Increased A2,6 Sialylation of N-Glycans in a Transgenic Mouse Model of Hepatocellular Carcinoma'

(CANCER RESEARCH 57. 4249-4256. October I, 19971 Increased a2,6 Sialylation of N-Glycans in a Transgenic Mouse Model of Hepatocellular Carcinoma'

Dominique Pousset, VÃ©roniquePiller,Nicole Bureaud, Michel Monsigny, and Friedrich Nller@ Glycobiologie, Centre de Biophysique MolÃ©culaire,F45071 OrlEans Cedex 02, France

ABSTRACT AFP3 levels and in alkaline phosphatase or a rapid deterioration of liver function may be a clue to the presence of the neoplasm (2, 3). Liver cancer is one of the most frequent and lethal malignancies Therefore, the identification of reliable markers for the early diagnosis worldwide. Early detection is hampered by the absence of reliable mark of hepatocellular carcinoma may increase the success rates of surgical era. Mice transgenic for the SV4Olarge T antigen under the control of a liver-specific promoter spontaneously develop well-differentiated hepato resection and other therapies. The search for carbohydrate changes in cellular carcinomas between 8 to 10 weeks of age. They are excellent hepatocellular carcinomas has focused primarily on AFP to distin models to investigate the alterations of protein expression in the early guish the liver-specific glycoprotein from glycoforms that are secreted stages of tumor development and to follow these changes during tumor in other malignancies. It was found that fucosylation of the aspar progressionS In the present study, we analyzed the glycosylation changes agine-linked glucosamine residue of N-glycans was significantly in occurring during tumor development in transgenic mice expressing the creased on AlP from human hepatocellular carcinomas (4, 5). An 5V40 T antigen under the control of the antithrombin ifi promoter. The other fucose-related glycan marker, the Le@epitope, has been reported analysis of serum and liver glycoproteins by an ELISA type assay, using on the cell membranes of poorly differentiated hepatocellular carci the lectin from Sambucusnigra (SNA) as a probe, revealed the presence of nomas in patients expressing high levels of AlP (6, 7). No glycosy increased levels of Neu5Aca2,6Gal@31,4GlcNAc on N-glycans in the hi mor-bearing transgenic mice as compared to controls. On serum glyco lation-related markers have been found for well-differentiated hepa proteins the increase In a2,6 sialylation followed tumor progression, tocellular carcinomas, which generally do not express AFP. reaching up to 10 tImes control levels. However, significantly higher SNA The absence of specific markers makes it almost impossible to binding (2-fold) could already be observed on serum glycoproteins from study the early stages of tumor development in human patients at risk mice exhibiting only microscopically small neoplastic foci. On liver mem for hepatocellular carcinoma. Therefore, the development of animal brane glycoproteins, the increase in a2,6 sialylation was less pronounced, models for the study of tumorigenesis is crucial for our understanding reaching two to three times control values in 6-month-old mice. Western of early events occurring during this process. The first animal models blottingofsenun and liverproteins withradiolabeledSNAshowedthat all were liver cancers induced by chemical carcinogens in the rat (8). glycoproteins that bind the lectin In controls exhibit larger amounts of Since the advent of transgenic techniques, the model of choice has Neu5Aca2,6Gal@31,4GlcNAcon N-glycans in the tumor-bearing mice. This general increase in a2,6 sialylation on all glycoproteins is due to become the mouse expressing a viral or mutated cellular oncogene in the increased activity of the galactosideux2,6 slalyltransferase (ST6GaI I), a strictly tissue-specific manner (9, 10). which specifically transfers Neu5Ac residues in a2,6 linkage to For the study of hepatocellular carcinoma, mice expressing the Galfil,4GIcNAc units on N-glycans. As for the structures synthesized by early region (T/t antigen or Tag) of the SV4O genome