Core3 O-glycan Synthase Suppresses Tumor Formation and Metastasis of

1Core3 O -glycan Synthase Suppresses Tumor Formation and Metastasis of Prostate Carcinoma PC3 and LNCaP Cells through Down-regulation ofα2β1 Integrin ComplexSeung Ho Lee 1, Shingo Hatakeyama 1, Shin-Yi Yu 2, Xingfeng Bao 1, Chikara Ohyama 3,Kai-Hooi Khoo 2, Michiko N. Fukuda 1, and Minoru Fukuda 11Glycobiology Unit, Tumor Microenvironment Program, Cancer Center, Burnham Institute for Medical Research, La Jolla California 92037, 2Institute of Biological Chemistry, Academia Sinica, Taipei, 11529, Taiwan and 3Department of Urology, Hirosaki University School ofMedicine, Hirosaki, 036-2562, JapanRunning Title: Core3 O -Glycan Suppresses Prostate Tumor MetastasisAddress correspondence to: Dr. Minoru Fukuda, Burnham Institute for Medical Research, La Jolla, California 92037, USA; Phone, 1-858-646-3144; Fax, 1-858-646-3193; E-mail, minoru@ While there are numerous reports of carbohydrates enriched in cancer cells, very few studies have addressed the functions of carbohydrates present in normal cells that decrease in cancer cells. It has been reported that core3 O -glycans are synthesized in normal gastrointestinal cells but are down-regulated in cancer cells. To determine the roles of core3 O -glycans, we transfected PC3 and LNCaP prostate cancer cells with β3-N -acetylglucosaminyltransferase-6 (core3 synthase) required to synthesize core3 O -glycans. Both engineered cell lines exhibited reduced migration and invasion through extracellular matrix components compared to mock-transfected cells. Moreover, we found that α2β1 integrin acquired core3 O -glycans in cells expressing core3 synthase with decreased maturation of β1 integrin, leading to decreased levels of the α2β1 integrin complex, decreased activation of focal adhesion kinase, and reduced lamellipodiaformation. Upon inoculation into the prostate of nude mice, PC3 cells expressing core3 O -glycans produced much smaller tumors without metastasis to the surrounding lymph nodes in contrast to robust tumor formation and metastasis seen in mock-transfected PC3 cells. Similarly, LNCaP cells expressing core3 O -glycans barely produced subcutaneous tumors in contrast to robust tumor formation by mock-transfected LNCaP cells. These findings indicate that addition of core3 O -glycans to β1 and α2 integrin subunit in prostate cancer cells suppresses tumor formation and tumor metastasis.Keywords : Core3 O -glycan; prostate cancer; α2β1 integrin; tumor metastasis Cancer cells often express surface carbohydrates different from normal cells {1}. One such change is expression of sialyl Lewis X and Lewis B blood group antigens in cancer cells {2, 3}. These structural elements are seen as capping oligosaccharides attached to the underlying glycan backbone where they likely function as ligands for cell adhesion molecules.The structure of underlying glycans also changes during malignant transformationand differentiation. In particular, there are several reports that an increase in the β1,6-N -acetylglucosaminyl branch inN -glycans synthesized by β1,6-N -acetylglucosaminyltransferase-V(GnT-V) is associated with oncogenic transformation {4-7}. Similar structural changes are seen in mucin-type O -glycans,/cgi/doi/10.1074/jbc.M109.010934The latest version is at JBC Papers in Press. Published on April 24, 2009 as Manuscript M109.010934Copyright 2009 by The American Society for Biochemistry and Molecular Biology, Inc. by guest, on October 9, 2010 Downloaded from2which have N -acetylgalactosamine at the reducing end linked to polypeptide threonine or serine residues. Addition of different carbohydrate residues to N -acetylgalactosamine confers a variety of backbone structures on mucin-type O -glycans; the most abundant of those are classified as core1, core2, core3, and core4 O -glycans {8} (Fig. 1). Among these O -glycans, the synthesis of the core2-branch has been extensively studied, particularly since conversion of core1 to core2 O -glycans was observed in T cell activation {9}. Expression of core2 branch apparently represents an onco-differentiation antigen, since core2 branched O -glycans are synthesized in early stages of T cell differentiation, downregulated in