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J Biol Chem, Vol. 274, Issue 35, 24641-24648, August 27, 1999


Expression of Core 2 beta -1,6-N-Acetylglucosaminyltransferase in a Human Pancreatic Cancer Cell Line Results in Altered Expression of MUC1 Tumor-associated Epitopes*

Paul V. BeumDagger , Jaswant SinghDagger §, Michael Burdick, Michael A. Hollingsworth, and Pi-Wan ChengDagger parallel

From the Dagger  Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska 68198 and the  Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Many tumor-associated epitopes possess carbohydrate as a key component, and thus changes in the activity of glycosyltransferases could play a role in generating these epitopes. In this report we describe the stable transfection of a human pancreatic adenocarcinoma cell line, Panc1-MUC1, with the cDNA for mucin core 2 GlcNAc-transferase (C2GnT), which creates the core 2 beta -1,6 branch in mucin-type glycans. These cells lack endogenous C2GnT activity but express a recombinant human MUC1 cDNA. C2GnT-transfected clones expressing different levels of C2GnT were characterized using monoclonal antibodies CC49, CSLEX-1, and SM-3, which recognize tumor-associated epitopes. Increased C2GnT expression led to greatly diminished expression of the CC49 epitope, which we identified as NeuAcalpha 2,6(Galbeta 1,3)GalNAcalpha -Ser/Thr in the Panc1-MUC1 cells. This was accompanied by the emergence of the CSLEX-1 epitope, sialyl Lewis x (NeuAcalpha 2,3Galbeta 1,4(Fucalpha 1,3)GlcNAc-R), an important selectin ligand. Despite this, however, the C2GnT transfectants could not bind to selectins. Increased C2GnT expression also led to masking of the SM-3 peptide epitope, which persisted after the removal of sialic acid, further suggesting greater complexity of the core 2-associated O-glycans on MUC1. The results of this study suggest that C2GnT could play a regulatory role in the expression of certain tumor-associated epitopes.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Many cancer cells are distinguished from their normal counterparts by the presence of certain cell surface epitopes. These tumor-associated epitopes are potential targets for diagnosis, imaging, and therapeutic treatment (reviewed in Refs. 1 and 2). The mechanism by which these epitopes arise is not well understood but may involve changes in the activity of glycosyltransferases, because carbohydrate is an essential component of many tumor-associated epitopes.

Mucin-type glycan structures are influenced by both the level of expression and the Golgi localization of glycosyltransferases, which compete with one another for common acceptor structures (3). When competing glycosyltransferases share the same Golgi localization, the final O-glycan structure is likely to be governed primarily by the relative activities of the enzymes. However, if the enzymes reside in different Golgi compartments, the earlier Golgi enzyme will have an advantage in dictating the oligosaccharide structure.

Glycosyltransferase competition can occur following the creation of the core 1 acceptor structure Galbeta 1-3GalNAcalpha Ser/Thr. UDP-GlcNAc:Galbeta 1-3GalNAc (GlcNAc to GalNAc) beta -1,6GlcNAc-transferase (C2GnT;1 EC 2.4.1.102) attaches GlcNAc in beta -1,6 linkage to GalNAc of the core 1 acceptor creating the core 2 beta -1,6 branch in the O-glycan chain.
Creation of the core 2 branch enables the O-glycan chain to be extended into complex structures such as polylactosamine chains (reviewed in Ref. 4). The sialyl transferases ST6GalNAc I and II compete with C2GnT for the core 1 acceptor substrate (5, 6) and direct the glycosylation pathway toward simpler structures lacking the core 2 branch, such as NeuAcalpha 2,6(Galbeta 1,3)GalNAc and NeuAcalpha 2,6GalNAc. The latter structures are recognized by the monoclonal antibody CC49 (7, 8), and therefore creation of the CC49 epitope should be influenced by the relative activities as well as the specific Golgi localization of both C2GnT and these sialyl transferases.

C2GnT activity is regulated under certain growth conditions, including maturation of granulocytes (9) and T cells (10) as well as T cell activation (11). Transgenic mice in which C2GnT was overexpressed showed normal T cell development but an impaired T cell immune response (12). The core 2 branched structure has been associated with the sialyl Lewis x (sLex) determinant (13-16), NeuAcalpha 2-3Galbeta 1-4(Fucalpha 1-3)GlcNAc-R, recognized by the monoclonal antibody CSLEX-1 (17). sLex acts as a ligand for binding of tumor cells (18-20) and leukocytes (reviewed in Refs. 21 and 22) to selectins on the surface of endothelial cells. The importance of C2GnT in vivo was recently shown in a study of mice in which the C2GnT gene was deleted from the germ line (23). Leukocyte interactions with selectins and endothelial cells were impaired, leading to a weakened inflammatory response.

The mucin MUC1 is a type I membrane-bound glycoprotein, which is aberrantly glycosylated in many cancer tissues (reviewed in Ref. 24), displaying several tumor-associated epitopes. For example, monoclonal antibodies SM-3 and HMFG-2 recognize peptide epitopes within the MUC1 tandem repeat region and react preferentially with MUC1 in cancer tissues (25-27) where aberrant glycosylation results in epitope exposure.