under the the enzyme, the increase of ST6GaI I activity in the serum as well as in control of the promoter regions of the albumin, cs-antitrypsin, and liver microsomes of the transgenic mice followed tumor progression. AT!!! genes have been created. All of these mice develop well Interestingly, the activity ofthe galactoslde:a2,3 sialyltransferase (ST3Ga1 differentiated hepatocellular carcinomas (reviewed in Refs. 11 and ifi), whichusesthesameacceptorsubstrate(Gal@1,4GlcNAc),wasun 12). The tumor develops first in microscopically small foci at multiple changed in the earlier stages of tumor development but decreased in the sites throughout the liver. It is characterized by the classical sequence serum and in liver microsomes from later stages. Using a rat ST6GaI I cDNA as a probe, Northern blots of total RNA extracted from the livers ofevents: hyperplasia, dysplasia, adenoma, and carcinoma. In contrast ofcontrol and transgenic mice revealed an increased (4-fold) expression of to other transgenic models of hepatocellular carcinoma, the ATIII the ST6GaJI gene. The single transcripts detected In both normal and SV4O-Tag transgene-induced tumors occasionally develop lung me cancerous liver showed identical size. tastasis and thus show all of the characteristics of an advanced tumor. The proliferating foci develop into nodules that grow until the liver INTRODUCTION reaches about 10 times its normal size. The tumors are composed exclusively of hepatocytes without any elements of the biliary epithe Hepatocellular carcinoma is the most frequent cancer in many parts hum. The tumors are well differentiated, secrete normal amounts of of the world, whereas it is relatively uncommon in Europe or in the albumin, and do not express AFP (1 1). United States. It is rarely cured by surgical resection, and other Recently, a similar transgenic model of rat hepatocellular carci therapies are usually administered as part of clinical trials (1). Success noma has been reported, which shows also all of the characteristics of rate is low, however, due to the absence of other reliable early a well-differentiated tumor. In addition, the comparison of several diagnostics, and the most common primary diagnostic is the detection tumor markers from focal and neoplastic lesions of the livers of rats of an increase in hepatic mass. In about one-half of the patients at risk treated with chemical carcinogens and from transgenic rats revealed (cirrhosis or hepatitis B or C infection), a progressive increase in many similarities between the two experimental neoplasms ( 12). All of these reports clearly show that animals expressing the 5V40- Received 5/19/97; accepted 7/30/97. Tag in a strictly liver-specific manner are excellent models for hep Thecostsof publicationofthisarticleweredefrayedinpartby thepaymentofpage charges. This article must therefore be hereby marked advertisement in accordance with atocellular carcinoma and may, therefore, serve as experimental sys 18 U.S.C. Section 1734 solely to indicate this fact. tems to identify markers of tumorigenesis. Carbohydrate changes on I This work was supported by grants from the Association pour Ia Recherche sur Ic Cancer, Biotechnocentre, the Conseil Regional de la Region Centre, and the MinistÃ¨rede l'Enseignement SupÃ©neurCtde Ia Recherche (to F. P.). D. P. is supported by a fellowship 3 The abbreviations used are: AFP, a-fetoprotein; SV4O-Tag, SV4O T antigen; ATIII, from the Association pour la Recherche sur le Cancer. antithrombin III; Neu5Ac. N-acetylneuraminic acid; Gal. galactose; GlcNAc, N-acetyl 2 To whom requests for reprints should be addressed, at Glycobiologie, Centre de glucosamine; ST6Ga1 I, CMP-Neu5Ac:Gal@3l,4GlcNAca2,6 sialyltransferase; ST3GaI Biophysique MolÃ©culaire,rueCharles Sadron, F4507l Orleans Cedex 02, France. Phone: III, CMP-Neu5Ac:Gal@l,3(4)GlcNAca2,3 sialyltransferase: SNA, Sambucus nigra ag (33) 238 25 76 43; Fax: (33) 238 69 00 94; E-mail: [email protected]. glutinin; AGP, a1-acid glycoprotein; PMSF, phenylmethylsulfonyl fluoride. 4249

