Aberrant O-glycosylation inhibits stable expression of dysadherin, a carcinoma-associated antigen, and facilitates cell–cell adhesion

Hitomi Tsuiji2, Seiichi Takasaki3, Michiie Sakamoto2, Tatsuro Irimura4 and Setsuo Hirohashi1,2

2 Pathology Division, National Cancer Center Research Institute, Chuo-ku, Tokyo 104-0045, Japan
3 Department of Biochemistry, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan
4 Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan

Received on December 2, 2002; revised on March 3, 2003; accepted on March 5, 2003


    Abstract
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 Abstract
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 Results
 Discussion
 Materials and methods
 References
 
Recently, we identified dysadherin, a novel carcinoma-associated glycoprotein, and showed that overexpression of dysadherin in human hepatocarcinoma PLC/PRF/5 cells could suppress E-cadherin-mediated cell–cell adhesion and promote tumor metastasis. The present study shows evidence that dysadherin is actually O-glycosylated. This was based on a direct carbohydrate composition analysis of a chimera protein of an extracellular domain of dysadherin fused to an Fc fragment of immunoglobulin. To assess the importance of O-glycosylation in dysadherin function, dysadherin-transfected hepatocarcinoma cells were cultured in a medium containing benzyl-{alpha}-GalNAc, a modulator of O-glycosylation. This treatment facilitated homotypic cell adhesion among dysadherin transfectants accompanied with morphological changes, indicating that the anti-adhesive effect of dysadherin was weakened. Modification of O-glycan synthesis also resulted in down-regulation of dysadherin expression and up-regulation of E-cadherin expression in dysadherin transfectants but did not affect E-cadherin expression in mock transfectants. Structural analysis of O-glycans released from the dysadherin chimera proteins indicated that a series of O-glycans with core 1 and 2 structures are attached to dysadherin, and their sialylation is remarkably inhibited by benzyl-{alpha}-GalNAc treatment. However, sialidase treatment of the cells did not affect calcium-dependent cell aggregation, which excluded the possibility that sialic acid itself is directly involved in cell–cell adhesion. We suggest that aberrant O-glycosylation in carcinoma cells inhibits stable expression of dysadherin and leads to the up-regulation of E-cadherin expression by an unknown mechanism, resulting in increased cell–cell adhesion. The carbohydrate-directed approach to the regulation of dysadherin expression might be a new strategy for cancer therapy.

Key words: benzyl-{alpha}-GalNAc / cell–cell adhesion / dysadherin / E-cadherin / O-glycosylation


    Introduction
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The cadherin-catenin cell–cell adhesion system is well known to be involved in animal morphogenesis and the maintenance of normal adhesion between cells and to play a crucial role in normal development as well as tumor development. Loss of E-cadherin function in tumors results in progression of relatively benign tumors to invasive, metastatic carcinomas. Inactivation of E-cadherin-mediated cell–cell adhesion in tumors has been reported to be caused by its genetic alteration, promoter methylation, and tyrosine phosphorylation of ß-catein that is associated with cytoplasmic domain of E-cadherin (Batlle et al., 2000Go; Grady et al., 2000Go; Graff et al., 2000Go; Hirohashi, 1998Go).

Dysadherin is a molecule that we identified as a tumor-associated antigen in many cancer tissues (Ino et al., 2002Go; Shimamura et al., 2003Go). The transfection of this molecule causes E-cadherin down-regulation and disturbs homotypic cell adhesion in human hepatocellular carcinoma cell line PLC/PRF/5 (Ino et al., 2002Go). The amino acid sequence of dysadherin predicted from cDNA sequence indicated that it is a type 1 transmembrane protein with features typical of membrane-associated mucins, including serine-, threonine-, and proline-rich ectodomains. There was no tripeptide sequence, Asn-X-Ser/Thr, known as a potential N-glycosylation site in the predicted amino acid sequence of dysadherin. Thus it has been suggested that dysadherin expresses only O-glycans.

