2Department of Medicine, University of Liverpool, Daulby Street, Liverpool L69 3GA, UK, 3School of Biological Sciences, University of Liverpool, Daulby Street, Liverpool L69 3BX, UK, 4Imperial Cancer Research Fund, Histopathology Unit, 35-43 Lincolns Inn Fields, London WC2A 3PN, UK, and 5Imperial College School of Science Medicine and Technology, Hammersmith Campus, Ducane Road, London W12 ONN, UK
Received on December 12, 2000; revised on February 12, 2001; accepted on February 12, 2001.
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Abstract |
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Key words: CD44/oncofetal carbohydrate/peanut agglutinin/sialyl-Tn/Thomsen-Friedenreich antigen
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Introduction |
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Enhanced expression of the TF antigen (galactose ß1-3 N-acetylgalactosamine -) by hyperplastic and neoplastic colon (Campbell et al., 1995
) has been shown to allow interaction with luminal TF-binding lectins of dietary origin, such as peanut agglutinin (PNA), resulting in increased proliferation (Ryder et al., 1992
). Previous studies have shown that PNA is mitogenic to HT29 but not Caco-2 colon cancer cells in vitro (Ryder et al., 1994
) and that peanut ingestion causes increased proliferation of rectal epithelia in colonoscopically normal individuals with mucosal expression of PNA receptor (Ryder et al., 1998
). Antibodies to TF antigen, which are universally present in human sera, also increase proliferation in human colon cancer cell lines in a dose-dependent manner (Yu et al., 1997
). Despite the diverse biological effects of PNA, its cell-surface receptors have not yet been identified in colon cancer. The fact that PNA is mitogenic to HT29 but not Caco2 colonocytes in vitro (Ryder et al., 1994
) also requires explanation because both cell lines express TF antigen on their cell surface.
In this study we have analyzed the profile of PNA-binding cell-surface glycoproteins in HT29 and Caco2 colon cancer cell lines. We report that PNA binds to TF antigen expressed by high molecular weight variant isoforms of CD44 (CD44v) in colon cancer. The standard CD44 from both normal and colon cancer tissues does not express TF. Both PNA and CD44v6 antibodies stimulate proliferation in HT29 cells, and their effect is not additive, suggesting that the proliferative effect of PNA is probably mediated via interaction with cell membrane CD44v. We also demonstrate coexpression of oncofetal carbohydrate antigens, TF with sialyl-Tn on CD44v. These findings provide a link between the altered glycosylation and alternative CD44 splicing that are commonly found in colorectal cancer and precancer.
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Results |
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Discussion |
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A 250-kDa CD44 containing the product of all known splice variant exons has been shown to be the major PNA-binding glycoprotein in human epidermal keratinocytes (Hudson et al., 1995). Selective antisense suppression of CD44 in mouse keratinocytes disrupts hyaluronate metabolism in skin and impairs keratinocyte proliferation (Kaya et al., 1997
). Because the proliferation of HT29 cells but not Caco2 colon cancer cells is stimulated by PNA (Ryder et al., 1998
) this suggests the possibility that the proliferative response to PNA may be dependent on expression of CD44v which is lacking in Caco2. This is further supported by the fact that although both PNA and monoclonal antibodies to CD44v6 enhance proliferation in HT29 colon cancer cells, PNA has no additive effect in the presence of mAb to CD44v. The proliferation increase seen in response to the CD44 antibody alone is assumed to be due to receptor clustering triggering protein kinase activity in a way analogous to that seen with antibodies to epidermal growth factor receptor (Spaargaren et al., 1991
).
CD44 is a transmembrane glycoprotein encoded by a complex gene composed of 20 exons, 10 of which are always expressed to produce a heavily glycosylated 8590-kDa isoform known as standard or hematopoetic CD44 (CD44s or CD44H). The remaining exons are alternatively spliced in various combinations to generate high molecular weight variant isoforms. CD44 is a receptor for hyaluronic acid and is involved in cellcell adhesion (Koopman et al., 1990), cellmatrix adhesion (Jalkanen and Jalkanen, 1992
), lymphocyte homing (Jalkanen et al., 1987
), and lymphocyte activation (Huet et al., 1989
).