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1997 American Association for Cancer Research. GLYCOSYLATIONINMURINEHEPATOCELLULARCARCINOMA glycoproteins and glycolipids are associated with tumor development The pellet was freeze-dried and extracted three times with methanol at 70Â°C (13, 14) and may be used as tumor markers (15, 16). However, very and dried in the vacuum at room temperature. The material was suspended in little is known about glycosylation changes in mouse hepatocellular 5 ml of Pronase buffer [150 mM Tris-HCI (pH 8.0), 75 mM NaCI, and 2 inst carcinoma. Therefore, we screened by lectin-ELISA serum and liver CaC12Iby vortexing and ultrasonication. A 50-pi aliquot of a solution of membrane glycoproteins from normal mice and from mice transgenic Pronase E (10 mg/mI in Pronase buffer) was added, and the mixture was kept at 37Â°Cfor20 h. After adjusting the pH to 8.0 with 2 si Tris-base, another for the T-antigen from SV4O under the dependence of the human aliquot of Pronase E was added, and the incubation continued for 24 h. The ATIII promoter and analyzed a variety of glycosyltransferases. The addition of Pronase and the incubation were repeated one more time. After specific activity of most glycosyltransferases tested was unchanged in centrifugation, the clear supernatant was heated in a boiling water bath for 5 the tumor-bearing mice, whereas the a2,6 sialyltransferase (ST6Gal I) mm and stored at 4Â°C.The Pronase digests were applied to a column of was dramatically increased. Because increased sialylation has been Sephadex 0-50 fine (1 X 100 cm) equilibrated and eluted in water. Fractions shown to be associated with malignant cancers in humans and in of 4 ml were collected and analyzed for absorbance at 280 nm, for neutral rodents (15), we analyzed the differences in glycosylation between sugars by the resorcinol-microtiter plate technique (21), and for sialic acid normal and tumor tissue. In the present study, we report the changes according to the fluorescence-based thiobarbituric acid method of Hammond in the sialylation of N-linked glycans, which are associated with the and Papermaster (22). development of hepatocellular carcinoma in Tag-transgenic mice. ELISA. Serial dilutions of serum samples in PBS (100 s.d) were incubated for 16 h at 4Â°Cin microwell plates and washed, and the free sites were quenched with 0.5% BSA in PBS. The plates were then incubated with 2 MATERIALS AND METHODS mg/mi biotinylated lectin for 30 mm at room temperature, and the bound lectin was revealed with peroxidase-coupled avidin (2 @tg/ml)accordingto standard Materials. CMP-['4C]NeuSAc (286 mCi/mmol), sodium['251]iodide (100 procedures (23). mCi/ml), [a-32P]dCTP (3000 Ci/mmol), and nylon transfer membrane Hy For the ELISA-inhibition assays, the plates were coated with 30 ng of fetuin bond-N@ were purchased from Amersham Corp. (Little Chalfont, United in 100 @lof PBS (the concentration was previously determined to give Kingdom). Nitrocellulose membrane filters were from Schleicher and Schuell half-maximal binding of the lectin), and the free sites were quenched with (Dassel, Germany). CMP-NeuSAc, 2,3-dehydro--2-deoxy-Neu5Ac,2,2'-azino 0.5% BSA in PBS. The biotinylated lectin (0.2 @g)was incubated with 100 gxl bis(3-ethylbenzthiazoline-6-sulfonic acid), benzyl 2-acetamido-2-deoxy-3-O- of the glycopeptides fractions and 10 @aloflOX concentrated PBS for 1 h at @3-n-galactopyranosyl-f3-D-glucopyranoside (GallJl,3GlcNAcf3-OCH2Ph), fe room temperature and then added to the fetuin-coated microwell plates. After tuin, Pronase E, pepstatin A, leupeptin, EDTA, sodium tetrathionate, Triton 1 h at room temperature, the plates were washed, and the bound lectin was CF-54, Triton X-lOO,DTT, 2-[N-morpholino]ethanesulfonic acid, and Seph revealed with avidin-peroxidase. The difference of inhibition between native adex G-50 were purchased from Sigma Chemical Co. (St. Louis, MO). PMSF and desialylated glycopeptides was taken as specific inhibition of SNA. Dc was from Serva (Heidelberg, Germany). Immuno plates Maxisorp were from sialylation of glycopeptides and serum glycoproteins was carried out as de Nunc (Roskilde, Denmark). The DNA random priming kit and spin columns scribed(24). were from Pharmacia Biotech, Inc. (Uppsala, Sweden). The neuraminidases Electrophoresis and Glycoprotein Staining. Crude membrane prepara from Vibrio cholerae and from Newcastle disease virus were purchased from tions were obtained by homogenization of 1 g of liver with the Ultra-turrax Boehringer (Mannheim, Germany). The Iodo-Beads iodination reagent was homogenizer in 5 ml of Tris-buffered NaC1 solution (TBS: 137 mM NaCl, 3 from Pierce