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The Journal of Immunology, 2002, 168: 1690-1696.
Copyright © 2002 by The American Association of Immunologists

Obligatory Requirement of Sulfation for P-Selectin Binding to Human Salivary Gland Carcinoma Acc-M Cells and Breast Carcinoma ZR-75-30 Cells1

Yan-Qing Ma and Jian-Guo Geng2

Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Stimulated endothelial cells and activated platelets express P-selectin, which reacts with P-selectin glycoprotein ligand-1 (PSGL-1) for leukocyte rolling on the stimulated endothelial cells and heterotypic aggregation of the activated platelets on leukocytes. P-selectin also binds to several cancer cells in vitro and promotes the growth and metastasis of human colon carcinoma in vivo. The P-selectin/PSGL-1 interaction requires tyrosine sulfation. However, it is unknown whether sulfation is necessary for P-selectin binding to somatic cancer cells. In this study, we show that P-selectin mediated adhesion of Acc-M cells, a cell line derived from a human adenoid cystic carcinoma of salivary gland. These cells had a moderate expression of heparan sulfate-like proteoglycans, but had no detectable expressions of PSGL-1, CD24, Lewisx, and sialyl Lewisx. Treatment with sodium chlorate (a sulfation biosynthesis inhibitor), but not 4-methylumbelliferyl-{beta}-D-xyloside (a proteoglycan biosynthesis inhibitor) or heparinases, reduced adhesion of these cells to P-selectin. Sodium chlorate also inhibited the P-selectin precipitation of the ~160-, ~54-, and ~36-kDa molecules from the cell surface of Acc-M cells. Furthermore, P-selectin could bind to human breast carcinoma ZR-75-30 cells in a sulfation-dependent manner. Our results thus indicate that sulfation is essential for adhesion of nonblood-borne, epithelial-like human cancer cells to P-selectin.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
P-selectin (CD62P) is a member of the selectin family of cell adhesion molecules. It is a presynthesized protein stored in the Weibel-Palade bodies of endothelial cells and the {alpha}-granules of platelets. Upon inflammatory and thrombogenic challenges, it translocates from these cellular granules to the cell surfaces of endothelial cells and platelets by exocytosis in seconds. Furthermore, it can be up-regulated by de novo synthesis in the stimulated endothelial cells in hours (1, 2, 3).

P-selectin interacts with P-selectin glycoprotein ligand-1 (PSGL-1)3 (CD162), a homodimeric mucin-like protein expressed on a majority of leukocytes. PSGL-1 is now generally believed as a principal leukocyte ligand for P-selectin. The interaction of P-selectin with PSGL-1 accounts for tethering (initial attachment), rolling, and weak adhesion of leukocytes on the activated endothelial cells and for heterotypic aggregation of the activated platelets to leukocytes (2, 4, 5). Structure-function studies have illustrated that the high-affinity binding of P-selectin to PSGL-1 requires tyrosine sulfation and an O-linked glycan containing sialyl Lewisx (SLex) and/or its derivatives. Both structures are located within an anionic region close to the amino terminus of the mature, processed PSGL-1 molecule (6, 7, 8, 9, 10).

Furthermore, P-selectin has been shown to bind to several human cancers and human cancer-derived cell lines, such as colon cancer, lung cancer (including small cell lung cancer), breast cancer, malignant melanoma, gastric cancer, neuroblastoma, and tongue squamous cancer (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23). We previously reported that NKI-4 cells, a cell line derived from a human malignant melanoma, and NCI-H345 cells, a cell line derived from a human small cell lung cancer, bound to P-selectin via the novel classes of glycoprotein ligands (13, 23). In addition, we showed that the cell surface heparan sulfate-like proteoglycans mediated adhesion of human malignant melanoma A375 cells and tongue squamous cancer Tca-8113 cells to P-selectin under flow (22).

Although the P-selectin/PSGL-1 interaction is well known to require sulfation, it remains to be determined whether sulfation is required for the binding of P-selectin to somatic cancer cells. In this study, we sought to investigate this using Acc-M cells, a cell line derived from a human adenoid cystic carcinoma of salivary gland (24), and ZR-75-30 cells, a cell line derived from a human breast carcinoma. Our results suggest that sulfation is necessary not only for leukocytes but also for nonblood-borne, epithelial-like human cancer cells, such as Acc-M cells and ZR-75-30 cells, to adhere to P-selectin under the physiological shear stress.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Proteins and Abs

P-selectin receptor globulin (Rg) was prepared as before (13, 25). G1 (a leukocyte adhesion-blocking IgG1 mAb against P-selectin) and PS1 (a leukocyte adhesion-nonblocking IgG1 mAb against P-selectin) were characterized as previously described (26, 27). The F(ab')2 fragments of G1 and PS1 were prepared using ImmunoPure F(ab')2 Preparation kit (Pierce, Rockford, IL). Rabbit preimmune IgG and an affinity-purified rabbit polyclonal Ab to a peptide corresponding to residues 41–55 of the amino acid sequence of PSGL-1 were prepared as before (13, 28).