mature T cells, and reappear in T cell leukemia and immune deficiencies such as AIDS and Wiskoff-Aldrich syndrome (for a review see {10}. In addition, overexpression of core2 O -glycans is seen in many cancers, including lung and breast carcinoma cells {11, 12}. By contrast, core3 and core4 O -glycans are synthesized in normal cells but apparently downregulated in gastric and colorectal carcinoma {13, 14}. Core3 O -glycans are synthesized by core3 synthase (β3GnT-6), which adds β1,3-linked N -acetylglucosamine to N -acetylgalactosamine at the reducing terminus {15} (Fig. 1). Iwai et al. showed that forced expression of core3 synthase in human fibrosarcoma HT1080 FP-10 cells resulted in significant reduction in the formation of lung tumor foci in mice after intravenous injection of tumor cells through a tail vein {16}. However, the same study did not address if the expression of core3 influences tumor metastasis since the cancer cells were intravenously injected and no primary tumor was formed to spread into the lung as metastasis in contrast to the other studies {17, 18}. Core4 O -glycan is synthesized by addition of β1,6-linked N -acetylglucosamine to a core3 acceptor by core2 β1,6-N -acetylglucosamine M type (C2GnT-M) or C2GnT-2 {18, 19} (Fig. 1). Huang et al. reported that C2GnT-M is downregulated in colonic carcinoma cells and that forced expression of C2GnT-M in HCT116 colonic carcinoma cells significantly decreased cell invasion and subcutaneous tumor formation {21}. How upregulation of core3 and core4 O -glycans influences the pathophysiology of cells expressing core3 and core4 O -glycans has not been addressed. Cell-extracelluar matrix (ECM) interaction play an essential role during acquisition of migration and invasive behavior of cancer cells. For example, α2β1 integrin is the major receptor for collagen {22}, and most abundantly expressed in prostate cancer cells {23}. Glycosylation on integrin is one of the important modulator of integrin functions and many glycan structures, mainly N -glycans, have been studied. An increase of bisecting GlcNAc structure on α5β1 integrin inhibits the cell spreading and migration {24}, and induced beta 1,6-GlcNAc sugar chains on N -glycans of β1 integrine results in stimulation of cell migration {25}. However, it has not been addressed if changes in O -glycans affect integrin maturation and functions. To determine the role of core3 O -glycans in tumor formation and metastasis, we analyzed PC3 and LNCaP human prostate cancer cells. We found these cell lines express only small amounts of detectable amounts of core3 synthase; thus we transfected the cell lines with core3 synthase. Core3 synthase-transfected PC3 and LNCaP cells expresse increased amounts of core3 O -glycans in α2β1 integrin, showed the reduced maturation of β1 integrin, and low levels of α2β1 integrin formation, and migrated less efficiently through collagen and other extracellular matrix components, and were less invasive than mock-transfected cells. Moreover, those cells exhibited decreased activation of focal adhesion kinase (FAK) compared to mock-transfected cells. by guest, on October 9, 2010 Downloaded from3Significantly, PC3 cells expressing core3 O -glycans produced almost no primary tumors in the prostate and formed much fewer metastases in the draining lymph nodes than mock-transfected cells. Similarly, LNCaP cells expressing core3 O-glycans produced much smaller subcutaneous tumors than mock-transfected LNCaP cells. These findings indicate that addition of core3 O -glycans to the α2β1 integrin leads to decreased cell migration and invasion, resulting in decreased prostate tumor formation and metastasis.Experimental and ProceduresCell culture and transfection- PC3 and LNCaP prostate cancer cell lines were obtained from American Type Culture Collection and cultured in RPMI-1640 supplemented with 10% fetal bovine serum. cDNA encoding core3 synthase (β3GlcNAcT-6) {15} was amplified by reverse transcription (RT)-PCR and cloned into pcDNA 3.1(N) as described {26}. pcDNA 3.1(N) was prepared by deleting the Zeocin resistance gene and f1 origin from pcDNA 3.1/Zeo as described {26}. PC3 and LNCaP cells were transfected with pcDNA 3.1(N) harboring core3-synthase