Some mucin-associated molecules have been reported to show anti-adhesive effects by steric hindrance owing to their size and/or by charge repulsion due to their negatively charged sialylated glycans. Episialin, also known as MUC1, is a glycoprotein shown in vitro to reduce E- cadherin-mediated cell–cell adhesion by steric hindrance (Wesseling et al., 1996Go). Using full-length or shortened episialin transfectants, it has been clearly shown that length of the episialin molecule is mainly responsible for its anti-adhesive effect. Sialidase treatment only partially restored the aggregation capacity of the transfectants (Ligtenberg et al., 1992Go; Wesseling et al., 1995Go). Podocalyxin is another membrane-associated mucin that has an anti-adhesion function (Takeda et al., 2000Go). In this case, a negative charge from sialic acid is primarily responsible for its anti-adhesive effect. Similarly, epiglycanin, which is highly expressed on the TA3/Ha mouse mammary tumor cell line, inhibits E-cadherin and integrin-mediated adhesion (Kemperman et al., 1994Go).

In this article, we show direct evidence of O-glycosylation of dysadherin and propose the structures of O-glycans. Then we evaluate the importance of glycosylation in dysadherin function by examining biological and biochemical changes resulting from treatment of dysadherin overexpressing hepatocellular cells with benzyl-{alpha}-GalNAc, a modifier of O-glycosylation.


    Results
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Carbohydrate composition analysis of dysadherin-Ig
The previous nucleotide analysis and apparent molecular weight suggested that a Ser-, Thr-, and Pro-rich extracellular domain of dysadherin is O-glycosylated. In this work, carbohydrate composition analysis of dysadherin-Ig was first performed to show direct evidence of its glycosylation. As expected, the chimera protein contained N-acetylgalactosamine (GalNAc) residues that are usually linked to Ser/Thr residues of proteins, suggesting that dysadherin is actually O-glycosylated (Table I). In addition, Gal and N-acetylglucosamine (GlcNAc) residues were detected as major constituents. Small amounts of mannose and fucose might be due to the presence of N-glycan at Fc fragment of Ig in the chimera protein because potential N-glycosylation sites (Asn-X-Ser/Thr) are not included in dysadherin. Carbohydrates of the protein account for approximately 40% (w/w).


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Table I. Carbohydrate composition analysis of dysadherin-Ig

 
Benzyl-{alpha}-GalNAc treatment results in enhance of Ca2+-dependent cell aggregation and induction of morphological change
To modify the O-glycosylation of dysadherin, mock- or dysadherin-transfected hepatocellular carcinoma cells were cultured for 72 h in a medium containing 4 mM benzyl-{alpha}-GalNAc or benzyl-{alpha}-GlcNAc as negative control from the next day of seeding on. The drug has been shown to change O-glycosylation of molecules in many cell lines (Huet et al., 1995Go, 1998Go; Kuan et al., 1989Go; Nakano et al., 1996Go; Ulloa et al., 2000Go). No effect on cell viability was observed as assessed by trypan blue exclusion. Cell aggregation capacity of dysadherin transfectant was significantly (P<0.01) lower than mock transfectant, as indicated previously (Ino et al., 2002Go). As shown in Figure 1A, this anti-adhesive effect was reduced after benzyl-{alpha}-GalNAc treatment in dysadherin-transfected cells (P<0.01).



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Fig. 1. Calcium-dependent cell aggregation capacity and morphology of cells treated or untreated with benzyl-{alpha}-GalNAc. Mock transfectants AV1 or dysadherin transfectants AL3-7 were cultured in a medium containing 4 mM benzyl-{alpha}-GalNAc or benzyl-{alpha}-GlcNAc (negative control for benzyl-{alpha}-GalNAc) for 72 h. (A) Cell aggregation assay was performed as described in Materials and methods. Aggregation after incubating 30 min with gyratory shaking at 100 rpm was presented. *, P<0.01. (B) Cell morphology was observed and photographed (original magnification 200x). Mock transfectant AV1 (a, b) or dysadherin transfectant AL3-7 (c, d) were cultured in a medium containing 4 mM benzyl-{alpha}-GalNAc (b, d) or benzyl-{alpha}-GlcNAc (a, c) for 72 h.