KPL1 (a leukocyte adhesion-blocking IgG1 mAb to PSGL-1) and ML5 (an IgG2a mAb to CD24) were purchased from BD PharMingen (Shanghai, China). SN3 (an IgG1 mAb against CD24) was purchased from DAKO (Shanghai, China). MMA, an IgM mAb against Lewisx (Lex), was purchased from BD Immunocytometry Systems (San Jose, CA). CSLEX, an IgM mAb against SLex, was prepared as before (27). The 10E4 (an IgM mAb against native heparan sulfate chains of proteoglycans) was a kind gift from G. David (Center for Human Genetics, University of Leuven, Leuven, Belgium) (29).

Cell lines

A human cell line derived from an adenoid cystic carcinoma of salivary gland (24) was purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). Human cell lines of promyeloid cells (HL-60; CCL 240), neuroblastoma (SK-N-SH; HTB 11), breast carcinoma (ZR-75-30; CRL 1504), and malignant melanoma (A375; CRL 1619) were purchased from American Type Culture Collection (Manassas, VA). They were cultured in RPMI 1640 medium (Life Technologies, Shanghai, China) supplemented with 10% heat-inactivated newborn bovine calf serum (BCS), 4 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in the presence of 5% CO2.

Flow cytometric assays

Adherent cells were detached by PBS, pH 7.4, containing 0.02% EDTA (Versene; Life Technologies). All cells were washed twice and resuspended in PBS/BCS (PBS supplemented with 1 mM CaCl2, 1 mM MgCl2, and 1% heat-inactivated BCS; 1 x 106 cells/ml). Each aliquot (0.5 ml) of cells was incubated with 1–3 µg of rabbit preimmune IgG, a rabbit anti-PSGL-1 peptide Ab, mouse IgG (Sigma, Shanghai, China), a ML5 mAb, a SN3 mAb, mouse IgM (Calbiochem-Novabiochem, La Jolla, CA), a MMA mAb, a CSLEX mAb, a 10E4 mAb, followed by 3 µg of FITC-conjugated Ab against rabbit IgG, mouse IgG, or mouse IgM (Sigma) at 22°C for 1 h with end-to-end rotation. For P-selectin cell surface-binding assay, each aliquot (0.5 ml) of cells was incubated with 3 µg of human IgG (Sigma) or P-selectin Rg, followed by 3 µg of FITC-conjugated Ab against human IgG (Pierce) at 22°C for 1 h with end-to-end rotation. Cells were sedimented (1500 rpm for 5 min), and supernatants were discarded. For Ab inhibition experiments, 3 µg of P-selectin Rg was preincubated with 10 µg of G1 F(ab')2 or PS1 F(ab')2 in 50 µl of PBS/BCS at 22°C for 30 min. Alternatively, cells were preincubated with 10 µg of mouse IgG or KPL-1 mAb in 0.1 ml of PBS/BCS at 22°C for 30 min. Each aliquot was then resuspended in 0.5 ml of PBS/BCS for immediate flow cytometric analysis (FACScan; BD Biosciences, Mountain View, CA).

Inhibition of proteoglycan and sulfate biosyntheses

For experiments of proteoglycan biosynthesis inhibition, cells were cultured in RPMI 1640 medium supplemented with 10% BCS with or without 1 mM 4-methylumbelliferyl-{beta}-D-xyloside ({beta}-D-xyloside; Sigma) for 1 wk (22, 30, 31, 32). For experiments of sulfate biosynthesis inhibition, cells were washed once with a sulfate-free RPMI 1640 medium (Life Technologies) supplemented with 10% dialyzed BCS with 100 mM sodium chlorate (Sigma) and cultured in the same medium for 2 h. After changing to the same fresh medium, these cells were further cultured for 16 h (13, 33). These treated cells were washed twice and resuspended in PBS/BCS for further experimentation.

Heparinase digestion

Cells were washed twice with an equal volume mixture of a DMEM-high glucose and a Ham’s F12 medium (both from Life Technologies) supplemented with 1 mM CaCl2 and 1 mM MgCl2. The washed cells were resuspended at 1 x 106 cells/ml in the same media. Each aliquot (0.5 ml) was digested with 1 U/ml heparinases I, II, and III (Sigma), in the presence of a mixture of protease inhibitors (10 µg/ml leupeptine, 10 µg/ml pepstatin, 20 µg/ml aprotinin, and 10 mM benzamidine; all from Sigma), at 37°C for 1 h with end-to-end rotation.