The purpose of this study was to examine the effects of C2GnT on the expression of certain MUC1 tumor-associated epitopes. We stably transfected the human pancreatic adenocarcinoma cell line Panc1-MUC1, which expresses a recombinant human MUC1 cDNA, with a bovine C2GnT cDNA. We found that increased expression of C2GnT resulted in de novo expression of the sLex epitope. However, this did not render the cells capable of binding selectins. C2GnT expression also led to the elimination of the CC49 epitope. Furthermore, the SM-3 and HMFG-2 tumor-associated MUC1 peptide epitopes were masked by high levels of C2GnT expression. In summary, introduction of C2GnT into a cancer cell line significantly altered the expression of MUC1 tumor-associated epitopes by shifting the mucin-type glycosylation pathway toward more complex O-glycans. This suggests a potential regulatory role for C2GnT in the generation of tumor-associated epitopes.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- The Panc1 cell line was purchased from the ATCC (Manassas, VA). S2-013 is a subline of a human pancreatic tumor cell line derived from a liver metastasis (28). UDP-[14C]GlcNAc, UDP-[3H]Gal, and CMP-[3H]NeuAc were from American Radiolabeled Chemicals (St. Louis, MO). Asialo ovine submaxillary mucin (aOSM) was prepared as described (29). FPDG was isolated as described (30) from the serum of the antarctic fish Dissostichus mawsoni, provided by Dr. Arthur DeVries at the University of Illinois at Champaign-Urbana. E-, P- and L-selectin/IgM chimeras were provided by Dr. John Lowe at the University of Michigan, Ann Arbor. Transferrin (iron-saturated) was from Collaborative Biomedical Products (Bedford, MA). Immobilon P polyvinylidene difluoride membrane was from Millipore (Bedford, MA). Bond Elut C18 cartridges were from Varian (Sunny Vale, CA). M2 anti-FLAG monoclonal antibody was from Kodak IBI. CC49 monoclonal antibody was a gift from Dr. David Colcher at the University of Nebraska Medical Center. CSLEX-1 monoclonal antibody was obtained from the ATCC. HMFG-2 and SM-3 monoclonal antibodies were gifts of Dr. Sandra Gendler at the Mayo Clinic, Scottsdale, AZ. C2GnT monoclonal antibody B5-1 was obtained as described (31). Biotin-conjugated lectins DBA and PNA were obtained from EY Laboratories (San Mateo, CA). Other chemicals were from Sigma unless otherwise noted.

Cell Culture-- Panc1 and Panc1-MUC1 cells were grown in minimal essential medium supplemented with 5% fetal bovine serum and antibiotics (50 units/ml penicillin and 50 µg/ml streptomycin). Panc1-MUC1 cells stably transfected with C2GnT cDNA were grown in minimal essential medium supplemented with 5% fetal bovine serum and 300 µg/ml Zeocin.

Stable Transfection of Panc1-MUC1 Cells with Bovine C2GnT cDNA-- A 1.6-kilobase fragment of bovine C2GnT cDNA (containing the complete open reading frame of C2GnT) (31) was subcloned into the mammalian expression vector pcDNA 3.1/Zeo+ (Invitrogen) using KpnI and NotI. Transfection of pcDNA 3.1-C2GnT into Panc1-MUC1 cells was performed using a transferrin-assisted lipofection protocol as described previously (32), and clones, which stably express C2GnT, were selected based on resistance to the antibiotic Zeocin.

Glycosyltransferase Enzyme Assays-- Enzyme assays were carried out on total cell homogenates prepared by washing confluent monolayers of the cells twice with cold PBS, scraping the cells off the flask in 0.25 M sucrose, and disrupting the cells by successive passage of the sucrose suspension through 18-, 20-, and 25-gauge syringe needles. Protein concentration was measured by the Bio-Rad assay (Bio-Rad) using BSA as standard. All assays were conducted at least in duplicate under conditions in which product formation was linear with respect to time and enzyme amount. An additional reaction without exogenous acceptor was performed to measure endogenous enzyme activity. Enzyme activity was calculated by subtracting endogenous activity from total activity and was expressed as nmol of sugar donor transferred/hour/mg protein. GalNAc TF, which catalyzes the attachment of GalNAc in alpha -linkage to serine or threonine of the mucin peptide, was assayed as described (33), using a synthetic 29-amino acid MUC2 peptide as acceptor having the sequence PTTTPITTTTTVTPTPTPTGTQTPTTTPI. Core 1 GalTF, which catalyzes attachment of galactose in beta -1,3 linkage to GalNAcalpha -Ser/Thr, was assayed as described (29) using aOSM as acceptor. C2GnT activity was assayed as described (34) using Galbeta 1-3GalNAcalpha -Bzl as acceptor. ST6GalNAc I, which catalyzes attachment of neuraminic acid in alpha -2,6 linkage to GalNAc in GalNAcalpha -Ser/Thr and Galbeta 1-3GalNAcalpha -Ser/Thr (6), was assayed as described (35) using aOSM as acceptor. ST6GalNAc II, which catalyzes attachment of neuraminic acid in alpha -2,6 linkage to GalNAc in Galbeta 1-3GalNAcalpha -Ser/Thr (but not in GalNAcalpha -Ser/Thr) (5), was assayed as described (35) using FPDG as acceptor. FPDG also acts as an acceptor for ST6GalNAc I and ST3Gal I, and therefore care must be taken in interpreting assay results using this acceptor. ST3Gal I, which catalyzes attachment of neuraminic acid in alpha -2,3 linkage to Gal of Galbeta 1-3GalNAcalpha -Ser/Thr (36), was measured in the same manner as ST6GalNAc I and II, except Galbeta 1-3GalNAcalpha -Bzl was used as acceptor. ST6GalNAc I and II cannot utilize Galbeta 1-3GalNAcalpha -Bzl as acceptor (5, 6), thus preventing their interference with the measurement of ST3Gal I activity.

Preparation of Cell Lysate for Western Blotting and Lectin Blotting-- Confluent cells were washed twice with cold PBS and scraped from culture flasks in lysis buffer (500 µl for a T25 flask) containing 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, and 1% Triton X-100, using a rubber cell scraper. Following a 40-min incubation on ice, lysates were centrifuged at 2000 rpm for 2 min in a microcentrifuge to pellet cell debris. Supernatant was then transferred to a new tube and stored at -20 °C.

Glycosidase Treatment of Cell Lysates-- Cell lysates (300 µg of total protein) prepared as described above were treated with 100 milliunits of Clostridium perfringens neuraminidase in a total volume of 200 µl for 3.5 h at 37 °C in 0.05 M sodium acetate, pH 5.5. One-third of the neuraminidase-treated lysates was exposed to 1 unit of peptide N-glycosidase F in 200 mM sodium phosphate, 10 mM EDTA, pH 7.2, at 37 °C for 18 h. Another third of the neuraminidase-treated lysates was treated with 10 milliunits of Diplococcus pneumoniae beta -galactosidase in 200 mM sodium cacodylate, pH 6.0, at 37 °C for 44 h. Samples were stored at -20 °C after treatment prior to SDS-polyacrylamide gel electrophoresis analysis and Western blotting or lectin blotting.