 
Our previous report also showed that dysadherin overexpression changed morphology of PLC/PRF/5 cells and that dysadherin-overexpressed cells had distinct cell–cell borders on phase-contrast micrographs and intercellular spaces filled with numerous microvilli observed by electron microscopy (Ino et al., 2002Go). As shown in Figure 1B, the morphology of the dysadherin transfectant after benzyl-{alpha}-GalNAc treatment was similar to that of mock transfectant, suggesting the cell–cell adhesion had been recovered.

Down-regulation of dysadherin and up-regulation of E-cadherin after benzyl-{alpha}-GalNAc treatment
It has been shown that E-cadherin was markedly decreased at protein level but not at mRNA level in dysadherin transfectants. After benzyl-{alpha}-GalNAc treatment, expression of dysadherin was significantly reduced (Figure 2A). Western blot analysis showed a slight increase of apparent molecular weight of dysadherin. Furthermore, E-cadherin expression, both in Triton-X 100–soluble fraction and Triton-X 100–insoluble fraction, was up-regulated. On the other hand, benzyl-{alpha}-GalNAc treatment did not affect E-cadherin expression in mock transfectants. To assay for a dose-dependent effect of benzyl-{alpha}-GalNAc, cells were treated with different concentrations of the drug. Down-regulation of dysadherin and up-regulation of E-cadherin were dependent on the concentration of benzyl-{alpha}-GalNAc (Figure 2B). These results suggest that there is an inverse relation between expressions of dysadherin and E-cadherin. Moreover, down-regulation of dysadherin expression caused by benzyl-{alpha}-GalNAc treatment was also observed in other human cell lines (AsPC-1; pancreas carcinoma, MKN28; stomach carcinoma, TE6; small cell lung carcinoma) (data not shown). These observations indicate that O-glycosylation is important for stable expression of dysadherin.



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Fig. 2. Western blot analysis of dysadherin and E-cadherin after benzyl-{alpha}-GalNAc treatments. (A) Mock transfectants (AV1 and AV11) and dysadherin transfectants (AL3-1, AL3-6, and AL3-7) were treated for 72 h in culture with 4 mM benzyl-{alpha}-GalNAc or benzyl-{alpha}-GlcNAc (negative control for benzyl-{alpha}-GalNAc). Equal amounts of protein in Triton X-100–soluble fraction (S; 20 µg) and–insoluble fraction (IS; 10 µg) were loaded into each lane. (B) Dysadherin transfectant AL3-7 cells were treated with different concentrations of benzyl-{alpha}-GalNAc before cells were lysed.

 
Analysis of O-glycans in dysadherin-Ig
To understand how O-glycosylation was affected by the benzyl-{alpha}-GalNAc treatment, composition analysis of neutral and amino sugars included in the benzyl-{alpha}- GalNAc-treated dysadherin-Ig sample was first performed, but the result was quite similar to that of the control dysadherin-Ig (data not shown). Next, O-glycans were released from the dysadherin-Ig samples by ß-elimination in the presence of sodium borohydride and analyzed by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). As shown in Figure 3A, the O-glycans from dysadherin-Ig produced in the absence of benzyl-{alpha}-GalNAc were shown to consist of multiple components. By comparing their elution positions with those of standard O-glycans, some of them were proposed to have structures as shown in Table II. Their identities were further confirmed by coinjection of the samples with standard glycans and analysis in the manner described previously: composition analysis, glycosidase digestion, desialylation with mild acid, and periodate oxidation (Kotani and Takasaki, 1997Go; Kotani et al., 1999Go). Briefly, the number of sialic acid was determined by partial desialylation; completely desialylated products of peaks 2, 4, 5, and 7 were eluted at the position of peak 2, and those of peaks 3 and 9 were at the position of peak 1. Peaks 1 and 2 were identified to core 1 disaccharide and core 2 tetrasaccharide by composition analysis, periodate oxidation, and glycosidase digestion. The result suggests that dysadherin-Ig is composed of a series of nonsialylated, monosialylated, and disialylated O-glycans with the core 2 structure, Galß1->3(Galß1->4GlcNAcß1->6)GalNAc-ol (peaks 2, 4, 5, and 7 in Figure 3A), and the core 1 structure, Galß1->3GalNAc-ol (peaks 1, 3. and 9 in Figure 3A). Unidentified glycans (peaks 6 and 8) were also included, but their structures could not be analyzed in detail.



