Cancer Research Grants Frontiers in Basic Cancer Research
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fukuda, M. N.
Right arrow Articles by Fukuda, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fukuda, M. N.
Right arrow Articles by Fukuda, M.
[Cancer Research 60, 450-456, January 15, 2000]
© 2000 American Association for Cancer Research

Tumor Biology

A Peptide Mimic of E-Selectin Ligand Inhibits Sialyl Lewis X-dependent Lung Colonization of Tumor Cells1

Michiko N. Fukuda2, Chikara Ohyama3, Kevin Lowitz, Osamu Matsuo, Renata Pasqualini, Erkki Ruoslahti and Minoru Fukuda

The Burnham Institute, Cancer Research Center, La Jolla, California 92037 [M. N. F., C. O., K. L., R. P., E. R., M. F.], and Department of Physiology, Kinki University School of Medicine, Osakasayama, Japan 589-8511 [O. M.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Selectins bind to carbohydrate ligands in a calcium-dependent manner and play critical roles in host defense and possibly in tumor metastasis. To isolate peptides that mimic E-selectin ligands, we screened a phage peptide library using E-selectin as a target molecule. This attempt unexpectedly failed, probably because the binding affinity of E-selectin to its ligand is low. We then took an approach that is analogous to the isolation of anti-idiotype antibodies and were able to isolate peptides that bound to anticarbohydrate antibodies recognizing E-selectin ligands. These peptides, enriched for their binding to anti-Lewis A antibody, were found to bind to E-, P- and L-selectins in a calcium-dependent manner. Phage harboring the identified peptide IELLQAR and synthetic peptides having the same sequence inhibited the binding of sialyl Lewis X or sialyl Lewis A oligosaccharides to E-selectin. The adhesion of HL-60 and B16 melanoma cells expressing sialyl Lewis X to E-selectin was also inhibited by the phage-displaying IELLQAR peptide. Moreover, i.v. injected IELLQAR peptide inhibited the lung colonization of mouse B16 melanoma and human lung tumor cells expressing sialyl Lewis X. These results demonstrate that it is possible to isolate peptides mimicking carbohydrate ligands by screening the peptides for binding to anticarbohydrate antibodies and then using them to inhibit carbohydrate-dependent experimental tumor metastasis.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell surface carbohydrates are characteristic of various stages of differentiation, and distinct carbohydrates are expressed in a tissue- and cell-specific manner during development and in adulthood (1, 2, 3) . These cell-type-specific carbohydrates are thought to play a role in cell-cell interactions. In particular, sialyl Lewis X, NeuNAc{alpha}2->3Galß1->4(Fuc{alpha}1->3)GlcNAc->R, whose presence was noted in neutrophils (4 , 5) , has been identified to be a ligand for selectins (6, 7, 8, 9) . Selectins contain a carbohydrate-binding domain at their NH2 terminus and belong to the C-type lectin gene family of which activity is dependent on Ca2+. E- and P-selectin are expressed on activated vascular endothelial cells and the binding of these selectins to sialyl Lewis X on leukocytes allows them to roll, which leads to extravasation of leukocytes (6, 7, 8, 9) . L-selectin, on the other hand, is present on lymphocytes and binds to sulfated sialyl Lewis X oligosaccharides present in L-selectin receptors restricted to high endothelial venules (10, 11, 12) . This interaction is critical for lymphocyte recirculation from the intravascular compartment to the lymphatic compartment. These results clearly indicate that sialyl Lewis X and its sulfated form play critical roles in the interaction between leukocytes and endothelial cells.

Certain epithelial cancer cells express sialyl Lewis X and sialyl Lewis A (NeuNAc{alpha}2->3Galß1->3(Fuc{alpha}1->4)GlcNAc->R), an isomer of sialyl Lewis X, as tumor-associated carbohydrate antigens (13, 14, 15, 16) . Sialyl Lewis A has also been shown to bind to E-selectin (15 , 17) , which suggests that sialyl Lewis X and sialyl Lewis A oligosaccharides may play a role in selectin-mediated adhesion in these cancer cells. It was shown that patients with colonic carcinomas expressing sialyl Lewis X exhibit a poorer prognosis than those negative for the same carbohydrate antigen (18) . Similarly, colonic tumor invasion was highly correlated with the expression of sialyl Lewis X in core 2 branched O-glycans (19) . In human lung and colon carcinomas, highly metastatic tumor cells express more sialyl Lewis X on the cell surface and bind more strongly to E-selectin than do their poorly metastatic counterparts (20 , 21) . Moreover, recent studies showed that the metastatic capability of B16 mouse melanoma cells are dramatically increased after acquiring sialyl Lewis X through transfection of Fuc-TIII (22) , and poorly metastatic human lung carcinomas become highly metastatic after transfection of {alpha}1,3-fucosyltransferase VII (23) . These results, combined together, indicate that sialyl Lewis X plays a critical role in the metastasis of certain tumor cells.