cDNA and pcDNA 3 harboring the neomycin-resistance gene using Lipofectamine. Transfected cells were selected first in 200 µg/ml Geneticin ®(Invitrogen) and maintained in 100 µg/ml of Geneticin. Clonal transfected cells were obtained by dilution and tested for expression of carbohydrates reactive to peanut agglutinin (PNA) after neuraminidase treatment. PNA reacts with Gal β1→3GalNAc α1→R core1 structure {27}. Core3 oligosaccharides do not react with PNA. For this assay, PC3 and LNCaP cells in cloning plates were dissociated into monodispersed cells using an enzyme-free dissociation solution (Hank’s balanced saline solution-based) purchased from Cell and Molecular Technologies. Dissociated cells were incubated with fluorescein isothiocyanate (FITC)-conjugated PNA and subjected to FACS analysis using FACScan flow cytometry (BD Biosciences) as described {26}. As controls, PC3 and LNCaP cells were transfected with empty pcDNA 3.1 (N) and pcDNA 3 and selected in geneticin. Mock-transfected and core3 synthase-expressing PC3 cells cultured on glass plates were stained with phalloidin to visualize F-actin as described previously {28}.Semi-quantitative RT-PCR analys - total RNA was isolated from PC3 and LNCaP cells using Trizol (Invitrogen). RT-PCR of core3 synthase (β3GnT-6) {15}, C2GnT-1 {29}, C2GnT-2 {19}, and C2GnT-3 {30} wasundertaken. First-strand cDNA was synthesized using Amplitag DNA polymerase (Applied Biosystems) and the following PCRprimers: C2GnT-1, 5’-tcggtggacacctgacgactatat-3’ (5’-primer) and 5’-aggtcataccgcttcttccacctt-3’ (3’-primer); C2GnT-2, 5’-agtccagggaatctcaaagccagt-3’ (5’-primer) and 5’-tgagctctggagcaagtcttccat-3’(3’-primer); C2GnT-3, 5’-gacatccagttctctagacctctg-3’ (5’-primer) and 5’-aaggcgaggtacttagggagtact-3’ (3’-primer);β3GnT-6, 5’-agcactgcagcagtggttc-3’ (5’-primer) and 5’-gaggaaggtgtccgcgaag-3’ (3’-primer) and glyceraldehyde-3-phosphatedehydrogenase (GAPDH),5’-cctggccaaggtcatccatgaca-3’ (5’-primer) and 5’-atgaggtccaccaccctgttgct-3’ (3’-primer).The PCR reaction was carried out at 94°C for 5 min followed by 35 cycles of 94°C for 30 sec, 56°C for 30 sec, and 72°C for 30 sec and by a single incubation at 72°C for 5 min. PCR products were separated by electrophoresis on 1% agarose gels. Similarly, the amounts of the transcripts for Cosmc {31} and core1 synthase were semi-quantitatively estimated by PCR. Twenty seven cycles of PCR reaction was done using 5-cactgtgacaaagcaga-3 and 5-ggttggggtgataagtca-3 primer for Cosmc and 29 cycles of PCR reaction usingby guest, on October 9, 2010 Downloaded from45-gtgggactgaaaaccaa-3 and 5-agatcagagcagcaacca-3 primers for core1 synthase. Expression levels were normalized by GAPDH expression . O-glycan structure analysis- PC3-, LNCaP-mock and PC3-, LNCaP-core3 cells were suspended in 0.1 M NH 4HCO 3, boiled for 10 min, and lyophilized. Dried samples were delipidated by chloroform-methanol (2:1 by volume) and then extracted by standard 6M-guanidine chloride protocol followed by reduction and alkylation with dithiothreitol/iodoacetic acid. After dialysis, the samples were digested with trypsin/chymotrypsin (Sigma) and then with N -glycanase F (Roche), and passed through a C18 Sep-Pak cartridge (Waters). De-N -glycosylated peptides were eluted stepwise from the C18 cartridge by 20-40 % 1-propanol in 5% acetic acid, and then treated with 0.05 M NaOH/1 M NaBH 4 at 37 °C for 3 days to release O -glycans. The samples were neutralized by acetic acid on ice until it stopped bubbling, followed by passing through Dowex 50x8 column in 5% acetic acid and dried. Borates were then removed by repeated co-evaporation with 10% acetic acid in methanol under a stream of nitrogen. An aliquot of the released and desalted O -glycans were permethylated and analyzed by MALDI-MS and MS/MS on a 4700 Proteomics Analyzer (Applied Biosystem, Farmington, MA), as described previously {32,33}. Immunoprecipitation and Western blot analysis - cells were solubilized in lysis buffer composed of 20mM Tris-HCl, pH 7.4, 150 mM NaCl, 5mM EDTA, 1% (w/v) Nonidet P-40, 5 mM sodium pyrophosphate, 10mM NaF, 1 mM sodium othovanadate, 10 mM β-glycerophophate, 1 mM phenylmethylsulfonyl fluoride and a protease inhibitor cocktail (Sigma). Equal amounts of cell