Immunoblotting-- For Western blot analysis of MUC1 and C2GnT, proteins in cell lysates were resolved by 6% SDS-polyacrylamide gel electrophoresis (with 3% polyacrylamide stacking gels) and 10% SDS-polyacrylamide gel electrophoresis (with 6% polyacrylamide stacking gels), respectively. Protein was electroblotted to Immobilon P polyvinylidene difluoride membrane overnight at 300 mA, then blocked in 5% nonfat milk in TBS (0.9% NaCl, 10 mM Tris, pH 7.5) at room temperature for 1 h. The MUC1 blots were then probed for 1 h at room temperature with various primary antibodies in 5% nonfat milk in TBS, whereas the C2GnT blot was probed with the C2GnT antibody B5-1 diluted 1:2500 in TBS, 1% BSA. Membranes were then washed 15 min in 5% nonfat milk in TBS (for MUC1 blots) or TBS, 1% BSA (for C2GnT blots) followed by two additional 5-min washes with fresh wash mixtures. The membranes were then exposed for 1 h at room temperature to peroxidase-conjugated goat anti-mouse IgG/IgM secondary antibody diluted 1:2000 in 5% nonfat milk in TBS (for MUC1 blots) or TBS, 1% BSA (for C2GnT blots). Washes were repeated as described above, and then ECL reagents (Pierce) were applied per the manufacturer's instructions; the blots were then exposed to ECL-sensitive film (Amersham Pharmacia Biotech).

Lectin Blotting-- Proteins in whole cell lysates were resolved by 6% SDS-polyacrylamide gel electrophoresis (with 3% polyacrylamide stacking gels). Protein was electroblotted to Immobilon P polyvinylidene difluoride membrane overnight at 300 mA and blocked in 2% BSA (fraction V) in PBS at room temperature for 1 h. The blots were probed for 1.5 h at room temperature with biotin conjugates of either DBA or PNA 1:500 in TBT (TBS + 0.1% BSA + 0.025% Tween 20). Membranes were washed three times, 5 min each, in TBT. Next, the membranes were exposed for 1 h at room temperature to peroxidase-conjugated streptavidin diluted 1:1000 in TBT. Washes were repeated as described above. ECL reagents were applied as per the manufacturer's instructions, and the blots were exposed to ECL-sensitive film.

Imunoprecipitation of MUC1 Using Antibody Against the MUC1 Tandem Peptide Repeat-- 500 µl of Panc1-MUC1 C2#5 total cell lysate was incubated with 200 µl of antibody HMFG-2 at 4 °C with mild agitation for 3 h. Protein G-Sepharose (150 µl) was then added, and the mixture was incubated for 16 h at 4 °C with mild agitation. The immunoprecipitate was washed three times with 1 ml of cold PBS. SDS-polyacrylamide gel electrophoresis sample buffer was added, and the mixture was boiled for 5 min; the supernatant was resolved on 6% SDS-polyacrylamide gel electrophoresis.

Flow Cytometric Analysis of Selectin Binding Ability of Panc1-MUC1 C2#7 Cells-- S2-013 cells and Panc1-MUC1 C2#7 cells were grown to approximately 90% confluence and released from the tissue culture flask by incubation for 30-60 min in PBS containing 0.5 mM EDTA and 0.1% BSA. Aliquots containing 5 × 105 cells were pelleted in wells of a 96-well plate and washed with staining medium (Dulbecco's modified Eagle's medium containing 0.1% BSA and 0.1% sodium azide). The cells were then stained for 1 h on ice with IgM-conjugated P-, E-, or L-selectin in staining medium with or without 5 mM EDTA added. Cells were washed twice with staining medium (with or without 5 mM EDTA, as appropriate) and stained 1 h on ice in the dark with 10 µg/ml fluorescein isothiocyanate-conjugated goat anti-human IgM in staining medium (with or without 5 mM EDTA, as appropriate). Cells were washed twice with staining medium and fixed for 10 min in 2% formaldehyde. The cells were resuspended in PBS containing 0.1% BSA and 0.1% sodium azide and analyzed on a Becton Dickinson FACScan or FACStarPlus.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Stable Transfection of C2GnT cDNA into Panc1-MUC1 Cells and Selection of Clones for Characterization-- MUC1-transfected Panc1 (Panc1-MUC1) cells were chosen as the host for stable transfection of C2GnT because this cell line lacks endogenous C2GnT expression but expresses core 1 Gal TF, which creates the acceptor substrate utilized by C2GnT. These cells also express a recombinant human MUC1 bearing a FLAG epitope (Fig. 1), which provides for ease of detection and purification of the MUC1. Furthermore, MUC1 in the Panc1-MUC1 cells has been previously characterized with a panel of antibodies recognizing specific carbohydrate antigens and was found to express the tumor-associated epitope recognized by CC49 (28), which does not possess a core 2 branch (7, 8).


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  		Fig. 1.   Schematic representation of MUC1 construct expressed by Panc1-MUC1 cells showing the N-terminal FLAG epitope inserted for ease of detection and purification (28). The tandem repeat domain consists of 40 copies of the MUC1 tandem repeat sequence.

Following transfection of the C2GnT cDNA into Panc1-MUC1 cells, stable clones were isolated and screened for expression of C2GnT. Three clones representing a wide range of C2GnT expression (Fig. 2) were chosen for characterization of the effects of C2GnT on MUC1. The three clones displayed an approximate 40-fold difference in C2GnT activity between the high expressing (C2#7) and low expressing (C2#5) clones (Fig. 2A). C2GnT expression levels in the clones assayed via Western blotting gave results consistent with those of the C2GnT enzyme assays (Fig. 2B).


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  		Fig. 2.   C2GnT expression levels in parental cells and selected C2GnT transfectants. A, C2GnT enzyme activities of Panc1, Panc1-MUC1, and C2GnT-transfected Panc1-MUC1 clones selected for characterization. B, Western blot against C2GnT in total cell lysates of Panc1, Panc1-MUC1, and C2GnT-transfectant clones selected for characterization.