Surface biotinylation, P-selectin precipitation, and streptavidin detection

Acc-M cells (1 x 107/ml) were biotinylated with 0.5 mg/ml sulfo-N-hydroxysulfosuccinimide-LC biotin (Pierce) on ice for 30 min with end-to-end rotation. After washing, these cells were lysed in the ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 3% (3-cholamidopropyl-dimethylammonio)-1-propanesulfonate, 1 mM CaCl2, 1 mM MgCl2, 10 mM benzamidine, and 2 mM PMSF). Following centrifugation at 12,000 x g for 10 min, the supernatants were collected and preincubated with the human IgG-bound protein A beads at 4°C overnight. They were then loaded onto a P-selectin Rg affinity column prepared as previously described (13, 23, 25, 27). After extensive washing with 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM CaCl2, and 1 mM MgCl2 (TBS/Ca/Mg), the bound molecules were eluted from the P-selectin columns with 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 10 mM EDTA. The eluates were recalcified by adding 1 M CaCl2 to the 20 mM CaCl2 final concentration. Each aliquot (equal to the materials isolated from 3 x 107 cells) of the recalcified eluates was then incubated with the 30-µg P-selectin Rg-bound protein A beads overnight with end-to-end rotation. For the Ab inhibition experiment, the P-selectin Rg-bound protein A beads were preincubated with 30 µg of G1 F(ab')2 or PS1 F(ab')2 for 4 h, followed by washing twice with cold TBS/Ca/Mg. After the incubation, they were washed five times with TBS/Ca/Mg. Reactants were boiled in the SDS sample buffer in the presence of 5% 2-ME, subjected to 7–15% SDS-PAGE, and transferred to Immobilon-P membranes. They were probed with the HRP-conjugated streptavidin, followed by a chemiluminescent detection system (25, 26, 27, 28).

Laminar flow assay

Polystyrene slides were coated with 2 ml (10 µg/ml) of human IgG or P-selectin Rg in 20 mM Tris-HCl, pH 9.5, 140 mM NaCl, 0.02% NaN3 at 4°C overnight and blocked with 3% human serum albumin at 22°C for 2 h. Slides were fitted into a parallel plate laminar flow chamber (22, 34) and mounted on the stage of an inverted phase-contrast Olympus microscope (Olympus Optical, Tokyo, Japan), which was connected to a time-lapse videocassette recorder STLV-24P (Samsung Electronics, Suwon, Korea) using a Panasonic color CCTV camera wv-GP410/G (Matsushita Communication Industrial, Okasa, Japan). Acc-M cells were resuspended at 0.5 x 106/ml in PBS supplemented with 10 mM HEPES, pH 7.4, and 2 mM CaCl2 and injected through the flow chamber at 22°C using a syringe pump. The wall shear stress was 1 dyne/cm2. The numbers of bound cells were quantified from videotape recordings of 10–20 fields of view obtained (3 min after flowing cells through the chamber) while scanning the lower plate of the flow chamber using a x10 objective lens. Adhesive interactions between potential cellular FcR and the Fc domain of P-selectin Rg were eliminated by preincubation of the cells with 10 µg/ml human IgG at 22°C for 20 min. For Ab inhibition experiments, the immobilized P-selectin Rg was preincubated with 10 µg/ml G1 (Fab')2 or PS1 (Fab')2 at 22°C for 20 min. Treatment of sodium chlorate was conducted exactly as above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
P-selectin binding to Acc-M cells

Using a cell surface-binding assay, we examined the interaction of recombinant P-selectin Rg with Acc-M cells. In this assay, an FITC-conjugated Ab to human IgG was used to report the binding of P-selectin Rg to these cells by flow cytometry. Fig. 1Go shows that, compared with human IgG, P-selectin Rg bound to HL-60 cells (a cell line of human promyeloid cells) and Acc-M cells (a cell line of human adenoid cystic carcinoma of salivary gland). Preincubation of P-selectin Rg with G1 F(ab')2 (a leukocyte adhesion-blocking mAb against P-selectin), but not PS1 F(ab')2 (a leukocyte adhesion-nonblocking mAb against P-selectin), inhibited this binding, indicating the specificity of this binding.



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  		FIGURE 1. The binding of P-selectin to Acc-M cells. HL-60 cells and Acc-M cells were incubated with human IgG or P-selectin Rg (designated as P-Rg) and a FITC-conjugated Ab against human IgG. For Ab inhibition experiments, P-selectin Rg was preincubated with G1 F(ab')2 (a leukocyte adhesion-blocking mAb to P-selectin) or PS1 F(ab')2 (a leukocyte adhesion-nonblocking mAb to P-selectin). The binding events were analyzed by flow cytometry. Results were presented as histograms of the log fluorescence intensities from 104 cells from the representative of three to five independent experiments.

 
Absence of PSGL-1 and CD24

We then decided to determine whether Acc-M cells expressed the currently known ligands, such as PSGL-1 for leukocytes and CD24 for certain cancer cells, using Abs specific to PSGL-1 and CD24. Fig. 2GoA shows that the affinity-purified rabbit anti-PSGL-1 peptide Ab, but not rabbit preimmune IgG, bound to HL-60 cells. However, the PSGL-1 peptide Ab did not react with Acc-M cells. Similar results were found using KPL-1, a leukocyte adhesion-blocking mAb to PSGL-1 (data not shown). Furthermore, we tested whether the KPL-1 mAb could block the binding of P-selectin to these cells. Fig. 2GoB shows that compared with human IgG, P-selectin Rg bound avidly to HL-60 cells and Acc-M cells. Preincubation of these cells with the KPL-1 mAb abrogated the binding of P-selectin Rg to HL-60 cells but did not interfere with the binding of P-selectin Rg to Acc-M cells.



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  		FIGURE 2. Expression and function of PSGL-1. HL-60 cells and Acc-M cells were incubated with rabbit preimmune IgG and rabbit anti-PSGL-1 peptide Ab, followed by a FITC-conjugated Ab against rabbit IgG (A). Alternatively, these cells were incubated with human IgG or P-selectin Rg (designated as P-Rg) and a FITC-conjugated Ab against human IgG. For Ab inhibition experiments, cells were preincubated with mouse IgG (data not shown) or KPL-1 (a leukocyte adhesion-blocking mAb against PSGL-1). B, The binding events were analyzed by flow cytometry, as described above. Results were the representative of three independent experiments.