lysates were separated by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes. Membranes were incubated separately with polyclonal anti-β1 integrin antibody (Ab1952, Chemicon) {34}, rabbit anti-α2 integrin antibody (Ab1936, Chemicon), rabbitanti-ERK antibody (Cell Signaling), mouse anti-FAK antibody (BD Biosciences), and anti-FAK [pY397] phospho-specific antibody (44-625G, BD Biosciences), and then incubated with HPR-conjugated goat anti-mouse IgG or HRP-conjugated goat anti-rabbit IgG. In parallel, aliquots of the lysate were treated with N -glycanase (Calbiochem) as described {33} before separation on SDS-gel electrophoresis. Alternatively, cell lysates were incubated with polyclonal anti-β1 integrin antibody followed by protein A-agarose. Immunoprecipitates were dissociated from protein A-agarose by boiling 5 min in sample buffer containing 1% SDS before electrophoresis. Solubilized proteins were separated on gels and blotted to a PVDF membrane. The blot was incubated with anti-α2-integrin antibody followed by HRP-conjugated anti-rabbit IgG. ECL reagents (Amersham Biosciences) were used to detect signals. The membrane was then stripped by incubation with 1 N NaOH for 1-2 min followed by washing three times with 10 mM Tris-HCl buffer, pH7.4 containing 0.14 M NaCl and 0.05% Tween 20 (TBS-T), and blocking with TBS-T containing 5% skim milk again. This membrane was then reacted with anti-α2 integrin antibody. The membrane was also reacted with biotin-conjugated GS-II and visualized usinga Vectastain ABC kit (Vector Laboratories Inc., Burlingame, CA) and, in parallel, with mouse monoclonal anti-β1 integrin antibody (610467, BD Biosciences) followed by HRP-conjugated anti-mouse IgG.Flow cytometry analysis- cells in semi - confluent conditions were detached from 10-cm culture dishes using enzyme free cell dissociation solution (Chemicon) and resuspended in 50 µl of PBS. The suspended by guest, on October 9, 2010 Downloaded from5cells (5–10 x 106 cells) were incubated with and without a primary antibody (rabbit anti-α1integrin polyclonal antibody(Chemicon), rabbit anti-human integrin α2polyclonal (Chemicon), and rat anti-human monoclonal antibody for α6 integrin (BD (Pharmingen) at a final concentration of 4 µg/ml for 1 h on ice. The cells were washed three times with PBS, then resuspended in PBS containing fluorescein isothiocyanate-conjugated secondary antibody,and further incubated for 1 h on ice. After washing three times with PBS, flow cytometry analyses were performed using a FACScan instrument (BD Biosciences) operated with CELLQuest software. Migration and invasion assay - cell migration was assayed using the 3 µm pore size of Transwell ® Permeable Supports (Corning). The bottom part of the transwell membrane was coated with Human laminin mixture (Chemicon), Rat laminin-5 (Chemicon), collagen I, or fibronectin (Sigma) with the concentration of 0.5 µg/ml in PBS at 4°C overnight. 5 x 104cells were added to the upper chamber, and 6 h later at 37°C on CO 2 incubator, cells reaching the bottom layer were stained with 0.5 % crystal violet and counted under a microscope. Cell invasion was assayed using an ECM Invasion Chamber (Chemicon) in which the upper layer of the transmembrane was coated with Matrigel. 2.8x105 cells for PC3 and 5 x105 for LNCaP were loaded in the upper chamber. After 24 h, cells reaching the bottom layer were visualized by 0.5 % crystal violet and counted. To determine the contribution of different integrins to invasion, cells were pre-incubated with 10 µg/ml of mouse anti-human integrin α1 I domain monoclonal antibody (Chemicon), mouse anti-human α2-integrin monoclonal antibody (Chemicon),rat anti-human monoclonal antibody GoH3 for α6 blocking (BD Pharmingen), and mousemonoclonal 4B4 anti-β1 integrin neutralizing antibody {34} (Beckman Coulter). In parallel, cells were incubated with control mouse IgG (10 µg/ml).To exclude the possibility that clonal varian contributes to the difference in cell migration, the parent PC3 cells and LNCaPcells were transiently transfected with core3synthase or empty vector. Three days after the transfection, cell migration was measured in the same way as described above.Orthotopic tumor cell inoculation- balb/c