Expression of C2GnT Leads to the Appearance of sLex but Does Not Confer Selectin Binding Ability on the Cells-- The presence of the core 2 branch in mucin-type glycans has been implicated in the generation of the sLex epitope (13-16), and transfection of C2GnT cDNA into a cell line, which expresses the P-selectin ligand P-selectin glycoprotein ligand-1, rendered the cells capable of binding to P-selectin (37, 38). Therefore, we examined whether expression of C2GnT in Panc1-MUC1 cells could generate the sLex epitope. Immunoblotting with CSLEX-1, which recognizes sLex (NeuAcalpha 2-3Galbeta 1-4(Fucalpha 1-3)GlcNAc-R) (17), revealed that the sLex epitope was not present in Panc1-MUC1 parental cells or in the low C2GnT-expressing clone C2#5. However, the epitope appeared in clones C2#14 and C2#7, which have higher C2GnT expression (Fig. 3A). MUC1 expression levels in the clones varied little (Fig. 3B), showing that the changes in sLex detection were not because of different amounts of MUC1 in the samples.


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  		Fig. 3.   Effect of C2GnT transfection on expression of the sialyl Lewis x epitope recognized by CSLEX-1. Total cell lysates (100 µg of protein/well) from Panc1, Panc1-MUC1, and C2GnT-transfected Panc1-MUC1 clones were fractionated by 6% SDS-polyacrylamide gel electrophoresis followed by immunoblotting against (A) the CSLEX-1 epitope sLex and (B) FLAG epitope present on the recombinant MUC1 expressed by the cells. C2GnT-transfectants are displayed in order of increasing C2GnT expression.

Because we detected sLex on MUC1 in the high C2GnT-expressing transfectants, we tested these clones for their ability to bind to P-, E-, and L-selectins using IgM-selectin fusion proteins followed by staining with a fluorescein isothiocyanate-conjugated anti-IgM secondary antibody and subsequent flow cytometry analysis. Despite the presence of the sLex epitope, no binding of the C2GnT transfectants to the selectins was detectable under conditions in which the positive control cell line, S2-013, was found to bind to the selectins (data not shown).

The CC49 Epitope on MUC1 in Panc1-MUC1 Cells Consists of a Trisaccharide Which Lacks the Core 2 Branch-- Because CC49 can recognize both NeuAcalpha 2,6(Galbeta 1,3)GalNAc and NeuAcalpha 2,6GalNAc (7, 8), we sought to identify which of these was present on MUC1 in the Panc1-MUC1 parental cells, as detected previously by Burdick et al. (28). We performed lectin blotting using DBA and PNA on Panc1-MUC1 cell lysates treated or untreated with neuraminidase. As shown in Fig. 4, A and B, lanes 1 and 2, PNA reacted intensely with MUC1 in neuraminidase-treated lysate, whereas DBA showed no reaction. Because DBA is specific for alpha -linked GalNAc, whereas PNA recognizes Galbeta 1-3 GalNAc, these results strongly suggest the presence of Galbeta 1-3 (NeuAc alpha  2-6) GalNAc, rather than sialyl Tn (NeuAcalpha 2-6GalNAc), on MUC1 in the Panc1-MUC1 parental cells. To rule out the possibility that the structure recognized by PNA is located on asparagine-linked glycans on MUC1 rather than on mucin-type glycans, we treated desialylated lysate with peptide N-glycosidase F to remove N-linked chains. No change was seen in the intensity of PNA recognition (Fig. 4, A and B, lane 3), confirming that the recognized structure is present on O-glycans. Finally, because PNA can react with terminal beta 1,4-linked galactose in addition to Galbeta 1-3 GalNAc, we treated desialylated lysate with D. pneumoniae beta -galactosidase, which cleaves beta -1,4-linked galactose (but not beta -1,3-linked galactose). Once again, no change was seen in PNA recognition (Fig. 4, A and B, lane 4), further supporting Gal beta -1-3GalNAc as the structure recognized by PNA. From these results, we conclude that the structure detected by CC49 in the Panc1-MUC1 parental cells consists mainly of the trisaccharide Gal beta -1-3 (NeuAcalpha 2-6) GalNAc. This conclusion is supported by our detection of substantial core 1 GalTF enzyme activity in Panc1 cells (Table I), which synthesizes Galbeta -1-3GalNAc.


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  		Fig. 4.   Lectin blotting characterization of oligosaccharide structure on MUC1 in parental Panc1-MUC1 cells. 30 µg of Panc1-MUC1 total cell lysates treated with nothing (lane 1), neuraminidase (lane 2), neuraminidase + peptide N-glycosidase F (lane 3), or neuraminidase + D. pneumoniae beta -galactosidase (lane 4) were separated by 6% SDS-polyacrylamide gel electrophoresis followed by lectin blotting using (A) DBA, which recognizes alpha -GalNAc, and (B) PNA, which recognizes Galbeta 1-3GalNAc. 30 µg of Panc1 total cell lysate (lane 5) and 10 µg of aOSM (lane 6) were used as controls.

                              
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Table I
Glycosyltransferase activities in Panc1 and Panc1-MUC1 C2#7 cells
Enzyme activities are shown as the values of donor substrates incorporated into the exogenous acceptor. Assays were performed at least in duplicate as described under "Experimental Procedures."

C2GnT Expression Reduces the Expression of the Tumor-associated Epitope Recognized by CC49-- CC49 was raised against the tumor antigen tumor-associated glycoprotein-72 (39). CC49 is highly tumor-selective, reacting with a high percentage of malignant cells from various carcinomas but with very few normal tissues (39, 40). Because the CC49 epitope lacks a core 2 branch, we hypothesized that C2GnT expression could lead to altered expression of this epitope. CC49 immunoblotting showed heavy expression of the CC49 epitope in the Panc1-MUC1 cells (Fig. 5A), confirming the previous report of Burdick et al. (28). Increased C2GnT expression led to decreased expression of the CC49 epitope, and the epitope was abolished in the highest C2GnT-expressing transfectant, C2#7. Again, these results were not because of differences in the amount of MUC1 present in the samples, as shown in Fig. 5B. Analysis of MUC1 O-glycans via beta -elimination and gel filtration chromatography using Bio-Gel P-4 (Bio-Rad) showed that transfection with C2GnT led to a nearly complete disappearance of the lower-MW MUC1 O-glycans (data not shown). This suggests that the reduction in the CC49 epitope was caused by a complete substitution of the O-glycans by C2GnT, instead of by only a partial substitution causing reduced access of the antibody to the unsubstituted O-glycans. These findings support the idea that C2GnT was able to utilize C6 on GalNAc in the core 1 acceptor to a greater extent than ST6GalNAc I and II, shifting the O-glycosylation pathway away from the structure recognized by CC49 (Fig. 6).