Several studies have suggested a link between CD44 splicing, altered cell surface glycosylation, and tumor cell behavior. Thus, transfection of a poorly tumorigenic rat colon cancer cell line with human H blood group antigen-forming (1-2) fucosyltransferase cDNA resulted in cell-surface expression of H antigens selectively born on CD44v6 and conferred increased mobility and tumorigenicity to the transfected cells (Goupille et al., 1997
). Studies in melanoma cell lines have shown that treatment with benzyl-alpha-N-acetylgalactosamine resulted in increased PNA binding on CD44 (Nakano et al., 1996
) and treatment with ginsenoside Rh2 (Rh2) increased PNA binding to cell-surface glycoproteins (Ota et al., 1993
); both reagents also caused an increase in experimental metastatic ability. Human melanoma cell populations enriched for PNA-binding cells also generated a higher frequency of metastases when xenografted into immune-suppressed neonatal rats (Dore et al., 1994
). Neuraminidase treatment of tumor cells (mouse sarcoma-1) alters in vivo (Balb/c-mice) the organotropic distribution of metastases; instead of being found exclusively in the lung, they are found both in lung and liver. However, preinjection and regular application of D-galactose or arabinogalactan prevents the setting of metastases in the liver (Uhlenbruck et al., 1987
). Terminal expression of galactose by CD44 variants, as shown by their PNA reactivity, might therefore contribute to organotropic distribution of metastases in colon cancer. High molecular weight CD44 variants have also been found in inflammatory bowel disease (Rosenberg et al., 1995
), a condition that is also associated with increased expression of TF (Campbell et al., 1995
) and sialyl-Tn (Karlen et al., 1998
) and with increased cancer risk (Podolsky, 1991
).
The demonstration that cell-surface TF antigen expression by colon cancer epithelial cells largely depends on the presence of high molecular weight splice variants of CD44 provides a link between the extensive evidence for disease-related alterations in CD44 splice variants and the equally extensive evidence for disease-related alteration in cell surface expression of TF antigen. Furthermore, it suggests that the nature of O-glycosylation may be determined, at least in part, by the amino-acid sequence of the protein (CD44) undergoing glycosylation.
TF antigenexpressing CD44v is also shown to bear the related oncofetal antigen sialyl-Tn. Mucin-associated sialyl-Tn (STn) antigen is a marker of poor prognosis for patients with colon and gastric cancer and of cancer risk for those with ulcerative colitis (Ma et al., 1993; Yamada et al., 1995
; Itzkowitz et al., 1995
, 1996). Furthermore, mucins bearing sialyl-Tn have been shown to mediate inhibition of natural killer cell cytotoxicity, thereby providing protection for cancer cells (Ogata et al., 1996
).
The coexpression of oncofetal carbohydrate blood group antigens on CD44 has functional consequences that seem highly likely to contribute to the malignant potential of epithelial cells thus confirming oncofetal carbohydrate antigens as appropriate targets for immunotherapy (Springer et al., 1995; MacLean et al., 1996
).
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Materials and methods |
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Cell culture
The HT29 and Caco-2 cell lines were obtained from the European Collection of Animal Cell Cultures at the Public Health Laboratory Service (Porton Down, Wiltshire, UK). The cell lines were grown in monolayer and maintained at 37°C in a humidified atmosphere of 5% CO2 95% air. The cells were maintained in Dulbeccos modified Eagles medium (DMEM) supplemented with 5% (v/v) fetal calf serum (FCS), 100 units/ml penicillin, 100 µg/ml streptomycin, 4 mM glutamine, and 50 ng/ml insulin. DNA synthesis assays were performed as described (Ryder et al., 1998). Briefly, cells were seeded at a density of 2 x 104/well into 24 well plates and 48 h later the culture medium was replaced with DMEM containing 1% (v/v) FCS alone (control) or containing either PNA (15 µg/ml), anti-CD44v6 antibody BBA13 (10 µg/ml) or PNA plus BBA13. After 24 h, 0.5 µCi [3H]-thymidine was added to each well for 3 h and the cells were prepared for scintillation counting. Statistical analysis was performed using one-way ANOVA followed by multiple comparisons of means (Newman and Keuls). Differences were considered significant when P < 0.05.
Preparation of human tissue extracts
Tissues obtained from the Cancer Tissue Bank (Department of Pathology, University of Liverpool) were taken from human colon cancers and macroscopically normal colon at least 10 cm distant from tumor, and homogenized in 50 mM Tris (pH 8) containing 150 mM NaCl, 0.02% (w/v) sodium azide, 100 µg/ml phenyl methylsulfonyl fluoride (PMSF), 100 µg/ml aprotinin, and 1% (v/v) Triton X-100. Total protein concentration was measured with the bicinchoninic acid assay (Sigma).