nude (nude/nude) mice (6- to 8-week old males) obtained from Tacomic were used for orthotopic tumor cell injection {35}. Mice were anesthetized with Avertin ® and laparotomy was performed; 2x106 of PC3-core3 and mock-transfected PC3 cells were suspended in 20 µl of serum-freeRPMI-1640 medium and inoculated into the posterior lobe of the prostate. The wound was then closed with surgical clips. Eight weeks later, mice were sacrificed, prostates and surrounding lymph nodes were removed, and organs were weighted. Specimens were preserved by fixation in neutral-buffered formalin.RESULTS Core3 synthase-expressing prostate cancer cell lines exhibit abnormal lamellipodia. Previously, it was shown that core3 synthase is downregulated in colonic carcinoma cells relative to normal tissues {14}. Expression of core3 synthase in a human fibrosarcoma cell line also resulted in decreased lung tumor foci formation compared to mock-transfected cells {16}. However, these studies did not address the mechanisms how core3 synthase expression results in decreased tumor formation and decreased tumor metastasis. To address these questions, we analyzed human PC3 andLNCaP prostate cancer cell lines, since these cells metastasize to the lymph nodes.RT-PCR analysis of mRNAs encoding different glycosyltransferases showed that both PC3 and LNCaP cells express a by guest, on October 9, 2010 Downloaded from6significant amount of C2GnT-1 but only negligible or small amounts of core3 synthase (β3GnT-6). PC3 cells express only a small amount of C2GnT-2, which is much less than that expressed in gastric cancer AGS cells (Fig. 2A).Individual vectors harboring core3 synthase (β3GnT-6) cDNA {15} and the neomycin (geneticin) resistance gene were co-transfected into PC3 and LNCaP cells, and cells were selected in geneticin. Transfected cells were subjected to Arthrobacterureafaciens sialidase treatment and peanutagglutinin (PNA) staining followed by flow cytometry analysis. PNA binds to core1 O -glycan, Gal β1→3GalNAc α1→Thr/Ser,which can be formed by desialylation of sialylated core1 O -glycan. As shown in Fig. 3A, core3 synthase-transfected PC3 cell lines showed weaker PNA staining than did mock-transfected cells, since some of Gal β1→3GalNAc must be replaced byGlcNAc β1→3GalNAc in the core3synthase-transfected cells. Similarly, LNCaP cells (clone 2) exhibited less PNA staining after transfecting with core3 synthase, though their level of PNA staining was much higher than those of PC3 cells for both mock-transfected and core3synthase-transfected LNCaP cells. These cell lines transfected with core3 synthase weredesignated PC3-core3 and LNCaP-core3, respectively. Hereafter, we studied clones 2 of PC3-core3 and LNCaP-core3 cells.RT-PCR analysis of PC3-core3 and LNCaP-core3 mRNAs showed that both cell lines expressed high levels of core3 synthase (Fig. 2B). By contrast, the expression level of Core1 synthase and Cosmc {31} was not changed after Core3 synthase transfection (Fig. 2C and D). We also noted that transfected LNCaP and PC3 cells differed morphologically from controls in that they showed abnormal lamellipodia (arrows in Fig.3B). Indeed, F-actin visualized by phalloidinwas decreased in core3 synthase-expressing PC3 cells compared to mock-transfected PC3 cells (Fig. 3C). By mass spectrometry analyses we confirmed the acquisition of core3 O -glycans after transfection with core3 synthase. As shown in Fig 4A, core3-containing O -glycans (ions at m/z 779.3,1140.5) were seen only in core3-transfected PC3 cells and absent in mock-transfected PC3cells. The deduced structures for these molecular ion signals were further confirmed by MS/MS analyses (Fig. 4B). LNCaP-core3 cells displayed much more core3 O -glycansthan mock-transfected LNCaP, and much more than PC3-core3 cells (Fig. 4B). The latter finding is consistent with the difference in PNA labeling between PC3 and LNCaPcells as seen in Fig. 3A. The amount of core3 O -glycans in PC3-core3 cells is almost equivalent to that in colon cells of wild-type mice {36} while that of LNCaP-core3 cells apparently represents an overexpressed level of core3 O-glycans.PC3 and LNCaP cells expressing core3 O-glycans exhibit reduced migration and invasion. To determine