 
We also used two CD24 mAbs to determine whether Acc-M cells expressed CD24. Fig. 3Go shows that CD24 mAbs, ML5 and SN3, bound avidly to SK-N-SH cells, a human neuroblastoma cell line known to express CD24 for P-selectin recognition (14). However, both of them did not recognize Acc-M cells. Thus, these findings appeared to exclude the possibility of PSGL-1 or CD24 as a candidate ligand for P-selectin on Acc-M cells.



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  		FIGURE 3. Expression of CD24. SK-N-SH cells and Acc-M cells were incubated with mouse IgG, ML5, or SN3 (both mAbs against CD24), followed by a FITC-conjugated Ab against mouse IgG. The binding events were analyzed by flow cytometry, as described above. Results were the representative of three independent experiments.

 
Cell surface expression of heparan sulfate-like proteoglycans

Because SLex and its derivatives (1, 2, 3, 4, 5) as well as heparan sulfate-like proteoglycans (22, 35, 36, 37, 38, 39) were previously reported to mediate the binding of P-selectin to leukocytes and certain cancer cells, we examined the carbohydrate structures expressed on Acc-M cells using carbohydrate-specific mAbs. Fig. 4GoA shows that compared with mouse IgM, both MMA (a mAb to Lex) and CSLEX (a mAb to SLex) bound to HL-60 cells but not to Acc-M cells. Furthermore, 10E4 (a mAb to heparan sulfate-like proteoglycans) bound avidly to A375 cells; it also bound to Acc-M cells (Fig. 4GoB). Our findings indicated that Acc-M cells had no apparent cell surface expression of Lex and SLex detectable to the MMA mAb and the CSLEX mAb. However, a moderate amount of heparan sulfate-like proteoglycans was expressed on Acc-M cells.



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  		FIGURE 4. Expression of Lex, SLex, and heparan sulfate-like proteoglycans. HL-60 cells, Acc-M cells, and A375 cells were incubated with mouse IgM, MMA (an IgM mAb against Lex), CSLEX (an IgM mAb against SLex), or 10E4 (an IgM mAb against heparan sulfate-like proteoglycan chains), followed by a FITC-conjugated Ab against mouse IgM. The binding events were analyzed by flow cytometry, as described above. Results were the representative of three independent experiments. A, Expression of Lex and SLex. B, Expression of heparan sulfate-like proteoglycans.

 
Assessment of functional activity for heparan sulfate-like proteoglycans

In literature, heparin and its analog, heparan sulfate, have been shown to bind to P-selectin, to inhibit leukocyte adhesion mediated by P-selectin, and to directly mediate adhesion of certain nonblood-borne, epithelial-like cancer cells to P-selectin (22, 35, 36, 37, 38, 39). Given the expression of heparan sulfate-like proteoglycans on the cell surface of Acc-M cells (Fig. 4GoB), we investigated whether they might mediate adhesion of these cells to P-selectin. We used the following experiments, in which biosynthesis of proteoglycans was inhibited by {beta}-D-xyloside and the cell surface expression of heparan sulfate-like proteoglycans was digested by heparinases, before the binding of P-selectin to them. Fig. 5Go shows that compared with human IgG, P-selectin Rg bound to A375 cells and Acc-M cells. {beta}-D-xyloside and heparinases (heparinase I, II, and III) had no inhibitory effects on the binding of P-selectin to Acc-M cells, even though they markedly neutralized the binding of P-selectin to A375 cells in the parallel experiments. These results indicated that the moderate amount of heparan sulfate-like proteoglycans expressed on Acc-M cells did not contribute significantly to the binding of these cells to P-selectin.



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  		FIGURE 5. Effects of {beta}-D-xyloside and heparinases on P-selectin binding. Acc-M cells and A375 cells were cultured in the presence of {beta}-D-xyloside (a heparin biosynthesis inhibitor; designated as {beta}-DX) or treated with heparinases (heparinase I, II, and III). After washing, they were incubated with human IgG or P-selectin Rg (designated as P-Rg) and a FITC-conjugated Ab against human IgG. The binding events were analyzed by flow cytometry, as described above. Results were the representative of three independent experiments.

 
Sodium chlorate inhibition of P-selectin binding

Because sulfation was required for P-selectin-mediated leukocyte adhesion (6, 7), we explored whether sulfation was necessary for the binding of P-selectin to Acc-M cells. In these experiments, sulfate biosynthesis was inhibited by sodium chlorate before the binding of P-selectin to them. Fig. 6GoA shows that sodium chlorate abolished the binding of P-selectin to Acc-M cells (A). Furthermore, P-selectin precipitated ~160-, ~54-, and ~36-kDa molecules from Acc-M cells following the cell surface biotinylation (B). These precipitated proteins, separated by SDS-PAGE and transferred to blotting membrane, were visualized by probing with the HRP-conjugated streptavidin. Preincubation of P-selectin with G1 F(ab')2 (a leukocyte adhesion-blocking mAb to P-selectin), but not PS1 F(ab')2 (a leukocyte adhesion-nonblocking mAb to P-selectin), abolished the interactions of P-selectin with these molecules. As expected, pretreatment of Acc-M cells with sodium chlorate significantly inhibited the bindings of the ~160-, ~54-, and ~36-kDa molecules to P-selectin. Our findings thus indicated that sulfation was critically involved in the binding of P-selectin to Acc-M cells.



