acids and fucoses, this beinggenerally accompanied by an increase in accessibility to ßgalactoseresidues. The slight increase in binding of SNA to some cell lines couldbe interpreted as an unmasking of preexisting
2,6sialylatedmolecules, possibly glycolipids. The precise mechanisms by whichthese changes occur are not defined as yet, although they couldbe explained at least in one instance by a loss of the carrier glycoproteinitself. The significance of these findings with respect to the apoptoticprocess and particularly to the disposal of apoptotic bodies byphagocytes will warrant further studies.
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Materials and methods
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Cell culture
PRO cells, obtained from Dr. F.Martin (Dijon, France) is a clonederived from a rat colon carcinoma cell line. Human colon carcinomacell lines HT-29, LS-174 T, SW-48, SW-707, and SW-1116 were obtainedfrom the American Type Culture Collection and cultured in RPMI 1640(GIBCO, Cergy Pontoise, France) supplemented with 10% FCSand 2 mM glutamine. For induction of apoptosis by UV, cells at near confluencywere submitted to a UV light for 1 min and cultivated in completemedium for 24 h. After a change of medium, cells were cultivatedagain for 24 h and floating cells were collected. For inductionof apoptosis by TNF
, cells at near confluencywere treated with 5 ng/ml human recombinant TNF
(Boehringer,Mannheim, Germany) for 24 h and floating cells were collected. Forinduction of apoptosis with anti-Fas, HT-29 cells at about 60% confluencywere treated with 1000 U/ml IFN
(BoehringerIngelheim, Gagny, France) in complete medium for 24 h. After removalof the medium, fresh medium containing 100 ng/ml of anti-Fasantibody clone CH-11 (BIOMOL Research Labs, Plymouth Meeting, PA)was added for 17 h and floating cells were collected.
DNA extraction and assessment of fragmentation
For analysis of DNA fragmentation, floating and adherent treatedor untreated cells were incubated for 2 h with proteinase K (20 µg/ml). The DNA was extractedwith phenol-chloroform and then precipitated overnight at 20°C following addition of ethanol. Afterincubation for 3 h at 37°C in Tris-EDTAcontaining 10 µg/ml RNase A,the DNA fragments were resolved by electrophoresis for 2 h at 40V on 1.8 % agarose gel and visualized under UV light afterethidium bromide staining.
Lectins and antibodies
Digoxigenin-labeled lectins from Maackia amurensis (MAA), Sambucus nigra (SNA) and Arachis hypogea (PNA)were obtained from Boehringer (Mannheim, Germany). FITC-labeledlectin from Phaseolus vulgaris (PHA-L) was obtained fromSigma (St. Louis, MO). MAbs 33A, 19-OLE, and 7-LE wereobtained from Dr. Bara (Paris, France). 33A recognizes alltypes of A determinants: types 1, 2, 3, 4 and difucosylated types1 and 2 (2
Bara et al., 1988).19-OLE is an anti-Lewis y and 7-LE is an anti-Lewis a (unpublishedobservations). MAb MBr1, obtained from Dr. Conaghi (Milan, Italy)is an anti-H types 3 and 4 specific antibody (4
Clausenet al., 1986). MAb 3E1 obtained from Dr.Blanchard (Nantes, France) is an anti-Lewis x specific antibody(unpublished observations). MAb LM137/276, obtained fromDr. Fraser (Glasgow, UK). This antibody reacts equally well withLewis b and H antigens (11
Good etal., 1992). MAb 1.1ASML was obtained from Dr. Herrlich(Karlsruhe, Germany). It recognizes a peptide epitope lying withinthe sequence of exon v6 of the rat CD44 molecule. MAb E4, obtainedfrom Dr. Douillard (Nantes, France), recognizes the glycoproteinTage 4 (3
Chadeneau et al.,1994) and was used as an irrelevant control antibody.FITC-labeled anti-mouse Ig and FITC-labeled anti-digoxigenin werepurchased from Sigma and Boehringer, respectively.
Cytofluorimetric analyses
Adherent cells were recovered by scrapping after two washingsand an incubation in Versene solution for 10 min at 4°C. Floatingcells obtained after treatments were washed three times in 0.1% gelatinin PBS. Treatments inducing apoptosis were set so that only a smallpercentage of floating cells were stained with trypan blue. Cellswere then incubated with the lectins or antibodies at the appropriateconcentrations in the same buffer for 30 min at 4°C.After washings, when required, cells were incubated with the FITC-labeledsecondary reagents, anti-digoxigenin or anti-mouse Ig. After finalwashings, fluorescence analysis was performed on a FACScan (Becton-Dickinson,San Jose, CA).
Assays for fucosyltransferases activities
Adherent cells were rinsed with ice-cold PBS, pH 7.2, then recoveredby scrapping. After washing again with ice-cold PBS, cells weresolubilized in 50 mM potassium phosphate pH 6.0, containing 2 % (v/v)Triton X-100 on ice for 30 min. Floating cells obtained after inductionof apoptosis were pelleted and solubilized in the same buffer asadherent cells. Following a centrifugation at 13,000 x g for 10 min, supernatants were collected and usedas crude enzyme preparations. Protein concentrations were determinedusing bicinchoninic acid obtained from Pierce (Rockford, IL).
The reaction mixtures contained 20 µMGDP-L-[14C]-Fucose(23 mCi/mmol, NEN Chemical Center, Dreieichenhain, Germany),26 mM Galß13GalNAc
1-sp-biotin (Synthesome,Munich, Germany) for the
1,2fucosyltransferaseactivity or Fuc
12Galß14-GlcNAcß1-sp-biotin for the
1,3fucosyltransferaseactivity, 10 mM L-fucose, 7.7 mM MgCl2,1.9 mM ATP, and 50 µg protein extractin a final volume of 33 µl. After anincubation at 37°C for 3 h, the reactionmixtures were quenched with 5 ml of distilled water and appliedto freshly conditioned C-18 Sep Pak cartridges (Waters-Millipore).The cartridges were washed with 20 ml water. The radiolabeled productswere then eluted with 5 ml methanol and counted in 10 ml scintillationliquid (Ready Safe, Beckman, Palo Alto, CA). Background levels ofradioactivity were obtained from controls without exogenous acceptor. Valuesobtained for the controls were then subtracted from those obtainedfor the assays.
Assay for fucosidase activity
Cell lysates prepared as for the fucosyltransferases assays containing25 µg of proteins in 0.2 M sodium acetatebuffer, pH 4.2 were incubated in 96 wells plates with 100 µl0.2 mM sodium 4-methylumbelliferyl-
-L-fucose (Sigma) in the same buffer for 90 minat 37°C. The presence of free 4-methylumbelliferonewas determined in a spectrofluorimeter (Fluorolite 1000, Dynatech,Guyancourt, France) using excitation light at 365 nm and fluorescenceemission at 450 nm.
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Acknowledgments
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We are grateful to the researchers who provided us with reagents.This work was supported by grants from the Ligue Départementalede Lutte Contre le Cancer de Loire Atlantique and from the Associationpour la Recherche sur le Cancer. Evgenia Rapoport was supportedby a grant from the Région des Pays de la Loire. We thankDrs. K.Meflah and N.Bovin for their continuous support.
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Footnotes
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a Presentaddress: Shemyakin Institute of Bioorganic Chemistry, Moscow, Russia 
b To whom correspondenceshould be addressed 
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