Selectins are also expressed on capillary endothelium during acute and chronic inflammation (6, 7, 8, 9, 10) . These include immune complex-dependent acute lung injury, cardiac allografts, rheumatoid arthritis, wound sepsis, and skin inflammation. The above results also suggest that unnecessary inflammatory response in diseases may be inhibited by using sialyl Lewis X oligosaccharides as antagonist. In fact, acute inflammatory response mediated by E- or P-selectin was inhibited by sialyl Lewis X oligosaccharides or glycopeptides containing sialyl Lewis X capping structure (24, 25, 26) . These attempts, however, have limitations inasmuch as the synthesis of such elaborate oligosaccharides involves many tedious steps and is time consuming. To overcome this problem, we searched for peptides that function as E-selectin ligands.

Recently, extensive studies have been carried out using phage peptide libraries to isolate and identify peptides that bind specifically to integrins or to receptors expressed in a tissue-specific manner (27, 28, 29) . These studies indicate that it is possible to identify relatively short peptides that bind to different proteins or different tissues. Moreover, a similar approach was successful in isolating peptides that bind to anti-Lewis Y antibody (30) and anti-GD1{alpha}4 antibody (31) , which recognize tumor-associated carbohydrate antigens. Encouraged by these findings, we began working on the identification of peptides that specifically bind to E-selectin. Our initial attempts failed when we screened peptides that directly bound to E-selectin. We then took a different approach, analogous to identifying carbohydrate epitope represented by anti-idiotype antibody (30 , 31) , and screened peptides for their binding to anticarbohydrate antibodies that recognize E-selectin carbohydrate ligands or related carbohydrates. Using this approach, we succeeded in isolating peptides that bind to E-selectin in a calcium-dependent manner and inhibit sialyl Lewis X-dependent experimental tumor metastasis.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Selection of Phage by Panning.
A flat-bottomed Linbro/Titertek 96-well plate (ICN Biomedicals) was coated with 10 µg of goat antimouse immunoglobulins, blocked with PBS containing 3% BSA and 0.02% Tween 20, and then incubated with 2–3 µg monoclonal anti-Lewis A antibody (clone 7LE; Ref. 15 ; mouse IgG1, Seikagaku Biochemicals). After washing with PBS containing 0.02% Tween 20, wells were blocked with PBS containing 3% BSA. A linear heptapeptide phage library (1011–12 transducing units; 32 ) was added to the well and incubated at room temperature for 1 h. After removing unbound phage, competent K91 kan Escherichia coli bacteria were added and incubated at room temperature for 1 h. Infected bacteria were then diluted in 20 ml of LB, and aliquots of the bacteria were plated on LB agar plates containing 100 µg/ml kanamycin and 20 µg/ml tetracycline as described previously (29) . Plates were incubated at 37°C overnight to allow bacteria to form colonies. The rest of the diluted bacteria was cultured in LB liquid overnight at 37°C, and phage was recovered by precipitating with 6% polyethyleneglycol in 0.32 M NaCl. Pelleted phage was dissolved in 1 ml of PBS and used for screening with anti-Lewis A antibody in the same manner as the first screen. After the third enrichment, individual colonies were subjected to DNA sequencing.

Assay of Phage Binding to Selectins.
The soluble chimeric forms of E-, L-, and P-selectin were prepared as described previously (33, 34, 35) . Briefly, COS-1 cells were transfected with a mammalian expression plasmid vector encoding each selectin-IgG chimera. Two days after transfection, culture supernatants were collected and selectin-IgG chimeras were purified by affinity chromatography with protein A (36) . A brevican lectin domain fused with IgG (37) was kindly provided by Dr. Yu Yamaguchi. Microtiter wells were coated with E-, L-, or P-selectin chimeras or brevican lectin chimera (10 µg/ml) at 4°C overnight, then blocked with TBS containing 1 mM CaCl2 and 3% BSA for 2 h. Phage (1 x 106 transducing units) was added to wells and incubated at room temperature for 1 h. After washing wells with TBS containing 1 mM CaCl2, bound phage was quantitated by transforming K91 kan bacteria, followed by colony counting, as described above.