Corp. (Rockford, IL). The Sep-Pak C-l8 cartridges were from mM KC1, and 15 mM Tris-HC1, pH 7.8) in the presence of protease inhibitors Waters (Milford, MA). Human a1-acid glycoprotein, a generous gift of Dr. M. and by centrifugation at 15,000 rpm for 15 mm at 4Â°C.The pellets were Steinbuch (Center National de Transfusion Sanguine, Paris, France) was suspended in four volumes of TBS containing 1%Triton X-lOOand inhibitors desialylated by mild acid hydrolysis in 0.1 M trifluoroacetic acid at 80Â°Cfor of proteases at 4Â°Cfor30 mm and centrifuged at 15,000rpm for 10mm at 4Â°C. I h. Elderberry bark (Sambucus nigra) lectin (SNA), biotinylated SNA, and The supernatants were stored in aliquots at â€”20Â°C.Theprotein concentration horseradish peroxidase-labeled avidin D were obtained from Vector Labora was determined after trichloroacetic acid precipitation (25) using the method of tories (Burlingame, CA). The cDNA encoding rat j3-galactoside a2,6 sialyl Lowry et aL (26). transferase (17) was kindly provided by Dr. J. C. Paulson (Cytel Corp., La SDS-PAGEwas performedon 8 and 10%acrylamidegels underreducing Jolla, CA), the cDNA encoding mouse (3-actin (18) was kindly supplied by Dr. conditions (100 mst DT1') using 25 @gofprotein from liver membranes and A. Legrand(Orleans,France). 50 @.tgofprotein from serum per lane. Elderberry bark (Sambucus nigra) lectin The transgenic mice expressing the early region of SV4Ounder the control (SNA) was radiolabeled by the lodo-Bead (Pierce) method according to the of a 700-bp promoter fragment from the human AT!!! gene were obtained from manufacturer's instructions. Dr. P. Briand (INSERM U380, Institut Cochin, Paris, France; Ref. 11). The Western blotting on nitrocellulose membranes was performed using stand oncogene was integrated in the Y chromosome, consequently only the male ard procedures (23). Blots were incubated with the iodinated lectin (0.8 x 106 mice are transgenic and develop an hepatocarcinoma, and females are healthy cpm/cm2 and 1.3 x 106 cprn/@xg) for 2 h. For desialylation, the blots were and serve as controls. The male transgenic mice are crossed with normal washed, saturated with 0.5% BSA in TBS, and incubated with 0.1 unit/mI female C57BL6 mice, and the normal and transgenic mice are kept under Vibrio cholerae neuraminidase in 50 inst NaOAc (pH 5.6), 1 mist CaCl2, 100 standard conditions. mM NaCl, and 0.1% BSA overnight at 37Â°C. Preparation of Microsomes. Microsomes were prepared essentially by the Blots were exposed to X-ray film (Hyper film MP; Amersham) and pho method described by Leelavathi et a!. ( 19). Mice were killed by cervical tographed with a Vilber Lourmat Bioprint (Wasserburg, Germany) system, and dislocation, and the livers were quickly removed and homogenized in homog the photographs were analyzed using the Image Quant (Molecular Dynamics, enization medium [0.5 Msucrose, 0.1 Mpotassium phosphate (pH 6.5), and 5 Sunnyvale, CA) software. mM MgC12] using a Ultra-turrax homogenizer (Ika-Werk, Germany) at 25,000 Sialyltransferase Assays. a2,6 and ct2,3 sialyltransferases were assayed rpm. The homogenate was centrifuged at 4000 X g for 15 mm at 4Â°C.The according to published protocols (27, 28). Incubations were performed for 1â€”4 supernatant was collected and centrifuged at 105,000 X g for 90 mm at 4Â°C. h at 37Â°Cina final volume of 25 @lunderthe following conditions: 80 @agof The pellet was washed twice with homogenization medium, suspended in 1ml liver microsomes, 50 mM 2-LN-morpholino]ethanesulfonic acid (pH 6.5), 15 of 25 m@sTris-HC1 (pH 8.3), 0.5 M sucrose, and inhibitors of proteases [I mM mM NaC1, 0. 1% Triton CF-54, 0.05% BSA, 1 inst 2,3-dehydro-2-deoxy PMSF,50 @ag/mlleupeptin,10 j.@g/mlpepstatinA, 5 mMEDTA (pH 8), and5 NeuSAc,0.25 mst CMP-['4C]Neu5Ac (25,000 cpm/nmol), and asialo-a1-acid mM sodium tetrathionate] and stored at â€”20Â°C. glycoprotein (about 10 inst terminal Gal residues) for the Galf3l,4GlcNAca2,6 Preparation of Glycopeptides. Glycopeptides were prepared as described sialyltransferase and 2 mlvi Galf3l,3GlcNAcj3-OCH2Ph for the Gal@l,3 (4)Gl (20). Briefly, crude membrane preparations were obtained by homogenization cNAccr2,3sialyltransferase. The â€˜4C-labeledreactionproducts were separated of 1 g ofliver in 5 ml phosphate buffer (PBS: 137 mMNaCl, 3 m'viKCI, 8 mM from CMP-['4C]Neu5Ac by gel filtration on a column (0.6 x 7 cm) of Na2HPO4, 15 mM @2PÂ°[email protected]) containing 5 mM EDTA and 1 mM PMSF Sephadex G-50 in 25 fist (NH4)HCO3buffer when asialo-a1-acid glycoprotein at 4Â°C.The homogenate was centrifuged for 15 mm at 15,000 X g in the cold. was the acceptor substrate. The amount of a6-linked sialic acid was deter 4250