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  		Fig. 5.   Effect of C2GnT transfection on expression of the tumor-associated epitope recognized by CC49. Total cell lysates (100 µg of protein/well) from Panc1, Panc1-MUC1, and C2GnT-transfected Panc1-MUC1 clones were fractionated by 6% SDS-polyacrylamide gel electrophoresis followed by immunoblotting against (A) the CC49 epitope Galbeta 1-3(NeuAcalpha 2-6)GalNAc and (B) FLAG epitope present on the recombinant MUC1 expressed by the cells. C2GnT transfectants are displayed in order of increasing C2GnT expression.


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  		Fig. 6.   Schematic diagram showing the postulated effect of C2GnT transfection on mucin-type O-glycosylation pathways in Panc1 cells. Pathways are shown for Panc1-MUC1 parental cells and C2GnT-transfected Panc1-MUC1 clones. Only structures that are involved in the generation of the CC49 and sLex epitopes are shown.

C2GnT Expression Masks the Tumor-associated Epitope Recognized by SM-3-- SM-3 and HMFG-2 react with the tandem peptide repeat domain of MUC1, and bind more strongly to MUC1 from cancer tissues than to the more heavily glycosylated MUC1 found in normal tissues (25-27). SM-3 was raised against a chemically deglycosylated form of the mucin recognized by HMFG-2 (25) and possesses a high degree of tumor specificity. SM-3 reacts with 92% of breast carcinoma samples tested, as well as several other carcinomas, and shows virtually no reactivity with normal resting breast, lactating breast, or other normal tissues (25, 26).

Because the CSLEX-1 and CC49 immunoblots indicated that C2GnT expression resulted in the synthesis of more complex O-glycans, we examined whether these O-glycans could mask the SM-3 tumor-associated peptide epitope. SM-3 immunoblotting showed strong recognition of MUC1 (~250 kDa) in the lysate of both the Panc1-MUC1 cells and the low C2GnT-expressing clone (C2#5) but virtually no recognition in the lysates of the clones expressing higher levels of C2GnT (Fig. 7). Similar results were seen in immunoblots probed with HMFG-2 (data not shown). This apparent masking effect further supports the hypothesis that introduction of C2GnT caused more complex MUC1 O-glycosylation.


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  		Fig. 7.   C2GnT transfection produces masking of the SM-3 tumor-associated peptide epitope. A, anti-MUC1 tandem repeat immunoprecipitate (from C2GnT transfectant C2#5, using antibody HMFG-2) or total cell lysates (100 µg of protein/well) and (B) conditioned medium (100 µl/well) from Panc1, Panc1-MUC1, and C2GnT-transfected Panc1-MUC1 clones were separated by 6% SDS-polyacrylamide gel electrophoresis followed by immunoblotting against the MUC1 peptide tandem repeat domain. C2GnT transfectants are displayed in order of increasing C2GnT expression. C, MUC1 expression levels are shown for normalization of blots in A and B. MUC1 was detected by immunoblotting using the M2 anti-FLAG antibody.

SM-3 also detected a series of lower molecular mass bands between ~100-140 kDa in the cell lysates of both the Panc1-MUC1 and the C2GnT clones (Fig. 7A). These fragments are likely derived from the tandem repeat region of MUC1, because they could be immunoprecipitated by HMFG-2 and were not detected in lysate from the negative control Panc1 cells (Fig. 7A), which do not express the recombinant MUC1. Furthermore, unlike full-length MUC1, the low molecular mass peptides were not masked in the C2GnT transfectants (Fig. 7A) and were not detected by the CC49 or CSLEX-1 antibodies (data not shown), which recognize specific carbohydrate structures. These results suggest that these peptides are poorly glycosylated, lacking even the core 1 acceptor structure on which C2GnT acts. SM-3 also detected full-length MUC1 in both the cell lysate and conditioned medium, whereas the low molecular mass MUC1 fragments were seen only in the cell lysate, suggesting that these peptides remain within the cell, whereas a fraction of full-length MUC1 is released into the medium.

Neuraminidase Treatment Does Not Reverse Masking of the MUC1 Tandem Peptide Repeat Epitope-- Removal of sialic acid has been shown to greatly increase access of anti-MUC1 tandem repeat antibodies such as SM-3 and HMFG-2 to the MUC1 peptide epitope on underglycosylated MUC1 in several cancer cell lines (41). Therefore, we treated cell lysates with neuraminidase to determine whether removal of sialic acid could overcome the effects of introducing C2GnT and expose the MUC1 peptide epitope. We performed immunoblots using the HMFG-2 antibody, because heavier glycosylation is required to mask the HMFG-2 epitope than the SM-3 epitope (25, 42). As shown in Fig. 8, removal of sialic acid dramatically decreased the SDS-polyacrylamide gel electrophoresis mobility of full-length MUC1 in the Panc1-MUC1 cells and the clone expressing low C2GnT (C2#5), consistent with heavy sialylation of MUC1. However, desialylation did not enhance MUC1 peptide epitope recognition by HMFG-2 in the clones expressing higher levels of C2GnT. This suggests that the nonsialic acid portion of the MUC1 O-glycans in the high C2GnT-expressing clones is sufficient to mask the peptide epitope, further indicating greater complexity of the MUC1 O-glycans in the C2GnT transfectants. Fig. 8 also shows that the low molecular mass MUC1 fragments were unaffected by neuraminidase treatment, both in their electrophoretic mobility and antibody recognition. This further supports the idea that these fragments are poorly glycosylated, if at all, because mucin-type glycans are often terminated with sialic acid.


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  		Fig. 8.   Removal of sialic acid does not increase MUC1 peptide epitope exposure in C2GnT-transfected Panc1-MUC1 cells. Total cell lysates from Panc1, Panc1-MUC1, and C2GnT-transfected Panc1-MUC1 clones were incubated at 37 °C for 1.8 h with or without 100 milliunits of neuraminidase and fractionated by 6% SDS-polyacrylamide gel electrophoresis followed by immunoblotting against the MUC1 tandem repeat using the antibody HMFG-2. C2GnT transfectants are shown in order of increasing C2GnT expression.