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Fig. 3. HPAEC of O-glycans obtained from dysadherin fused with Fc fragment of IgG. O-glycans were released from purified dysadherin-Ig by ß-elimination and analyzed by HPAEC-PAD as described in Materials and methods. (A and B) Dysadherin-Ig preparations produced in the absence or presence of benzyl-{alpha}-GalNAc, respectively.

 

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Table II. Proposed structures of O-glycans released from dysadherin-Ig produced by PLC/PRF/5 cells in the presence and absence of benzyl-{alpha}-GalNAc

 
On the other hand, O-glycans released from dysadherin-Ig produced in the presence of benzyl-{alpha}-GalNAc were quite differently eluted from the column (Figure 3B). A remarkable change was that most glycans were not sialylated. Instead, two neutral glycans, peaks 1 and 2, corresponding to the nonsialylated core 1 disaccharide and the core 2 tetrasaccharide, were detected. In addition, peak 1 gave a symmetrical peak coresponding to Galß1->3GalNAc-ol by HPAEC-PAD with a lower alkaline concentration (22 mM NaOH), in which a clear peak separation of GalNAc-ol and Galß1->3GalNAc-ol can be achieved, suggesting the absence of substantial amounts of Tn antigen. Thus the result indicates that sialylation was almost completely inhibited by the benzyl-{alpha}-GalNAc treatment.

Sialidase treatment did not affect cell aggregation
To investigate whether the altered sialylation per se causes the decrease in cell aggregation, the mock and dysadherin transfectants were treated with sialidase for 30 min before cell aggregation assay. As shown in Figure 4, no significant effect on either cell was observed (P>0.2). Apparent molecular weight of membrane-associated dysadherin in western blot analysis of whole cell lysate was slightly increased as in the case of dysadherin-Ig (data not shown), suggesting that sialidase treatment was effective.



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Fig. 4. Calcium-dependent aggregation index of cells treated with sialidase. Single-cell suspensions of mock (AV1) and dysadherin (AL3-7) transfectants were prepared in accordance with an E-cadherin-saving procedure and incubated with 0.1 U/ml of sialidase in HCMF/BSA at 37°C for 30 min. Cell aggregation assay was performed as described in Materials and methods. Aggregation after incubating 30 min with gyratory shaking at 100 rpm was presented.

 

    Discussion
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The results reported herein indicate that dysadherin-Ig produced by hepatocellular carcinoma PLC/PRF/5 cells is heavily glycosylated and composed of a series of O-glycans having the core 2 structure, Galß1->3(Galß1-> 4GlcNAcß1->6)GalNAc-ol, and the core 1 structure, Galß1->3GalNAc-ol, as backbones. Large parts of them are sialylated. We do not know exactly whether O-glycans of membrane-associated dysadherin are the same as those of soluble dysadherin-Ig chimera protein. However, lectin blot analysis after sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of immunoprecipitates of dysadherin shows that the membrane-associated dysadherin carries T and sialyl T antigens (data not shown).

To assess the importance of O-glycans expressed on the cell surface dysadherin, dysadherin transfectant of PLC/PRF/5 cells was treated with benzyl-{alpha}-GalNAc, which is known to modify O-glycosylation. The anti-adhesive effect of dysadherin, which was shown by the previous observation that dysadherin transfectants were less aggregated than mock transfectants (Ino et al., 2002Go), was significantly reduced after treatment. Benzyl-{alpha}-GalNAc treatment of dysadherin transfectants also resulted in a remarkably decreased expression of cellular dysadherin with a concomitant increase of E-cadherin expression. As expression of E-cadherin in mock transfectant was not affected by benzyl-{alpha}-GalNAc treatment, the theory that benzyl-{alpha}-GalNAc directly affects E-cadherin expression is excluded.