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1997 American Association for Cancer Research. GLYCOSYLATION IN MURINE HEPATOCELLULARCARCINOMA mined after digestion ofthe ct3-linkedsialic acid by the specific neuraminidase from New Castle disease virus as described (20). When Galf3l,3GlcNAc@3- OCH2Phwas used as the acceptorsubstrateforthe ct2,3sialyltransferase,the 1.5 product was isolated by reverse phase chromatography on Sep-Pak C-l8 cartridges. The reaction mixture was stopped by the addition of 1 ml of cold E 0.1 M(NH4)HCO3and applied to a Sep-Pak cartridge. After washing with 10 ml 0.1 M(NH4)HCO3,the bound material was eluted with 3 ml of methanol 31.0 and counted in a liquid scintillation counter. RNAPreparation and Northern Blot Analysis. TotalRNAwas isolated (3 from liver by the method of Chirgwin et a!. (29). Northern blot experiments :@ were performed as described (30), with 25 @goftotal RNA/lane. The rat a) J3-galactosidea2,6 sialyltransferase cDNA and mouse (3-actincDNA probes z were radiolabeled using the random priming kit (Pharmacia) according to the manufacturer's instructions. 0.0 RESULTS 100 A simple and noninvasive way to detect glycosylation changes in liver diseases is the analysis of serum glycoproteins (31). For the 80 screening of a large number of samples, the ELISA assay is most C0 suitable and requires only small amounts of serum. Biotinylated SNA, .4-, 60 which specifically recognizes Neu5Aca2,6Gal(31,4GlcNAc-R moi -U eties on N-glycans, reacted with serum glycoproteins from normal and -C transgenic mice (Fig. 1). The binding was found to be specific because .E @o it could be dramatically reduced by treatment of the serum samples with V. cholerae neuraminidase (data not shown) or completely abol 20 ished by treatment with 0.1 Mtrifluoroacetic acid at 80Â°C(Fig. 1). The amount of SNA-binding glycoproteins was consistently higher in sam plea from tumor-bearingmice as compared to controls. In serum samples 0 from normal mice, no gender- or age-related differences could be 10 15 20 25 30 35 detected (data not shown). The increase in Neu5Aca2,6Gal@31,4GlcNAc Fraction number on serum glycoproteins was already significantly higher in Tag transgenic animals at 2 months of age, when the liver appeared Fig. 2. Sialic acid analysis of liver membrane glycopeptides. Sialic acid contents (A) and SNA inhibition of liver membrane glycopeptides (B) from control mice (â€¢)and macroscopically normal and the neoplastic foci were still microscop transgenic mice developing hepatocellular carcinoma (0) are shown. Glycopeptides were ically small. The amount of glycoproteins recognized by SNA in isolated by gel filtration chromatography. The values obtained for the glycopeptides from creased during the development of the tumor and reached up to 10 human AGP are shown in the inset. times the level of control sera in some animals. The results obtained with serum glycoproteins were corroborated by an analysis of membrane-derived glycopeptides from normal and 1.2 cancerous livers (Fig. 2). Because glycopeptides from Pronase digests of glycoproteins do not adhere to microtiter wells, we developed an ELISA-inhibition assay where the wells were coated with bovine 1.0 fetuin and the binding of biotinylated SNA was inhibited with the E glycopeptide fractions obtained by size-exclusion chromatography of C the Pronase digests on a Sephadex G-50 column. Specific inhibition to 0.8 0 (Fig. 2B) could be observed in the fractions corresponding to the elution position of N-glycans as determined with control glycopep 0.6 tides derived from human a1-acid glycoprotein (Fig. 2B, inset). Fur Q) C) thermore, the fractions inhibiting the binding of SNA to fetuin con C a tamed most of the sialic acid in the glycopeptide preparation (Fig. 2A). @0 0.4 I- The N-linked sialic acid content is about 2-fold higher in the tumor 0 tissue (Fig. 14), and the capacity of the N-linked glycans to inhibit the U) .0 binding of SNA to fetuin (Fig. 2B) is also twice as high in the tumor 0.2 as compared to the normal liver. These findings indicate that most of the increase in sialic acid observed in the cancerous liver is due to an increase in Neu5Aca2,6Gal@l,4GlcNAc-R bearing N-glycans. Mu 3 4 5 6 cm-type proteins with a high degree of O-glycosylation are partly Serum dilutions (log) resistant to Pronase, and mucin-derived sialoglycopeptides would elute in the void volume of the gel filtration column (20). However, Fig. 1. SNA binding to serum glycoproteins from normal mice and from Tag transgenicmicedevelopinghepatocellularcarcinoma.Microwellplateswerecostedwith these fractions contained large amounts of glycogen and DNA, which serial dilutions of untreated serum (filled symbols) or with dilutions of serum treated with interfere with the thiobarbituric acid determination of sialic acid and, 0.1Mtrifluoroaceticacidfor60mmat80Â°C(opensymbols).Thecirclesrepresentthesera therefore, may mask the contribution of mucin-type O-glycans to the fromthecontrolmiceandtheinvertedtriangles,squares,andtrianglesrepresentthesera from tumor-bearing transgenic animals at 2, 4, and 6 months of age, respectively. All tests total sialic acid content. were done in triplicate and the means of three independent experiments are shown. The increase in SNA binding in the serum and in the liver of the 4251