Inhibition of the Binding between Sialyl Lewis X and Selectin Chimeras by Phage and Peptides.
Five hundred ng of polymeric sialyl Lewis X (Ref. 38 ; sLex-PAA-biotin, GlycoTech, Rockville, MD), dissolved in TBS containing 1 mM CaCl2, was added in the presence or absence of a phage or peptide to the well coated with a selectin chimera. After incubating at room temperature for 1 h, wells were washed with TBS containing 1 mM CaCl2, and sLex-PAA-biotin bound to each selectin was determined using a peroxidase-conjugated avidin and peroxidase substrate (Pierce). A405 nm was read in an ELISA reader. Synthetic peptides were made by AnaSpec, Inc. (San Jose, CA). One of the synthetic peptides was an octameric peptide containing (Lys)4->(Lys)2->Lys backbone.

In Vitro Cell Binding.
Mouse B16 melanoma cells were transfected with pcDNAI-Fuc-TIII (39) , and resultant B16-FTIII cells were obtained as described previously (22) . In the present study, B16-FTIII cells expressing moderate amounts of sialyl Lewis X, B16-FTIII·M cells (22) , were used. A cell-binding assay was conducted as described previously (35) using microtiter wells coated with E-selectin chimera. After preincubating the wells with TBS containing 1 mM CaCl2 containing inhibitor (phage or peptide) for 20 min, HL-60 cells or B16-FTIII cells were incubated (1 x 104 cells/well) at 4°C for 1 h (35) . Unbound cells were removed by washing the wells with TBS. Numbers of those remaining in the well were counted under a microscope.

Lung Colonization Assay.
A total of 2 x 105 of B16-FTIII·M cells (>90% viability) were suspended in 100 µl of serum-free DMEM and injected into a tail vein of each C57BL/6 mice. After 3 weeks, the mice were killed, and tumor nodules in various organs were counted under a dissecting microscope as described previously (22) . The majority of tumors were formed in the lung; therefore, this organ was selected to evaluate tumor formation. Because melanoma cells have pigment, even small tumor nodules (less than 2-mm diameter) could be counted. Similarly, human lung adenocarcinoma HAL-8Luc cells (5 x 105) were injected i.v. into BALB/C nude (null/null) mice (21 , 40) . After 10 weeks, the mice were killed, and the weights of the lungs were measured. To test the inhibitory activity of IELLQAR peptide, phage harboring the peptide (1 x 108 transforming units/100 µl/mouse) or a synthetic IELLQAR peptide (500 µg/100 µl/mouse) was injected through a tail vein. After 20 min, metastatic tumor cells were injected into a second tail vein. Three (for B16-FTIII·M) or 10 (for HAL-8Luc cells) weeks later, numbers of metastatic foci in the lungs were counted or weights of the lungs measured.

Immunocytochemical Detection of Tumor Cells.
The lungs of mice injected with tumor cells were removed 1 h after injection and fixed. Tissue was embedded in paraffin, and 5-µm thick sections were cut and mounted on glass slides. After deparaffination and rehydration, the sections were incubated with mouse antimelanoma antibody (HMB-45, Dako), followed by biotinylated ant-mouse IgG and avidin-biotin reagent (Vectastain ABC Kit, Vector) as described previously (19 , 22) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Isolation of Peptides That Bind to E-Selectin Using Anticarbohydrate Antibodies.
Initially we attempted to isolate peptides that bind to E-selectin coated on plastic plates. We could not, however, isolate peptides that mimic E-selectin ligand. None of the peptides that were marginally enriched bound to E-selectin in a calcium-dependent manner, nor did they inhibit the binding of sialyl Lewis X oligosaccharides to E-selectin. Similar results were reported previously when E-selectin was used as a target molecule during screening (41) .

We reasoned that this outcome is probably related to the relatively weak affinity of E-selectin to sialyl Lewis X (IC50 = ~750 µM; Refs. 42 , 43 ). On the other hand, it has been reported that peptides mimicking carbohydrates could be isolated using anticarbohydrate antibodies (30 , 31 , 44, 45, 46) . To overcome the above issues, we chose various monoclonal antibodies as targeting molecules that recognize the entire, or a part of, sialyl Lewis X or sialyl Lewis A oligosaccharides. Attempts were, thus, initially made using anti-Lewis X, MMA (47) , PMN6 (48) , SSEA-1 (49) antibodies and antisialyl Lewis X, CSLEX-1 antibody (14) . However, none of the peptides enriched for binding to those antibodies bound to E-selectin in a specific manner. We then unexpectedly found that peptides enriched for binding to anti-Lewis A antibody (7LE;, Ref. 15 ) were best to mimic E-selectin ligands.