Considering these results and the previous observation that dysadherin transfection resulted in decreased protein expression of E-cadherin without affecting its expression on the mRNA level, it is likely that benzyl-{alpha}-GalNAc treatment primarily inhibits dysadherin expression and then induces E-cadherin expression by unknown mechanisms. Presently we do not know how O-glycans regulate expression of dysadherin. However, it will be important to consider the possible role of O-glycosylation in metabolic fate or stability of dysadherin as suggested in the cases of other glycoprotein receptors. For example, it has been shown that inhibition of O-glycosylation of very-low- density lipoprotein receptors results in a rapid cleavage from the cell and release of a large amino-terminal fragment into the culture medium (Magrane et al., 1999Go). Similarly, in the absence of O-glycosylation, the decay-accelerating factor is proteolytically cleaved soon after reaching the cell surface with release of its large fragment into the culture medium, resulting in low expression of this molecule on the cell surface (Reddy et al., 1989Go).

Benzyl-{alpha}-GalNAc was initially reported to selectively inhibit O-glycosylation through its ability to compete with GalNAc-O-Ser/Thr for the ß1,3-galactosyltransferase involved in the biosynthesis of O-glycan (Kuan et al., 1989Go). However, in mucin-secreting HT-29 cells, it was shown that the major step inhibited by treatment with this sugar analog is sialylation rather than the transfer of Gal to GalNAc-{alpha}-O-Ser/Thr (Hennebicq-Reig et al., 1998Go; Huet et al., 1998Go, 1995Go). In this article, we also indicate that sialylation of O-glycan attached to dysadherin produced in PLC/PRF/5 cells is almost completely inhibited by benzyl-{alpha}-GalNAc treatment. It is suggested that sialylation of O-glycans is involved in stable expression of dysadherin. However, the direct contribution of sialic acid to the anti-adhesive function of dysadherin expressed on the cell surface is not likely because sialidase digestion of dysadherin transfectant does not significantly change cell–cell adhesion.

There are several mucins affecting cell adhesion of cancer cells. Epiglycanin, which is highly expressed on the TA3/Ha mouse mammary tumor cell line, inhibits E-cadherin and integrin-mediated adhesion (Kemperman et al., 1994Go). MUC1 mucin (episialin) is expressed in a wide variety of tumors, and its anti-adhesion ability and important role in tumor development have been shown in various cells. A normal mammary epithelial cell line and a melanoma cell line do not aggregate efficiently after transfection of cDNA encoding MUC1 (Ligtenberg et al., 1992Go). MUC1 transfectants of human gastric cancer cells (Suwa et al., 1998Go) and pancreatic cancer cells (Satoh et al., 2000Go) exhibit enhanced in vitro invasion, increased motility, and decreased binding to extracellular matrix components. Adhesive properties of the transfected cells are abolished by benzyl-{alpha}-GalNAc treatment in both cases (Satoh et al., 2000Go; Suwa et al., 1998Go), suggesting an important role of O-glycans in MUC1 function. Decreased MUC1 expression in breast cancer cell lines induced by an antisense oligonucleotide induces E-cadherin-mediated cell adhesion (Kondo et al., 1998Go). Sialomucin complex (SMC), a rat homolog of the human mucin MUC4, is a large, membrane-bound mucin complex originally isolated from highly metastatic ascites 13762 (Komatsu et al., 1997Go). Overexpression of SMC induces morphology changes, cell detachment, and cell–cell dissociation of transfected A375 human melanoma cells in culture. Intravenous injection of SMC-overexpressing cells also results in substantially greater lung metastasis than injection of SMC-repressed cells (Komatsu et al., 2000Go). As previously demonstrated with MUC1 (Wesseling et al., 1996Go), these mucins seem to sterically disrupt molecular interactions for cell adhesion. There is a relationship between expression of MUC1 and functional down-regulation of E-cadherin. However, there is no evidence showing that MUC1 regulates expression of E-cadherin molecules at protein level, and this fact contrasts with dysadherin which expression down-regulates E-cadherin expression (Ino et al., 2002Go).