how the expression of core3 synthase affects cell migration, we utilized transwell migration assay. For this assay, the bottom side of the transwellmembrane was coated with extracellular matrix components. Cells were then loadedon the upper side of the chamber and migration to the bottom part of the membrane was determined 6 or 24 hrs later. Migration ofPC3-core3 cells was much less efficient than that mock-transfected PC3 cells (Fig. 5A).This reduction was observed on collagen-, fibronectin-, a mixture of laminin, or laminin-5-coated membranes (Fig. 5A, lower panels). Similar results were obtained whenLNCaP-core3 cells were tested (Fig.5B).These results indicate that forced expression of core3 results in reduced migration on various components of the extracellularmatrix. To determine how expression of core3 O -glycans influences invasion by prostate cancer cells, mock and core 3 transfected PC3and LNCap cells were seeded on the transwell by guest, on October 9, 2010 Downloaded from7membrane coated with Matrigel in the upper chamber of a Boyden invasion chamber, and cells reaching the bottom layer were counted 24 h later. First, we found that PC3- and LNCaP-core3 cells invade much less than mock-transfected cells. (Fig. 6A and B) Since the integrin family is well known heteromeric receptor for extracellular matrix reported to have biological functions in protection against apoptosis {37}, and malignant transformation {38, 39}, we used functional blocking antibodies for several integrins to determine the major target integrin for core3 synthase. Invasiveness of PC3 cells was completely inhibited by treatment with anti-β1 integrin-blocking 4B4 antibody {34}. Additionally, significant decrease of invasion was shown when treated with α2 blocking antibody compared with α1or α6 blocking antibody (Fig. 6C). These results show that decreased invasion by PC3-core3 cells is likely due to decreases in α2β1-integrin-mediated adhesion andmigration.To expand the above studies further, PC3 cells were transiently transfected withCore3 synthase and cell migration was measured. The results showed clearly that PC3 cells migrated much slower after transfection with core3 synthase, compared to mock-transfected PC3 cells (Fig. 7A). Almost identical results were obtained on LNCaP cells (Fig. 7B). In addition, PC3-core3 and LNCaP-core3 cells shared all characteristics although two cell lines were independently obtained. These results indicate that the results obtained after core3 synthase transfection is not due to clonal variation of the transfected cells, and that core3 synthase is responsible for decreased migration and invasion, and impaired tumor formation Forced expression of core3 synthase decreases prostate cancer formation and lymph node metastasis. To determine how core3 expression influences tumor formation, PC3-mock and PC3-core3 cells were orthotopically inoculated into the prostate of nude mice as described {35}. The prostate and the surrounding lymph nodes were isolated 8 weeks later and analyzed for tumor formation. Mice receiving mock-transfected cells showed larger prostate tumors, and the surrounding lymph nodes contained metastatic tumors (Fig. 8A). By contrast, mice inoculated with PC3-core3 cells showed much smaller prostate tumors, and the tumor metastasis to the surrounding lymph nodes was not noticed. These results indicate that core3 synthase expression results in reduction of both primary tumor and metastasis to the lymph node. Similar results were obtained on subcutaneously inoculated LNCaP-Core3 cells. Compared to robust subcutaneoustumor formation by mock-transfected LNCaP cells, LNCaP-core3 cells barely formed subcutaneous tumor (Fig. 8B). These results demonstrate that expression of core3O-glycans suppress tumor formation and metastasis.Maturation and cell surface expression of β1 integrin is attenuated in PC3-core3 cells. Among different integrin subunits, we examined glycosylation status of β1 integrin, since this protein has been shown to havemucin-typeO -glycans {40}. The N -acetylglucosaminyl terminus of core3 O -glycan, GlcNAc β1→3GalNAc α1→Thr/Ser, can be detected by Griffonia simplifolia lectin II (GS-II) {41}. Western blot analysis of β1-integrin