GLYCOSYLATIONINMURINEHEPATOCELLULARCARCINOMA

Coomauie blue Â°â€˜I-SNA Sialidaic 50 kDa, the difference between tumor and normal liver membranes + - appears to be accentuated. However, this may be due to degradation Wa 195 in necrotic follicles observed frequently in the late stages of the cancer @ 112- (1 1). However, the overall increase in SNA binding to tumor mem @ brane glycoproteins was measured by scanning of the autoradiographs @ at different exposures. For all blots analyzed, the membrane prepara tion from tumor-bearing mice bound about two times (between 1.9 35 - and 2.4) more â€˜251-labeledSNA than those from controls. 32- The biosynthesis of Neu5Aca2,6Gal@l,4GlcNAc-R structures is catalyzed by the galactoside cr2,6 sialyltransferase, which transfers Neu5Ac from CMP-Neu5Ac to glycoproteins and oligosaccharides exposing N-acetyllactosamine at the nonreducing end (27). The a2,6 I 2 3 4 1 2 3 4 F sialyltransferase activity was found to be much higher in the micro Fig. 3. Lectin blots of serum proteins. Duplicate serum samples were loaded onto 10% somal preparations from the livers of 6-month-old Tag-transgenic SDS-PAGE gels. After electrophoresis, the gel was cut in half; one-half was stained with Coomassie Brilliant Blue, and the other half was electroblotted onto a nitrocellulose mice than those from controls (Fig. 5). In both preparations, the a2,6 membrane. The molecular mass markers are shown on the left of the Coomassie Blue sialyltransferase activity is dependent on the incubation time and on stained gel. Lanes 1. normal serum; Lanes 2â€”4,serum from transgenic mice developing the amount of microsomal protein added, and there appears to be no hepatocarcinoma at 6, 4, and 2 months of age, respectively. Lane F. fetuin as a positive control for the SNA binding. The sialidase treatment was performed on the Western blot difference in their affinities for their substrates asialo-AGP or CMP of a serum sample from 4-month-old transgenic mice, and the glycoprotein was revealed Neu5Ac. The observed increase in enzymatic activity may, therefore, with â€˜251-labeledSNA. be due only to the higher enzyme concentration. In normal mice, no age- or sex-related differences on a2,6 sialyltransferase activity could transgenic mice could be due either to the highly increased expression be detected (data not shown). of one or a few cancer-specific glycoproteins or to an increased a2,6 The a2,6 sialyltransferase activity was then determined in mi sialylation of all glycoproteins. To resolve this question, we analyzed crosomal preparations from the livers and in the serum of mice at either serum glycoproteins or hepatocyte membrane glycoproteins on different stages of tumor development (Fig. 6). The a2,6 sialyl Western blots developed with â€˜25I-labeledSNA (Figs. 3 and 4, re transferase activity was higher in the Tag-transgenic mice than in spectively). All samples contained equal amounts of protein, as shown the control animals, both in the liver microsomes and in the serum. by Coomassie staining (left panels in Figs. 3 and 4). However, the The increase followed the development of the tumor and was autoradiogram in Fig. 3 revealed increased binding of labeled SNA to already 2-fold in transgenic mice at 2 months of age, when no all glycoproteins from the serum of animals developing a tumor (Fig. macroscopic alterations of the liver could be detected. However, 3, Lanes 2â€”4)as compared to control (Fig. 3, Lane 1). Some glyco these crude enzyme preparations contain also the galactoside a2,3 proteins were even almost undetectable in the normal serum, whereas sialyltransferase, which can use the same acceptor substrate as the they were predominant in the serum of the Tag-transgenic mice. This a2,6 sialyltransferase, even under conditions that favor the transfer is the case for a glycoprotein with apparent molecular mass of about in a2,6 linkage. To distinguish between the products of these two 40 kDa, which most likely is a1-acid glycoprotein. In accordance with enzymes, the reaction mixtures were treated with neuraminidase the ELISA assay, the increased SNA binding could be observed from Newcastle disease virus, which specifically removes only the already in those animals that had only microscopically small tumor a2,3-linked sialic acid. The values given in Fig. 6, A and C, are foci, and the difference with the healthy individuals was accentuated thus corrected to represent only the transfer in a2,6 linkage. With during the tumor development. When analyzed by the same tech microsomes as well as with serum as enzyme source, the New niques, the membrane glycoproteins from cancerous livers exhibit castle disease virus-neuraminidase treatment removed about 10â€” almost uniformly higher levels of Neu5Aca2,6GalfJl,4GlcNAc-R 15% of the radioactive sialic acid transferred to AGP; however, in structures than those from normal liver (Fig. 4). In the region below mice at the terminal stages of tumor development, this ratio de

Coomassie blue â€˜@I-SNA

kD* 195-

I12 - Fig. 4. Lectin blots of hepatocyte membrane proteins. Duplicate samples were loaded onto an 8% SDS-PAGE gel and treated as de 84- scribed for Fig. 3. Lane 1. normal control; Lane 2, hepatocellular carcinoma at 6 months; Lane F, fetum control. Left lane, molecular weight markers. The histogram shows the scans of the autoradiograms. 63 - Dotted line, scan of Lane 1; solid line, scan of Lane 2. 52.5 -

35- 32- â€¢.a@i

1 2 F I 2

4252

I, I' -1j5 -C 0 >â€˜0@

> O@3

0 E @ 2 >â€˜ 100 0 E1 2: 50 C L__I Q 0 30 60 90 120 0 50 100 150 200 Time [mm] Protein [@i1g]

r@@:13 I,

-@ Ã·@800 L@ 0 o_ 27' â€˜) Â°600 @ .â€” >@ > -@J @ -I-, _> 400 Â°1 _I. <@1 0 0 -@200 E E L_@@JC@ @J L@_Jo_ 0 0 50 100 150 200 0 10 20 30 40 50 AsiaIoâ€”a1AGP[pg] CMPâ€”NeuAc [MM]