In conclusion, this article reveals that dysadherin is a heavily O-glycosylated mucin-like molecule of which cellular expression is regulated depending on its glycosylation status. Aberrant O-glycosylation caused by benzyl-{alpha}- GalNAc treatment inhibits stable expression of dysadherin and leads to up-regulation of E-cadherin by an unknown mechanism, resulting in increased cell–cell adhesion. Therefore regulation of dysadherin expression might be a new strategy for cancer therapy, and the carbohydrate-directed approach is expected to aid its development.


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 Abstract
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 Results
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 Materials and methods
 References
 
Cell lines and cell culture
Dysadherin-transfected PLC/PRF/5 human hepatocellular carcinoma cells (AL3-1, AL3-6, and AL3-7) and mock transfectants (AV1 and AV11) were prepared as described elsewhere (Ino et al., 2002Go). They were maintained in Dulbecco's modified eagle's medium (Gibco, Paisley, Scotland) supplemented with 500 µg/ml G418 (Life Technologies, Gaithersburg, MD), 250 IU/ml penicillin, 100 µg/ml streptomycin, and 10% fetal bovine serum.

To prepare stable dysadherin-Ig transfectant, pCDNA3 vector containing an extracellular domain of dysadherin fused with Fc fragment of human immunoglobulin G was transfected into PLC/PRE/5 cells using LipofectAMINE (Life Technologies). Stable transfectants were isolated by growing them in G418-containing medium.

Preparation of dysadherin-Ig
Dysadherin-Ig transfectants were cultured subconfluently, and after washing three times with phosphate buffered saline (PBS), the media were changed into Opti-MEM (Life Technologies). Cells were cultured for another 3 days, and dysadherin-Ig molecules secreted into the media were collected and purified using Protein-G Sepharose column (Life Technologies) according to the manufacturer's instructions. Its purity was checked using PAGE followed by silver staining.

Analysis of carbohydrate composition and O-glycans
Dysadherin-Ig was heated in 0.5 ml 4 N trifluoroacetic acid at 100°C for 3 h for analysis of neutral and amino sugars, or in 0.01 N HCl at 100°C for 20 min for analysis of sialic acid. The hydrolysates were freed from acid by repeated evaporation with water and analyzed by HPAEC-PAD using a Bio-LC system (Dionex, Sunnyvale, CA) equipped with a CarboPac PA-1 column (4x250 mm). PAD response was monitored by a Power Chrom system (AD Instruments, Tokyo) connected to a personal computer. An isocratic elution with 22 mM NaOH was used for analysis of neutral and amino sugars (Hardy et al., 1988Go), and an elution with 150 mM sodium acetate in 100 mM NaOH was used for analysis of sialic acid. The flow rate was 1 ml/min under both conditions.

O-glycans were liberated from dysadherin-Ig by ß-elimination (Iyer and Carlson, 1971Go) and purified as previously described (Kotani and Takasaki, 1997Go). HPAEC-PAD of the O-glycans was carried out by elution with a linearly increasing concentration of 0–400 mM sodium acetate in 100 mM NaOH from 0–30 min. The flow rate was 1 ml/min.

Benzyl-{alpha}-GalNAc treatment
Benzyl-{alpha}-GalNAc and benzyl-{alpha}-GlcNAc, negative control for benzyl-GalNAc (Sigma, St. Louis, MO), were dissolved in dimethylsulfoxide to 800 mM. Cells were seeded; the following day the mediums were changed to ones containing various concentrations of the drug and cultured for 72 h.