immunoprecipitated from PC3- and LNCaP-core3 cells showed a strong band detected by GS-II. By contrast, β1 integrin from mock-transfected cells did not react with GS-II, although β1-integrin levels did not differ between the two cell types (Fig. 9A). α2 integrin of LNCaP cells but not PC3 cells apparently acquired core3 O -glycan while α2integrin from PC3 cells barely acquired it. In support of this finding, PNA-binding to β1 integrin was slightly decreased in PC3-core3 cells compared to the mock-transfected PC3 by guest, on October 9, 2010 Downloaded fromcells. By contrast, PNA binding to β1 integrin and α2 integrin was significantly decreased in LNCaP-core3 cells. (Fig. 9B). The results confirmed that β1 integrin from PC3-core3 cells and β1 and α2 integrin from LNCaP-core3 cells express core3 O-glycans. The results are consistent with the conclusions obtained by mass spectrometric analysis (Fig.4). It is possible that Arthrobacter ureafaciens sialidase treatment did not efficiently remove sialic acid from core1 O-glycans of PC3 cells yielding weak signals by PNA staining (Figs. 3A), while the same treatment efficiently removed sialic acid from LNCaP cells.The immunoblotting using anti-β1 integrin antibody detected two forms of β1 integrin, and core3 expressing PC3 cells expressed lower levels of β1 integrin with the higher molecular weight (arrowhead in Fig. 9C) than did the mock transfected cells, while the amount of α2 integrin was equivalent in both cell types (Fig. 9C). It was reported that the higher molecular weight form represents the mature form of β1 integrin {42}. Two differently glycosylated forms of β1 integrin resolved to one lower molecular weight band after N-glycanase treatment (Fig. 9C).We then examined the cell surface expression of several integrin subunits. Interestingly, surface expression of β1 integrin is significantly reduced without changing that of α2 integrin expression in PC3-core3 cells (Fig. 9D). For LNCaP cells, reduced surface expression of α2 and α6 integrins as well as β1 integrin was detected. These results suggest that adding the core3 O-glycan could affect the maturation and surface expression for β1 integrin for PC3 cells and β1-, α2-integrin, and possibly for α6-integrin for LNCaP cells.Association of α2 and β1 integrin is attenuated in PC3-core3 and LNCaP-Core3 cells. Previously, it was reported that PC3 cells express primarily α2β1 integrin {23},which is consistent to our data (Fig. 9B).The assays described in Fig. 6 indicate that invasion of PC3 cells is largely dependent onα2β1-integrin. Since the above results suggestthat α2β1 integrin may not function well inPC3-core3 and LNCaP-core3 cells, we analyzed the amount of β1 integrin complexed with α2 integrin in those cells and compared them with mock-transfected cells.We thus immunoprecipitated β1 integrin fromtwo cell types using rabbit anti-β1 integrinand reacted immunoprecipitates sequentiallywith anti-α2 integrin and anti-β1 integrin antibodies. As a complimentary experiment,α2 integrin was immunoprecipitated followedby blotting with anti-β1 antibody andα2-antibody. Levels of α2-integrinco-immunoprecipitated with β1 integrin were significantly decreased in PC3 cells expressing core3 O-glycans compared tomock-transfected PC3 cells (Fig. 10A).Almost identical results were obtained forLNCaP cells (Fig. 10B). These results suggest that expression of core3 O-glycans inα2β1 integrin led to decreased heterodimerization. In this experiment, β1integrin was immunoprecipitated by rabbitanti-β1 integrin antibody, and then immunoblotted with the monoclonal anti-β1integrin antibody or anti-α2 integrin antibody. Consistent with the previous report {42}, the polyclonal anti-β1 antibody can detect twoforms of β1 integrin by immunoblotting (Fig.9C), while the same antibody and the monoclonal antibody immunoprecipitatesmostly a major β1 integrin with lower molecular weight (Fig. 10) as shown previously {43}.Reduction in integrin-mediated activation of PC3-core3 cells. The aboveresults suggested that PC3 and LNCaP cells expressing core3 synthase express lowerlevels of functional α2β1 integrin than domock transfected PC3 cells. To support thisby guest, on October 9, Downloaded from8。