Fig. 5. Characterization of ST6Gal I in normal and tumor liver microsomes. A, time dependence of sialic acid transfer to asialo-AGP. B, dependence of transfer on microsomal protein concentration. C and D, transfer rate dependence on acceptor and donor substrate concentrations, respectively. All experiments were carried out in triplicate. and the means are shown. Symbols are as in Fig. 2.

creased to less than 5% (data not shown). These results prompted DISCUSSION us to examine also the a2,3 sialyltransferase activity using a specific acceptor substrate and assay conditions that are optimized Cell surface carbohydrates play important roles in cellular functions for the a2,3 sialyltransferase. In contrast to a2,6 sialyltransferase and in maintaining the essential interactions between cells in a given activity, which increases constantly over the whole time of the tissue. Consequently, the glycan structures on cell surface glycocon development of the tumor in both serum and hepatocyte micro jugates change during cell development and differentiation when the somes, the a2,3 sialyltransferase remains at the same level in the functions of the cells as well as the interactions between cells within early stages of tumor formation and decreases in the late stages of a tissue undergo critical modifications. Because functions and/or the disease (Fig. 6, B and D). cellular interactions are seriously altered in tumors, it is not surprising To determine whether the expression of the a2,6 sialyltransfer that carbohydrate changes are associated with tumor development in ase was controlled at the level of the transcription of the gene, we many tissues. The most frequently encountered changes are increased probed Northern blots of 25 @tgoftotal RNA from normal liver and branching and increased sialylation of N-glycans, incomplete synthe from the liver of transgenic mice at 6 months of age with a sis of O-glycans, and the expression of fetal-type glycans such as the 32P-labeled probe derived from the cDNA of rat liver a2,6 sialyl increase in linear polylactosamine chains or the loss of blood group transferase. In both normal and tumor tissue, the probe revealed antigens (13â€”16).In the liver, carbohydrate changes such as increased only one transcript at around 4 kb, but the amount of a2,6 sialyl branching and increased fucosylation have been reported for hepato transferase mRNA was higher in the tumor tissue than in the cellular carcinomas in the rat (4, 32). In human liver cancer, increased normal liver (Fig. 7). Scanning of the autoradiogram indicated an fucosylation of a-fetoprotein has been described, and recently, it has about 4-fold increase over control of a2,6 sialyltransferase mRNA been shown that in some cases, L9-positive cells may be found in the in the liver of transgenic animals exhibiting a hepatocellular car tumor foci (5â€”7).The mouse has become a model system for exper cinoma. The levels of mRNA in normal and tumor tissue are in imental liver cancer only through the advent of transgenic techniques good agreement with the observed changes in sialyltransferase that allow the tissue-specific expression of various oncogenes (9). activity and N-glycan structures, indicating that an increased Iran In this study we screened, by a lectin-based ELISA assay, serum scription of the sialyltransferase gene may be responsible for these glycoproteins from mice developing hepatocellular carcinoma caused alterations. by the liver-specific expression of the T-antigen from SV4O, and we 4253