Cell aggregation assay
The assay used was a modification of a previously described method (Shimoyama et al., 1992Go). Cells were cultured on 100-mm dishes overnight in Dulbecco's modified eagle's medium supplemented with 10% fetal bovine serum. Single-cell suspensions were prepared in accordance with an E-cadherin-saving procedure described previously (Takeichi, 1977Go). Subconfluent cell layers were rinsed twice with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered saline (HCMF; 8.0 g/L NaCl, 0.4 g/L KCl, 0.12 g/L Na2HPO412H2O, 1.0 g/L glucose, 2.3 g/L HEPES in 0.005 M NaOH, pH 7.4) containing 5 mM CaCl2 and were incubated in HCMF containing 0.01% trypsin (Trypsin type IX; Sigma) and 5 mM CaCl2 at 37°C with gyratory shaking at 100 rpm for 15 min. Reaction was immediately stopped by adding trypsin inhibitor (Sigma). After washing twice in HCMF containing 1% bovine serum albumin (BSA) (HCMF/BSA), cells were resuspended (5x105 cells/ml) in HCMF/BSA containing 40 ng/ml DNAase (Sigma) and 1 mM MgCl2 by 10 passages through a 26-gauge needle. For removal of cell surface sialic acids, a cell suspension was incubated with 0.1 U/ml of neuraminidase from Streptococcus sp. (Seikagaku, Tokyo) in HCMF/BSA at 37°C for 30 min.

The cell suspension (0.5 ml/well) was seeded in a 24-well plate previously coated with 2% BSA in HCMF and allowed to aggregate for 30 min in the presence or absence of 5 mM CaCl2 at 37°C with gyratory shaking at 100 rpm. Aggregation was quantified by counting representative aliquots from each sample on a hematocytometer using phase-contrast optics. At least 500 cells were counted from each sample. Quantification of aggregation was estimated by the following formula: aggregation index=(N0-Nt)/N0, where Nt is the total number of particles at the incubation time t, and N0 is the total number of cells. Statistical analyses were performed by Mann- Whitney U test.

Cell extraction and western blot analysis
Triton X-100 soluble and insoluble lysates were isolated in accordance with the protocol described by Hinck et al. (1994)Go with a minor modification. The subconfluent cell layer was washed three times with PBS, followed by incubation at 4°C in 50 mM Tris buffer containing 50 mM NaCl, 3 mM MgCl2, 0.5% Triton X-100, 300 mM sucrose, 1 mM phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail tablet (Boehringer Mannheim, Mannheim, Germany) with gentle shaking at 800 rpm. Cell lysates were collected and centrifuged at 15,000 rpm for 30 min, and the supernatant was prepared for Triton X–soluble faction. The remaining pellet was resuspended in SDS buffer (15 mM Tris, pH 7.3, 1% [w/v] sodium dodecyl sulfate, 5 mM ethylenediamine tetra-acetic acid). After boiling, the solution was used as Triton X–insoluble fraction. The total protein concentration of each lysate was measured by DC protein assay kit (BioRad, Hercules, CA). An equal amount of protein was separated in ready-made 4–12% gradient SDS–PAGE gel (Invitrogen, Carlsbad, CA) and electroblotted onto a polyvinylidene difluoride membrane (Immobilon, Millipore, Bedford, MA). Membranes were soaked in PBS containing 5% (w/v) nonfat dry milk followed by overnight incubation with primary antibody in PBS containing 2% (w/v) normal swine serum at 4°C. After washing three times with PBS containing 0.1% Tween20, the membrane was incubated with horseradish peroxidase–conjugated secondary antibodies (Amersham, Buckinghamshire, UK) in PBS containing 2% (w/v) normal swine serum at room temperature for 90 min. Proteins were visualized with enhanced chemiluminescent western blotting detection reagents (Amersham). NCC-M53 antibody is a mouse monoclonal antibody recognizing dysadherin (Shimamura et al., 2003Go), and its hybridoma culture supernatant was used at 1:5 dilution. Purified HECD-1 antibody (a mouse monoclonal antibody against human E-cadherin) was from Takara Shuzo (Kyoto, Japan) and was used at 1:200 dilution.


    Acknowledgements
 
This work was supported by a Grant-Aid for the Second Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labor, and Welfare of Japan. H.T. is a domestic research fellow supported by Japan Science and Technology Corporation.

1 To whom correspondence should be addressed; e-mail: shirohas{at}ncc.go.jp Back


    Abbreviations
 
BSA, bovine serum albumin; HEPES, N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid; HPAEC-PAD, high-pH anion-exchange chromatography with pulsed amperometric detection; PBS, phosphate buffered saline; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SMC, sialomucin complex


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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