Transfection of C2GnT Did Not Significantly Alter the Activity of Other Glycosyltransferases Which Could Affect MUC1 Epitopes-- It is conceivable that the changes seen in expression of the tumor-associated epitopes could have been caused by unanticipated changes in the activities of glycosyltransferases other than C2GnT in the C2GnT transfectants. Therefore, we compared the activities of several glycosyltransferases, which could lead to altered MUC1 epitope expression in the original Panc1 cells, with those in the highest C2GnT-expressing transfectant, Panc1-MUC1 C2#7. As shown in Table I, transfection of C2GnT resulted in no apparent changes in activity of any of the enzymes tested. These results suggest that C2GnT activity was solely responsible for the changes in MUC1 epitope detection shown in Figs. 3, 5, 7, and 8.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this report we describe the effects of stable expression of C2GnT on tumor-associated epitopes in a human pancreatic adenocarcinoma cell line, Panc1-MUC1, which does not express endogenous C2GnT. Three C2GnT-transfected clones expressing varying levels of C2GnT, along with the original Panc1 cell line and parental Panc1-MUC1 cells, were compared in their expression of tumor-associated epitopes recognized by antibodies CSLEX-1, CC49, SM-3, and HMFG-2.

The appearance of the sLex epitope in the high C2GnT-expressing transfectants is consistent with previous studies, which have linked the sLex structure, NeuAcalpha 2-3 Gal beta 1-4 (Fuc alpha 1-3) GlcNAc beta 1-6, with the presence of the core 2 beta  1-6 branch (13-16). Interestingly, it was recently found that transfection of a cDNA for a specific alpha 1-3 fucosyltransferase into COS-1 cells, which express C2GnT activity,2 also resulted in the creation of sLex (43). This study, combined with our results, highlights the importance of both C2GnT and the alpha 1-3 fucosyltransferase in generating the sLex structure, as shown in Fig. 6. Our results imply that Panc1 cells express this fucosyltransferase, the alpha 2-3 sialyl transferase, and the beta 1-4 Gal transferase necessary for generating sLex and that C2GnT is the missing component in the sLex synthetic pathway in Panc1 cells.

The inability of our sLex-bearing C2GnT transfectants to bind to selectins may be explained by the known antiadhesive properties of MUC1, which can hinder cell-cell and cell-matrix interactions (44, 45). Such an antiadhesive effect may be sufficient to block the sLex-selectin interaction. Our results are also consistent with those described in a recent report by McDermott et al. (46), in which introduction of a MUC1 cDNA into the pancreatic cancer cell line S2-013 abolished the selectin binding ability possessed by the parental S2-013 cells, despite the presence of sLex on the MUC1 expressed by the cells. It is possible that the sLex structures on MUC1 are not presented in a favorable configuration for high affinity selectin binding. For example, it has recently been suggested that clustering of the epitope may play a key role in selectin binding (21).

The apparent shift in mucin-type glycosylation pathways caused by introducing C2GnT (Fig. 6) may be partially explained by differences in Golgi localization for C2GnT and the sialyl transferases ST6GalNAc I and II. C2GnT has been detected within the cis- to medial-Golgi compartments (47). The specific Golgi localization of ST6GalNAc I and II has not yet been reported. However, a localization beyond the cis-Golgi is suggested by the inability of ST6GalNAc I present in the cells (Table I) to generate NeuAcalpha 2,6GalNAc, as suggested by our DBA lectin blotting results (Fig. 4). Furthermore, the CC49 epitope was significantly reduced even in the lowest C2GnT-expressing transfectant, C2#5 (Fig. 5), in which the measured C2GnT activity was approximately equal to that of ST6GalNAc I and II (Table I). This result could be explained by earlier Golgi localization for C2GnT than for ST6GalNAc I and II, because this would enable C2GnT to utilize the core 1 acceptor before ST6GalNAc I and II.

C2GnT also competes for the core 1 acceptor structure with ST3Gal I, which attaches neuraminic acid in alpha -2,3 linkage to galactose of the core 1 structure (36). Evidence for such competition includes shortened MUC1 O-glycans seen in certain breast cancer cell lines, which were attributed to increased ST3Gal I activity and reduced C2GnT activity (48). Furthermore, Whitehouse et al. (49) found that transfection of a C2GnT-expressing normal breast cell line with a ST3Gal I cDNA resulted in decreased core 2 branching of MUC1 O-glycans. These investigators localized ST3Gal I to the medial- to trans-Golgi, and thus its localization overlaps with that reported for C2GnT (47). This suggests that partial overlap in Golgi localization is sufficient for efficient glycosyltransferase competition for a common acceptor substrate.

Immunoblots using the anti-MUC1 tandem peptide repeat antibodies SM-3 and HMFG-2 showed that increased C2GnT expression significantly obscured these tumor-associated peptide epitopes (Figs. 7 and 8). Interestingly, we found that SM-3 strongly recognized MUC1 in the Panc1-MUC1 parental cell line, which we showed to possess the sialylated core 1 carbohydrate structure (Fig. 4). This suggests that SM-3 is capable of recognizing certain glycosylated forms of MUC1, despite the fact that SM-3 was raised against a chemically deglycosylated form of MUC1 (25). Moreover, this finding is consistent with a recent study by Karsten et al. (50), which showed that glycosylation of MUC1 by GalNAc or Galbeta 1,3GalNAc can actually enhance recognition by SM-3. Together with the masking of the SM-3 epitope we observed upon introduction of C2GnT, these results suggest that SM-3 is capable of reacting with MUC1 possessing O-glycan structures below a certain threshold level of complexity.

Masking of the HMFG-2 epitope persisted following removal of sialic acid (Fig. 8). Because HMFG-2 recognition of MUC1 is known to be enhanced by desialylation of underglycosylated MUC1 present in certain cancer cell lines (41), this result strongly suggests significantly increased MUC1 glycosylation in the C2GnT transfectants, beyond a level at which desialylation can expose the epitope. These results are consistent with the more complex glycosylation of MUC1 in the C2GnT transfectants implied by the immunoblotting results using CC49 and CSLEX-1. We conclude that the core 2-associated O-glycans free of sialic acid are sufficient to block antibody recognition, underscoring the importance of the core 2 structure in regulating tumor epitope expression.