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  		FIGURE 6. Effects of sodium chlorate on P-selectin binding. A, Acc-M cells were cultured in the absence or presence of sodium chlorate (a sulfate biosynthesis inhibitor). After washing, they were incubated with human IgG or P-selectin Rg (designated as P-Rg) and a FITC-conjugated Ab against human IgG. The binding events were analyzed by flow cytometry, as described above. Results were the representative of three to five independent experiments. B, Acc-M cells without or with the sodium chlorate treatment were surface biotinylated and lysed. The lysates were precipitated with P-selectin in the absence (designated as +) or presence of G1 F(ab')2 and PS1 F(ab')2. The precipitated proteins were fragmented on SDS-PAGE and transferred to Immobilon-P membranes, followed by visualization with the HRP-conjugated streptavidin. Results were the representative of three independent experiments.

 
Neutralization of Acc-M cell adhesion to P-selectin under flow by sodium chlorate

In an attempt to correlate the above findings of the cell surface-binding assay with a more physiologically relevant assay, we used a laminar flow assay to measure adhesion of Acc-M cells to P-selectin under flow. Using this assay, we tested the effects of sodium chlorate on adhesion of Acc-M cells to P-selectin. Fig. 7Go shows that Acc-M cells adhered to immobilized P-selectin Rg but not to immobilized human IgG, under shear stress similar to those of capillary venules (1 dyne/cm2). G1 F(ab')2 (a leukocyte adhesion-blocking mAb to P-selectin), but not PS1 F(ab')2 (a leukocyte adhesion-nonblocking mAb to P-selectin), neutralized adhesion of Acc-M cells to P-selectin, demonstrating the specificity of this adhesion. Treatment with sodium chlorate (an inhibitor for sulfate biosynthesis) markedly reduced the adhesion of Acc-M cells to P-selectin. Together with our observations obtained using the cell surface-binding assay, these data indicated that sulfation was necessary for interaction of P-selectin with Acc-M cells.



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  		FIGURE 7. Inhibition of Acc-M cell adhesion to P-selectin under flow. Adhesion of Acc-M cells to immobilized human IgG or immobilized P-selectin Rg (designated as +) was measured under flow. For Ab inhibition experiments, P-selectin Rg was preincubated with G1 F(ab')2 (a leukocyte adhesion-blocking mAb against P-selectin) or PS1 (a leukocyte adhesion-nonblocking mAb against P-selectin). For biosynthesis inhibition experiments, Acc-M cells were pretreated with sodium chlorate (designated as Chlorate). All results were expressed as the mean ± SD number of adherent Acc-M cells in 10–20 fields of view using a x10 objective lens from five to six separate experiments.

 
Sulfation requirement for P-selectin binding to breast carcinoma ZR-75-30 cells

To determine the significance of the above findings, we also investigated whether sulfation was required for P-selectin binding to other cell lines of human somatic cancers with distinct tissue and organ origins. We found that sulfation was indeed required for P-selectin binding to ZR-75-30 cells, a cell line of a human breast carcinoma. As summarized in Table IGo, P-selectin Rg, but not human IgG, bound avidly to ZR-75-30 cells. This binding was abolished by G1 F(ab')2 (a leukocyte adhesion-blocking mAb to P-selectin) but not by PS1 F(ab')2 (a leukocyte adhesion-nonblocking mAb to P-selectin). Probing with specific mAbs, such as the PSGL-1 peptide Ab, ML5 and SN3 (mAbs to CD24), MMA (a mAb to Lex), and CSLEX (a mAb to SLex), failed to detect apparent expressions of PSGL-1, CD24, Lex, and SLex on ZR-75-30 cells. Notably, although the high expression of heparan sulfate-like proteoglycans was detected by 10E4 (a mAb to native heparan sulfate chains), pretreatment with {beta}-D-xyloside and heparinases could not reduce the binding of P-selectin to these cells. Instead, preincubation with sodium chlorate could significantly neutralize the binding of P-selectin to ZR-75-30 cells. Our data thus indicated the obligatory requirement of sulfation for P-selectin recognition of certain nonblood-borne, epithelial-like cancer cells, such as Acc-M cells derived from a human adenoid cystic carcinoma of salivary gland and ZR-75-30 cells derived from a human breast carcinoma.


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  		Table I. Sulfation requirement for P-selectin binding

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we have shown that Acc-M cells (a cell line of human adenoid cystic carcinoma of salivary gland) and ZR-75-30 cells (a cell line of human breast carcinoma) bind specifically to P-selectin. The binding of P-selectin to these cells is sensitive to the treatment of sodium chlorate. Our results thus indicate that sulfation is essential to the P-selectin recognition of these cells. It is, to our best knowledge, the first demonstration for requirement of sulfation for the binding of P-selectin to nonblood-borne, epithelial-like cancer cells, even though it is well known that sulfation, along with sialylation and fucosylation, is necessary for the high-affinity binding of P-selectin to leukocyte PSGL-1 (6, 7, 8, 9, 10).