PNA-agarose affinity chromatography of radioiodinated cell surface proteins
Iodination of cell surface proteins in HT29 and Caco2 cells was performed by the method of Bresalier et al. (1990) using lactoperoxidase and glucose oxidase. A confluent 75-cm2 culture plate was washed three times in phosphate buffered saline (PBS), pH 7.4, and once with PBS containing 3 mM glucose. Tyrosine residues of cell surface proteins were labelled in situ by the addition of 5 U lactoperoxidase, 0.5 mCi carrier-free Na[125I], and 10 U glucose oxidase in 2 ml PBS containing 3 mM glucose. The flask was then incubated at 25°C for 30 min. Cells were then washed four times with PBS containing 1 mM unlabeled sodium iodide and 5 mM PMSF. Cells were then scraped into 4 ml PBS-PMSF and centrifuged at 2000 x g for 10 min. The cell pellet was resuspended in 1 ml PBS-PMSF, sonicated on ice using three 20-s pulses to lyse the cells, and centrifuged at 100,000 x g for 1 h. The membrane-rich cell pellet was solubilized by sonication in PBS containing 1% (v/v) Nonidet P40 (NP40) and 5 mM PMSF using five 20-s pulses, left on ice for 2 h, and then centrifuged at 100,000 x g for 1 h. The supernatant was collected and loaded onto a 2-ml PNA-agarose column, which was then washed with PBS until the absorbance of the wash-through at 280 nm returned to the baseline and the bound proteins were then eluted with PBS containing 0.2 M galactose. The eluate was then desalted on a PD-10 Sephadex GM25 column (Amersham-Pharmacia Biotech), and the void volume fraction was freeze-dried. This fraction, containing PNA-affinity-purified, radiolabeled, cell-surface glycoproteins was then subjected to SDSpolyacrylamide gradient (415%) gel electrophoresis followed by autoradiography using Hyperfilm. Of the HT29 cell preparations, 2.3% of cell-surface radiolabeled material adhered to the PNA agarose column and 0.3% was eluted with galactose; of the Caco2 cell preparations, 14.7% of cell-surface radiolabeled material adhered to the PNA agarose column and 0.9% was eluted with galactose. Protein bands were quantified after scanning using Quantity One software (Biorad, Hemel Hemstead, UK).
Metabolic labeling with [35S]-sulfate and preparation of extracts
Subconfluent cultures of HT29 and Caco-2 cells were incubated for 18 h in culture medium containing 50 µCi/ml [35S]-sulfate. The medium was removed and cells were rinsed three times with PBS. Cells were then left on ice for 15 min in 1 ml 10 mM Tris, 66 mM EDTA, 1 mM PMSF (pH 7.4) containing 1% (v/v) NP40 and 0.4% (w/v) sodium deoxycholate. The extracts were centrifuged at 16,000 x g for 15 min at 4°C and the pellets discarded.
Immunoprecipitation and western blotting
Labeled or unlabeled cell extracts were precleared with 20 µl Protein A-agarose beads to remove nonspecific binding material. The supernatants were then incubated for 3 h with pan CD44 antibody (BBA10) at 4°C on a shaker. Protein A-agarose beads were then added and the mixture incubated in an end-over-end shaker overnight at 4°C. Beads and associated immune complexes were collected by centrifugation and washed once in PBS containing 0.5% (v/v) Triton X-100, 0.1% (w/v) SDS; once in PBS containing 0.5% (v/v) Triton X-100 and 0.5 M NaCl; and twice more in the former buffer. Pellets were resuspended in SDSPAGE sample buffer (62.5 mM Tris [pH 6.8] containing 2% [w/v] SDS, 10% [v/v] glycerol, 5% [v/v] mercaptoethanol, and 0.001% [w/v] bromophenol blue) and boiled for 5 min. Immunoprecipitated polypeptides were then separated on a 7.5% or 415% gradient polyacrylamide gels. The separated polypeptides were electrotransferred to nitrocellulose membrane for 1 h at 100 V in 25 mM Tris, 192 mM glycine, and 20% (v/v) methanol. After transfer the membrane was blocked with PBS containing 1% (w/v) bovine serum albumin (BSA), 0.1% (v/v) Tween 20 for 1 h and then incubated with primary antibody, followed by peroxidase-conjugated rabbit anti-mouse IgG, each for 1 h at room temperature in PBS with 1% (w/v) BSA. Bound antibody was detected using an enhanced chemiluminescence kit (Amersham-Pharmacia Biotech).
Selective removal of TF antigen and N-glycans
Selective removal of TF antigen was achieved by treatment of PNA-agarose-purified HT29 membrane extract with S. pneumoniae endo--N-acetylgalactosaminidase (O-GlycanaseTM). Briefly, lyophilized PNA-agarose-purified HT29 membrane extract (25 µg protein) was incubated with 2 mU O-GlycanaseTM in 50 µl 100 mM citrate/phosphate buffer, pH 6, containing 100 µg/ml BSA and 0.02% (w/v) sodium azide for 18 h at 37°C. Enzymatic release of TF was confirmed using asialofetuin (Sigma) as a control TF-expressing substrate.
N-linked oligosaccharides were removed using Peptide-N-Glycosidase F (N-GlycanaseTM). PNA-agarose purified HT29 membrane extract (20 µg protein) was dissolved in 20 µl of 20 mM sodium phosphate, pH 7.5, containing 50 mM EDTA, 0.02% (w/v) sodium azide, 0.5% (w/v) SDS, 5% (v/v) ß-mercaptoethanol, and denatured by heating at 100°C for 2 min. To the denatured extract were added 10 µl 5% (v/v) NP-40, 20 µl incubation buffer and 6 µl (3 U) N-GlycanaseTM and the mixture was incubated for 18 h at 37°C.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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