In conclusion, we have found that the stable expression of C2GnT in a human pancreatic cancer cell line was able to greatly reduce the expression of tumor-associated epitopes recognized by monoclonal antibodies SM-3 and CC49, although inducing expression of sialyl Lewis x. These findings imply that alterations in C2GnT activity could play a key role in regulating the expression of these epitopes on cancer cells. In particular, our results suggest that C2GnT activity may be suppressed in certain cancer cells, which deserves further exploration.

    ACKNOWLEDGEMENTS

We thank James Seberger for providing instructions for lectin blotting and Kim McDermott for providing instructions for selectin binding assays. We also thank Dr. Charles Kuszynski of the Cell Analysis Facility, University of Nebraska Medical Center, for assistance with flow cytometric analysis, and the Monoclonal Antibody Facility, University of Nebraska Medical Center, for assistance in production of monoclonal antibodies.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants RO1 HL48282, RO1 CA57362, RO1 CA69234, and P30 CA36727.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Current address: Regional Research Laboratory, Jammu-Tawi-180001, India.

parallel To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Nebraska Medical Center, 984525 Nebraska Medical Center, Omaha, NE 68198-4525. Tel.: 402-559-5776; Fax: 402-559-6650; E-mail: pcheng@unmc.edu.

2 P.-W. Cheng, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: C2GnT, UDP-GlcNAc:Galbeta 1-3GalNAc (GlcNAc to GalNAc) beta 1-6GlcNAc-transferase; GalNAc TF, UDP-GalNAc:Polypeptide GalNAc transferase; Core 1 GalTF, UDP-Gal:GalNAc beta 1-3Gal transferase; ST6GalNAc I, CMP-NeuAc:GalNAc/Galbeta 1-3GalNAc (NeuAc to GalNAc) alpha 2-6NeuAc transferase; ST6GalNAc II, CMP-NeuAc:Galbeta 1-3GalNAc (NeuAc to GalNAc) alpha 2-6NeuAc transferase; ST3Gal I, CMP-NeuAc:Galbeta 1-3GalNAc (NeuAc to Gal) alpha 2-3-NeuAc transferase; DBA, Dolichos biflorus agglutinin; PNA, Arachis hypogaea agglutinin; Gal, galactose; Panc1-MUC1, Panc1 cells transfected with FLAG epitope-tagged MUC1 cDNA; sLex, sialyl Lewis; Bzl, benzyl; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; BSA, bovine serum albumin; TF, transferase; FPDG, freezing point-depressing glycoprotein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Farah, R. A., Clinchy, B., Herrera, L., and Vitetta, E. S. (1998) Crit. Rev. Eukaryotic Gene Expression 8, 321-356[Medline] [Order article via Infotrieve]
2. Rowlinson-Busza, G., and Epenetos, A. A. (1992) Curr. Opin. Oncol. 4, 1142-1148[Medline] [Order article via Infotrieve]
3. Brockhausen, I. (1996) in New Comprehensive Biochemistry (Montreuil, J. , Vliegenthart, J. F. G. , and Schachter, H., eds), Vol. 29a , pp. 201-259, Elsevier Publishing Co., Inc., New York
4. Dennis, J. W. (1993) Glycobiology 3, 91-96[Free Full Text]
5. Kurosawa, N., Kojima, N., Inoue, M., Hamamoto, T., and Tsuji, S. (1994) J. Biol. Chem. 269, 19048-19053[Abstract/Free Full Text]
6. Kurosawa, N., Hamamoto, T., Lee, Y.-C., Nakaoka, T., Kojima, N., and Tsuji, S. (1994) J. Biol. Chem. 269, 1402-1409[Abstract/Free Full Text]
7. Hanisch, F.-G., Uhlenbruck, G., Egge, H., and Peter-Katalinic, J. (1989) Biol. Chem. Hoppe-Seyler 370, 21-26[Medline] [Order article via Infotrieve]
8. O'Boyle, K. P., Markowitz, A. L., Khorshidi, M., Lalezari, P., Longenecker, B. M., Lloyd, K. O., Welt, S., and Wright, S. E. (1996) Hybridoma 15, 401-408[Medline] [Order article via Infotrieve]
9. Fukuda, M., Carlsson, S. R., Klock, J. C., and Dell, A. (1986) J. Biol. Chem. 261, 12796-12806[Abstract/Free Full Text]
10. Baum, L. G., Pang, M., Perillo, N. L., Wu, T., Delegeane, A., Uittenbogaart, C. H., Fukuda, M., and Seilhamer, J. J. (1995) J. Exp. Med. 181, 877-887[Abstract/Free Full Text]
11. Piller, F., Piller, V., Fox, R. I., and Fukuda, M. (1988) J. Biol. Chem. 263, 15146-15150[Abstract/Free Full Text]
12. Tsuboi, S., and Fukuda, M. (1997) EMBO J. 16, 6364-6373[CrossRef][Medline] [Order article via Infotrieve]
13. Maemura, K., and Fukuda, M. (1992) J. Biol. Chem. 267, 24379-24386[Abstract/Free Full Text]
14. Heffernan, M., Lotan, R., Amos, B., Palcic, M., Takano, Y., and Dennis, J. W. (1991) J. Biol. Chem. 268, 1242-1251[Abstract/Free Full Text]
15. Wilkins, P. P., McEver, R. P., and Cummings, R. D. (1996) J. Biol. Chem. 271, 18732-18742[Abstract/Free Full Text]
16. Ohmori, K., Takada, A., Yoneda, T., Buma, Y., Hirashima, K., Tsuyoka, K., and Kannagi, R. (1993) Blood 81, 101-111[Abstract/Free Full Text]
17. Fukushima, K., Hirota, M., Terasaki, P. I., Wakisaka, A., Togashi, H., Chia, D., Suyama, N., Fukushi, Y., Nudelman, E., and Hakomori, S.-I. (1984) Cancer Res. 44, 5279-5285[Abstract/Free Full Text]