During this study, we found that Acc-M cells and ZR-75-30 cells apparently did not express Lex and SLex detectable to MMA and CSLEX mAbs, respectively (Fig. 4Go and Table IGo). The absence of Lex and SLex detectable to MMA and CSLEX mAbs was also reported for human small cell lung cancer NCI-H345 cells (11), human malignant melanoma A375 cells, and human tongue squamous cancer Tca8113 cells, all of which were well recognized by P-selectin (22). On the basis of these findings, we speculate that SLex and its derivatives may not participate in the interaction of P-selectin with these cancer cells.

Studies presented in this work strongly indicated the requirement of sulfation for the interaction of P-selectin with Acc-M cells and ZR-75-30 cells. Along this line of experimental observations, the sulfate-containing molecules, such as heparin and sulfatides, were reported to directly react with P-selectin. Furthermore, heparin and sulfatides were shown to potently inhibit adhesion of leukocytes and cancer cells to P-selectin (35, 36, 37, 38, 39). According to these findings, we propose that the sulfated moieties may function as the key determinant for P-selectin recognition, especially for somatic cancer cells, such as Acc-M cells and ZR-75-30 cells.

In the earlier study, we have shown that the cell surface heparan sulfate-like proteoglycans can mediate adhesion of human malignant melanoma A375 cells and human tongue squamous cancer Tca-8113 cells to P-selectin (22). Notably, they had a moderate expression (Acc-M cells) and a high expression (ZR-75-30 cells) of heparan sulfate-like proteoglycans on their cell surfaces (Fig. 4GoB and Table IGo). However, preincubation with {beta}-D-xyloside (an inhibitor for proteoglycan biosynthesis) and pretreatment with heparinases I, II, and III failed to alter the binding of P-selectin to Acc-M cells and ZR-75-30 cells, although they clearly attenuated the binding of P-selectin to A375 cells in the parallel experiments (Fig. 5Go). These data suggest that P-selectin apparently does not interact with the heparan sulfate-like proteoglycans expressed on the cell surface of Acc-M cells and ZR-75-30 cells.

Consequently, these results raise a question as to why P-selectin reacts with the cell surface heparan sulfate-like proteoglycans on A375 cells and Tca-8113 cells but does not react with those on Acc-M cells and ZR-75-30 cells. We infer that P-selectin may recognize a minor constituent of heparan sulfate-like proteoglycans that have specific modification(s) by sulfate(s) to fit exactly into the binding pocket. In this regard, the density, length, and specific modifications of heparan sulfate-like proteoglycan chains along with their cellular topologies may all contribute to the biological activity for P-selectin recognition. In addition, the protein or other carbohydrate component(s), along with heparan sulfate proteoglycans, may also be necessary for P-selectin recognition.

Results from previous and current investigations indicate that P-selectin apparently can react with the following structures: 1) the sialylated and fucosylated carbohydrate structures (such as SLex and its derivatives) and the tyrosine sulfation of PSGL-1 expressed on leukocytes (1, 2, 3, 4, 5, 6, 7, 8, 9, 10); 2) the SLex and its derivatives and the sulfated structures of CD24 expressed on human small cell lung cancer SW-2 cells (14); 3) the heparan sulfate-like proteoglycans expressed on human malignant melanoma A375 cells and on human tongue squamous cancer Tca-8113 cells (22); and 4) the non-PSGL-1- and non-CD24-derived sulfated moieties expressed on human salivary gland carcinoma Acc-M cells and breast carcinoma ZR-75-30 cells in this study. Notably, the SLex and its derivatives and the sulfated moieties all contain negative charges as the common feature, attesting to the functional importance of the anionic ions in P-selectin recognition. This conceptual understanding is reminiscent of our previous observations that chemicals with strong positive charges and/or a higher concentration of salt, such as 0.3 M NaCl, completely abolished adhesion of human neutrophils and human promyeloid HL-60 cells to P-selectin (data not shown). We believe that a better understanding of the molecular mechanisms for P-selectin recognition of leukocytes and cancer cells may facilitate the development of novel antiadhesion medicines for treatment of inflammation and growth and metastasis of cancer.


   Acknowledgments
 
We thank Dr. Guido David (Center for Human Genetics, University of Leuven, Leuven, Belgium) for 10E4 mAb.