ST6GaI I ST3GaI III linked sialic acid on the serum glycoproteins of mice with hepatocel lular carcinoma reflects more closely the amount of enzyme present in 0.6 the liver microsomes. 5 A The activity of the a2,6 sialyltransferase is high in the liver of most 4 0.4 mammals. So far, a change in the expression of this enzyme has not @ .E C been reported for rat or human hepatocellular carcinomas, which a) a) 4-, represent the two species most widely studied. However, cr2,6 sialyl 0 2 0 0.2 transferase is significantly increased in human colon cancer, and the o_ 0@ @r'-i @@-/C change in a2,6-linked Neu5Ac expression can also be observed dur ing the migration of normal colon epithelial cells from the crypts to E 0.03 E the top of the villi (35â€”37).In colon tumors as well as in cell lines -C @ 0.15 derived from colon tumors, the expression level of the a2,6 sialyl 0 0.02 0 transferase can be correlated to the invasive potential of the tumor E o.io E cells (38, 39). The most common localization of these metastases is C / C the host liver (40â€”42).Recenfly, it has been shown that the expression 0.01 @ 0.05 j/@D@j/ of oncogenes like c-Ha-ras in rat fibroblasts result in elevated expres sion of sialic acid on cell surface glycoconjugates due to the increased r'-i@1/ transcription of the galactoside:a2,6 sialyltransferase gene (43). 6 2 4 6 6 2 6 Moreover, the presence of large amounts of cs2,6-linked sialic acid on Age [month] the cell surface of the ras-transfected fibroblasts correlates with a high invasive potential (44). In the hepatocellular carcinoma that develops Fig. 6. Sialyltransferase activities in hepatocyles and serum from normal and Tag transgenic mice developing hepatocellular carcinoma. A and C, ST6GaI I; B and D, in the transgenic animals, only about 10% of the mice shows metas ST3Gal III; A and B, hepatocyte microsomes; C and D, serum. The values shown are the tases (11). In the present study, we found only 1 mouse among the 27 means of three experiments; bars. SE. Open bars, control mice, hatched bars, transgenic mice. animals sacrificed at 5â€”6months of age that developed macroscopi cally detectable lung metastases, but the level of a2,6 sialyltransferase was not different from other tumor-bearing mice of the same age. found significantly higher levels of SNA binding compared to control In liver and cultured hepatoma cells, a2,6 sialyltransferase expres mice. Western blots of the serum samples revealed that all glycopro sion can be influenced by cytokines and glucocorticoids, which induce teins reacting with SNA in control sera contained increased amounts the acute phase reaction (45). Therefore, it has been suggested that the of Neu5Aca2,6Galf3l,4GlcNAc-R structures on their N-glycans in the enzyme may be considered as an acute phase protein (46), notwith Tag-transgenic animals, which all develop hepatocellular carcinoma. standing that it is a membrane protein that is released into circulation A few glycoproteins were undetectable in the normal sera but showed only by proteolysis. However, it has been shown that although a2,6 strong SNA binding in the transgenic mice. We have not yet deter mined whether this increase is only due to elevated levels of a2,6 sialylation or if the protein itself is expressed at higher levels. The 12 latter may be the case for a1 -acid glycoprotein that, as a stress-related protein, may well be expressed in larger amounts in the liver exhib iting multiple neoplastic lesions. The development of hepatocellular carcinoma in the transgenic mice did not only increase the sialylation of the secreted glycoproteins but also the sialylation of the membrane glycoproteins. The analysis of glycopeptides derived from hepatocyte membranes showed an overall increase in sialylation of N-glycans and that most of the excess sialic acid was a2,6 linked. Again, no selective A increase of the expression of tumor-specific glycoproteins could be found on Western blots of membrane proteins, but all of the glyco proteins reacting with SNA in normal hepatocytes bound more of the lectin on blots from tumor-derived membrane proteins. This increase in the specific a2,6 sialylation of glycoproteins synthesized in the liver of SV4O-Tag-expressing mice is due to the increased expression of the galactoside:a2,6 sialyltransferase. The 4-fold increase in a6 sialyltransferase mRNA gives rise to a 7-fold increase in enzymatic activity, which in turn causes a 7- and 2-fold increase of sialylation of N-glycans on secreted and membrane glycoproteins, respectively. The good concordance between these data is in agreement with earlier results, which show that the activity of a2,6 sialyltransferase in rat tissues is solely dependent on the mRNA level (33, 34). In hepatocyte membranes from transgenic mice, the amount of a2,6-linked sialic B acid is lower than expected from the elevated enzyme levels. This may be due to the already high degree of sialylation in liver membrane glycoproteins. The small increase in Neu5Aca2,6Galf3l,4GIcNAc-R concentration on the hepatocyte membranes made it impossible to Fig. 7. Northern blots of total RNA from mouse liver at 6 months. Lane 1, mRNA from detect differences by immunocytochemical staining between control the liver of a Tag-transgenic mouse with a hepatocellular carcinoma; Lane 2, mRNA from and Tag-transgenic animals. On the other hand, the quantity of a2,6- the liver of a normal mouse. 4254

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1997 American Association for Cancer Research. GLYCOSYLATION IN MURINE HEPATOCELLULARCARCINOMA sialyltransferase activity increases 1.5â€”2-foldin the serum of turpen lular carcinomas, the increased expression of the a2,6 sialyltransfer tine-treated rats, the specific activity in the liver remained unchanged ase in the tumor cells significantly changes the sialylation of most (47). In addition, acute phase glycoproteins like AGP have been serum glycoproteins, and this change can be easily detected by a lectin extensively studied for glycosylation changes associated with the ELISA assay. It will be of great interest to examine the a2,6 sialyl acute phase response, but neither in mouse, rat, or humans has an transferase activity and the sialylation status of serum glycoproteins in increase of a2,6 sialylation been described (48). Furthermore, the human hepatocellular carcinoma to determine whether the results glycosylation changes that have been found during the acute phase obtained with the mouse model may be useful in the diagnostics of response are increased branching and increased fucosylation of the human liver cancer. lactosamine units, which would reduce rather than augment the amount of a2,6-linked sialic acid on these glycoproteins (49). Among ACKNOWLEDGMENTS confirmed acute phase proteins (50), only a1-acid glycoprotein may be present at higher levels in the serum of the transgenic animals, but We thank Dr. Pascale Briand (INSERM U380, Paris, France) for providing the glycoprotein at 40 kDa could only be detected by staining with the transgenic mouse model and Dr. James C. Paulson for the rat ST6Ga1 I SNA and not by conventional protein or glycoprotein stains. 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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1997 American Association for Cancer Research. Increased α2,6 Sialylation of N-Glycans in a Transgenic Mouse Model of Hepatocellular Carcinoma

Dominique Pousset, Véronique Piller, Nicole Bureaud, et al.

Cancer Res 1997;57:4249-4256.

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