18. Walz, G., Aruffo, A., Kolanus, W., Bevilacqua, M., and Seed, B. (1990) Science 250, 1132-1135[Abstract/Free Full Text]
19. Majuri, M.-L., Mattila, P., and Renkonen, R. (1992) Biochem. Biophys. Res. Commun. 182, 1376-1382[CrossRef][Medline] [Order article via Infotrieve]
20. Takada, A., Ohmori, K., Yoneda, T., Tsuyuoka, K., Hasegawa, A., Kiso, M., and Kannagi, R. (1993) Cancer Res. 53, 354-361[Medline] [Order article via Infotrieve]
21. Varki, A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7390-7397[Abstract/Free Full Text]
22. McEver, R. P., Moore, K. L., and Cummings, R. D. (1995) J. Biol. Chem. 270, 11025-11028[Abstract/Free Full Text]
23. Ellies, L. G., Tsuboi, S., Petryniak, B., Lowe, J. B., Fukuda, M., and Marth, J. D. (1998) Immunity 9, 881-890[CrossRef][Medline] [Order article via Infotrieve]
24. Ho, S. B., and Kim, Y. S. (1991) Semin. Cancer Biol. 2, 389-400[Medline] [Order article via Infotrieve]
25. Burchell, J., Gendler, S. J., Taylor-Papadimitriou, J., Girling, A., Lewis, A., Millis, R., and Lamport, D. (1987) Cancer Res. 47, 5476-5482[Abstract/Free Full Text]
26. Girling, A., Bartkova, J., Burchell, J., Gendler, S., Gillett, C., and Taylor-Papadimitriou, J. (1989) Int. J. Cancer 43, 1072-1076[Medline] [Order article via Infotrieve]
27. Arklie, J., Taylor-Papadimitriou, J., Bodmer, W., Egan, M., and Millis, R. (1981) Int. J. Cancer 28, 23-29[Medline] [Order article via Infotrieve]
28. Burdick, M. D., Harris, A., Reid, C. J., Iwamura, T., and Hollinsworth, M. A. (1997) J. Biol. Chem. 272, 24198-24202[Abstract/Free Full Text]
29. Cheng, P.-W., and Bona, S. J. (1982) J. Biol. Chem. 257, 6251-6258[Abstract/Free Full Text]
30. Lin, Y., and DeVries, A. (1974) Biochem. Biophys. Res. Commun. 59, 1192-1196[CrossRef][Medline] [Order article via Infotrieve]
31. Li, C.-M., Adler, K. B., and Cheng, P.-W. (1998) Am. J. Respir. Cell Mol. Biol. 18, 343-352[Abstract/Free Full Text]
32. Cheng, P.-W. (1996) Hum. Gene Ther. 7, 275-282[Medline] [Order article via Infotrieve]
33. Nishimori, I., Johnson, N. R., Sanderson, S. D., Perini, F., Mountjoy, K., Cerny, R. L., Gross, M. L., and Hollingsworth, M. A. (1994) J. Biol. Chem. 269, 16123-16130[Abstract/Free Full Text]
34. Ropp, P. A., Little, M. R., and Cheng, P.-W. (1991) J. Biol. Chem. 266, 23863-23871[Abstract/Free Full Text]
35. Cheng, P.-W., Moeller, S. L., and Boat, T. F. (1980) Fed. Proc. 39, 2002
36. Rearick, J. I., Sadler, J. E., Paulson, J. C., and Hill, R. L. (1979) J. Biol. Chem. 254, 4444-4451[Abstract/Free Full Text]
37. Li, F., Wilkins, P. P., Crawley, S., Weinstein, J., Cummings, R. D., and McEver, R. P. (1996) J. Biol. Chem. 271, 3255-3264[Abstract/Free Full Text]
38. Kumar, R., Camphausen, R. T., Sullivan, F. X., and Cumming, D. (1996) Blood 88, 3872-3879[Abstract/Free Full Text]
39. Muraro, R., Kuroki, M., Wunderlich, D., Poole, D. J., Colcher, D., Thor, A., Greiner, J. W., Simpson, J. F., Molinolo, A., Noguchi, P., and Schlom, J. (1988) Cancer Res. 48, 4588-4596[Abstract/Free Full Text]
40. Molinolo, A., Simpson, J. F., Thor, A., and Schom, J. (1990) Cancer Res. 50, 1291-1298[Abstract/Free Full Text]
41. Ho, J. J. L., Cheng, S., and Kim, Y. S. (1995) Biochem. Biophys. Res. Commun. 210, 866-873[CrossRef][Medline] [Order article via Infotrieve]
42. Burchell, J., Durbin, H., and Taylor-Papadimiriou, J. (1983) J. Immunol. 131, 508-513[Medline] [Order article via Infotrieve]
43. Lowe, J. B., Stoolman, L. M., Nair, R. P., Larsen, R. D., Berhend, T. L., and Marks, R. M. (1990) Cell 63, 475-484[CrossRef][Medline] [Order article via Infotrieve]
44. Wesseling, J., van der Valk, S. W., Vos, H. L., Sonnenberg, A., and Hilkens, J. (1995) J. Cell Biol. 129, 255-265[Abstract/Free Full Text]
45. Makiguchi, Y., Hinoda, Y., and Imai, K. (1996) Jpn. J. Cancer Res. 87, 505-511[CrossRef][Medline] [Order article via Infotrieve]
46. McDermott, K. M., Crocker, P., Harris, A., Burdick, M. D., and Hollingsworth, M. A. Midwest Student Biomedical Research Forum, Omaha, February 1999, Abstract, University of Nebraska Medical Center and Creighton University, Omaha
47. Skrincosky, D., Kain, R., El-Battari, A., Exner, M., Kerjascki, D., and Fukuda, M. (1997) J. Biol. Chem. 272, 22695-22702[Abstract/Free Full Text]
48. Brockhausen, I., Yang, J.-M., Burchell, J., Whitehouse, C., and Taylor-Papadimitriou, J. (1995) Eur. J. Biochem. 233, 607-617[Medline] [Order article via Infotrieve]
49. Whitehouse, C., Burchell, J., Gschmeissner, S., Brockhausen, I., Lloyd, K. O., and Taylor-Papadimitriou, J. (1997) J. Cell Biol. 137, 1229-1241[Abstract/Free Full Text]
50. Karsten, U., Diotel, C., Klich, G., Paulsen, H., Goletz, S., Muller, S., and Hanisch, F.-G. (1998) Cancer Res. 58, 2541-2549[Abstract/Free Full Text]

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