   Footnotes
 
1 This study was funded by grants from the Chinese Academy of Sciences (KSCX2-2-02), National Natural Science Foundation of China (39925015, 30130090, and 39970794), Special Funds for Major State Basic Research of China (Grant G1999053907), Shanghai Commission on Science and Technology, and Chinese National Human Genome Center at Shanghai (CNCS-2000-M-04). Back

2 Address correspondence and reprint requests to Dr. Jian-Guo Geng, Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Room 505, 320 Yue-Yang Road, Shanghai, 200031, China. E-mail address: jggeng{at}sunm.shcnc.ac.cn Back

3 Abbreviations used in this paper: PSGL-1, P-selectin glycoprotein ligand-1; BCS, bovine calf serum; {beta}-D-xyloside, 4-methylumbelliferyl-{beta}-D-xyloside; Lex, Lewisx; Rg, receptor globulin; SLex, sialyl Lex. Back

Received for publication September 17, 2001. Accepted for publication December 6, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Springer, T. A.. 1994. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76:301.[Medline]
  2. McEver, R. P., K. L. Moore, R. D. Cummings. 1995. Leukocyte trafficking mediated by selectin-carbohydrate interactions. J. Biol. Chem. 270:11025.[Abstract/Free Full Text]
  3. Butcher, E. C., L. J. Picker. 1996. Lymphocyte homing and homeostasis. Science 272:60.[Abstract]
  4. Kansas, G. S.. 1996. Selectins and their ligands: current concepts and controversies. Blood 88:3259.[Free Full Text]
  5. Geng, J.-G.. 2001. Directional migration of leukocytes: their pathological roles in inflammation and strategies for development of anti-inflammatory therapies. Cell Res. 11:85.[Medline]
  6. Pouyani, T., B. Seed. 1995. PSGL-1 recognition of P-selectin is controlled by a tyrosine sulfation consensus at the PSGL-1 amino terminus. Cell 83:333.[Medline]
  7. Wilkins, P. P., K. L. Moore, R. P. McEver, R. D. Cummings. 1995. Tyrosine sulfation of P-selectin glycoprotein ligand-1 is required for high affinity binding to P-selectin. J. Biol. Chem. 270:22677.[Abstract/Free Full Text]
  8. Wilkins, P. P., R. P. McEver, R. D. Cummings. 1996. Structures of the O-glycans on P-selectin glycoprotein ligand-1 from HL-60 cells. J. Biol. Chem. 271:18732.[Abstract/Free Full Text]
  9. Aeed, P. A., J.-G. Geng, D. Asa, L. Raycroft, L. Ma, Å. P. Elhammer. 1998. Characterization of the O-linked oligosaccharide structures on P-selectin glycoprotein ligand-1 (PSGL-1). Glycoconjugate J. 15:975.[Medline]
  10. Somers, W. S., J. Tang, G. D. Shaw, R. T. Camphausen. 2000. Insights into the molecular basis of leukocyte tethering and rolling revealed by structures of P- and E-selectin bound to SLex and PSGL-1. Cell 103:467.[Medline]
  11. Stone, J. P., D. D. Wagner. 1993. P-selectin mediates adhesion of P-selectin to neuroblastoma and small cell lung cancer. J. Clin. Invest. 92:804.
  12. Kunzendorf, U., S. Kruger-Krasagakes, M. Notter, H. Hock, G. Walz, T. Diamantstein. 1994. A sialyl-Lex-negative melanoma cell line binds to E-selectin but not to P-selectin. Cancer Res. 54:1109.[Abstract/Free Full Text]
  13. Kaytes, P. S., J.-G. Geng. 1998. P-selectin mediates adhesion of the human melanoma cell line NKI-4: identification of glycoprotein ligands. Biochemistry 37:10514.[Medline]
  14. Aigner, S., C. L. Ramos, A. Hafezi-Moghadam, M. B. Lawrence, J. Friederichs, P. Altevogt, K. Ley. 1998. CD24 mediates rolling of breast carcinoma cells on P-selectin. FASEB J. 12:1241.[Abstract/Free Full Text]
  15. Dardik, R., N. Savion, Y. Kaufmann, D. Varon. 1998. Thrombin promotes platelet-mediated melanoma cell adhesion to endothelial cells under flow conditions: role of platelet glycoproteins P-selectin and GPIIb-IIIA. Br. J. Cancer 77:2069.[Medline]
  16. Giavazzi, R., M. Foppolo, R. Dossi, A. Remuzzi. 1993. Rolling and adhesion of human tumor cells on vascular endothelium under physiological conditions. J. Clin. Invest. 92:3038.
  17. Aruffo, A., M. T. Dietch, H. Wan, K. E. Hellstrom, I. Hellstrom. 1992. Granule membrane protein 140 (GMP140) binds to carcinomas and carcinoma-derived cell lines. Proc. Natl. Acad. Sci. USA 89:2292.[Abstract/Free Full Text]
  18. Goetz, D. J., H. Ding, W. J. Atkinson, G. Vachino, R. T. Camphausen, D. A. Cumming, F. W. Luscinskas. 1996. A human colon carcinoma cell line exhibits adhesion interactions with P-selectin under fluid flow via a PSGL-1-independent mechanism. Am. J. Pathol. 149:1661.[Abstract]
  19. Handa, K., T. White, K. Ito, H. Fang, S.-S. Wang, S.-I. Hakomori. 1995. P-selectin-dependent adhesion of human cancer cells: requirement for co-expression of a "PSGL-1-like" core protein and the glycosylation process for sialosyl-Lex or sialosyl-Lea. Int. J. Oncol. 6:773.
  20. Mannori, G., P. Crottet, O. Cecconi, K. Hanasaki, A. Aruffo, R. M. Nelson, A. Varki, M. P. Bevilacqua. 1995. Differential colon cancer cell adhesion to E-, P-, and L-selectin: role of mucin-type glycoproteins. Cancer Res. 55:4425.[Abstract/Free Full Text]
  21. Pottratz, S. T., T. D. Hall, W. M. Scribner, H. N. Jayaram, V. Natarajan. 1996. P-selectin-mediated attachment of small cell lung carcinoma to endothelial cells. Am. J. Physiol. 15:L918.
  22. Ma, Y.-Q., J.-G. Geng. 2000. Heparan sulfate-like proteoglycans mediate adhesion of human malignant melanoma A375 cells to P-selectin under flow. J. Immunol. 165:558.[Abstract/Free Full Text]
  23. Li, L., H. J. Short, K.-X. Qian, . P. Elhammer, J.-G. Geng. 2001. Characterization of glycoprotein ligands for P-selectin on a human small cell lung cancer cell line NCI-H345. Biochem. Biophys. Res. Commun. 288:637.[Medline]
  24. Guan, X. F., W. L. Qiu, R. G. He, X. J. Zhou. 1997. Selection of adenoid cystic carcinoma cell clone highly metastatic to the lung: an experimental study. Int. J. Oral Maxillofac. Surg. 26:116.[Medline]
  25. Asa, D., L. Raycroft, L. Ma, P. Aeed, P. S. Kaytes, . P. Elhammer, J.-G. Geng. 1995. The P-selectin glycoprotein ligand functions as a common human leukocyte ligand for P- and E-selectin. J. Biol. Chem. 270:11662.[Abstract/Free Full Text]
  26. Geng, J.-G., M. P. Bevilacqua, K. L. Moore, T. M. McIntyre, S. M. Prescott, J. M. Kim, G. A. Bliss, G. A. Zimmerman, R. P. McEver. 1990. Rapid neutrophil adhesion to activated endothelium mediated by GMP-140. Nature 343:757.[Medline]
  27. Ma, L., L. Raycroft, D. Asa, D. C. Anderson, J.-G. Geng. 1994. A sialoglycoprotein from human leukocytes functions as a ligand for P-selectin. J. Biol. Chem. 269:27739.[Abstract/Free Full Text]
  28. Geng, J.-G., T. J. Raub, C. A. Baker, G. A. Sawada, L. Ma, Å. P. Elhammer. 1997. Expression of a P-selectin ligand in zona pellucida of porcine oocytes and P-selectin on acrosomal membrane of porcine sperm cells: potential implications for their involvement in sperm-egg interactions. J. Cell Biol. 137:743.[Abstract/Free Full Text]
  29. Bai, X. M., B. van der Schueren, J.-J. Cassiman, H. van den Berghe, G. David. 1994. Differential expression of multiple cell surface heparan sulfate proteoglycans during embryonic tooth development. J. Histochem. Cytochem. 42:1043.[Abstract]
  30. Carey, D. J., C. M. Rafferty, M. S. Todd. 1987. Effects of inhibition of proteoglycan synthesis on the differentiation of cultured rat Schwann cells. J. Cell Biol. 105:1013.[Abstract/Free Full Text]
  31. Hamati, H. F., E. L. Britton, D. J. Carey. 1989. Inhibition of proteoglycan synthesis alters extracellular matrix deposition, proliferation, and cytoskeletal organization of rat aortic smooth muscle cells in culture. J. Cell Biol. 108:2495.[Abstract/Free Full Text]
  32. Lucas, M., T. Mazzone. 1996. Cell surface proteoglycans modulate net synthesis and secretion of macrophage apolipoprotein E. J. Biol. Chem. 271:13454.[Abstract/Free Full Text]
  33. Conrad, H. E.. 1998. Heparin-Binding Proteins 164. Academic Press, San Diego.
  34. Lawrence, M. B., T. A. Springer. 1991. Leukocyte roll on a selectin at physiologic flow rates: distinction from and prerequisite for adhesion through integrin. Cell 65:859.[Medline]
  35. Skinner, M. P., D. J. Fournier, J. J. Gorman, C. N. Chesterman, M. C. Berndt. 1989. Characterization of human platelet GMP-140 as a heparin-binding protein. Biochem. Biophys. Res. Commun. 164:1373.[Medline]
  36. Skinner, M. P., C. M. Lucas, G. E. Burns, C. N. Chesterman, M. C. Berndt. 1991. GMP-140 binding to neutrophils is inhibited by sulfated glycans. J. Biol. Chem. 266:5371.[Abstract/Free Full Text]
  37. Aruffo, A., W. Kolanus, G. Walz, P. Fredman, B. Seed. 1991. CD62/P-selectin recognition of myeloid and tumor cell sulfatides. Cell 67:35.[Medline]
  38. Nelson, R. M., O. Cecconi, W. G. Roberts, A. Aruffo, R. J. Linhardt, M. P. Bevilacqua. 1993. Heparin oligosaccharides bind L- and P-selectin and inhibit acute inflammation. Blood 82:3253.[Abstract/Free Full Text]
  39. Koenig, A., K. R. Norgard-Sumnicht, R. Linhardt, A. Varki. 1998. Differential interactions of heparin and heparan sulfate glycoproteins with the selectins: implications for the use of unfractionated and low molecular weight heparins as therapeutic agents. J. Clin. Invest. 101:877.[Medline]



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