As shown in Fig. 1Citation , among phages enriched, a phage containing the IELLQAR peptide exhibited the highest binding to E-selectin. This binding is entirely calcium-dependent inasmuch as no binding was observed in the absence of calcium. The same results also showed that peptides with a high affinity to E-selectin have the common sequence I(E/D)L(L/M)QAR (Fig. 1Citation ).



View larger version (32K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Binding of phage harboring different peptides to E-selectin. Phages were added to microtiter wells coated with an E-selectin chimeric protein. Bound phage were quantitated using K91 kan bacteria as described previously (29) . Peptides displayed on phage are: 1, ISLLQAR; 2, IDLMQAR; 3, IILLQAR; 4, IELLQAR; 5, ISLLGAR; 6, FSLLDAR; 7, IFLLWQR; 8, FAQLDWH. Each pair shows phage bound to E-selectin-IgG chimera in the presence (+) or absence (-) of 1 mM CaCl2. Experiments were carried out in duplicates; bars, SD. Almost identical results were obtained in two additional repeated experiments.

 
E-Selectin Binding to Sialyl Lewis X Oligosaccharides Can Be Inhibited by the IELLQAR Peptide.
We then tested whether the peptide inhibits the binding of sialyl Lewis X oligosaccharides to E-selectin. For this, we used polymeric sialyl Lewis X (38) , in which sialyl Lewis X oligosaccharides are attached to polyacrylamide gel (on average, 10 sialyl Lewis X oligosaccharides per gel).

As shown in Fig. 2ACitation , a phage harboring either the IELLQAR peptide or IDLMQAR peptide effectively inhibited the binding of polymeric sialyl Lewis X oligosaccharides to E-selectin. IELLQAR was more efficient than IDLMQAR in a similar way as seen in phage binding to E-selectin (Fig. 1Citation , Lanes 2, 4, and 2A). In contrast, a control phage harboring FAQLDWH peptide bound minimally to E-selectin (Fig. 1Citation , Lane 8) and barely had an effect on the binding of sialyl Lewis X oligosaccharides to E-selectin (Fig. 2ACitation ) These results indicate that these active peptides are recognized by E-selectin in a manner similar to sialyl Lewis X oligosaccharide recognition by E-selectin. Our results also demonstrated that these peptides do not bind to sialyl Lewis X oligosaccharides because phage harboring these peptides did not bind to sLeX-PAA coated on plates (data not shown). Because IELLQAR peptide was more active than IDLMQAR peptide, IELLQAR peptide and phage displaying the peptide was used hereafter.



View larger version (17K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Inhibition of E-selectin binding to sialyl Lewis X or sialyl Lewis A oligosaccharides by individual phage or synthetic peptides. A, the inhibition of sialyl Lewis X binding to E-selectin by selected phage. Polymeric sialyl Lewis X was added to E-selectin-coated wells in the presence of various amounts of phage. B, the inhibition of polymeric sialyl Lewis A binding to E-selectin by IELLQAR phage. C, the inhibition of polymeric sialyl Lewis X binding to E-selectin by synthetic (linear, cyclic, and multivalent) IELLQAR peptides. CaCl2 (1 mM) was added to the buffer in all of the experiments. Similar results were obtained in two additional repeated experiments.

 
To corroborate the above results, peptides were synthesized according to the amino acid sequence. In this experiment, we also took advantage of the fact that a circular peptide CIELLQARC and multivalent (octameric) peptide can be synthesized. The results shown in Fig. 2CCitation demonstrate that linear and circular monovalent peptides inhibit the binding of sialyl Lewis X oligosaccharides to E-selectin with the IC50 of 2 x 10-4 and 1 x 10-4 M for the linear and circular peptide, respectively. The IC50 values reported previously for monovalent sialyl Lewis X oligosaccharides range from 3 x 10-3 to 1 x 10-4 M (42 , 43) . Thus, our monomeric peptides bind to E-selectin with at least as high an affinity as monomeric carbohydrate ligands. Notably, the octameric peptide displayed a substantially higher affinity, with a IC50 value of 1 x 10-5 M (Fig. 2CCitation ). These results show that a multivalent peptide is more potent as a ligand than a monovalent ligand, consistent with previous findings showing multivalent carbohydrates serve better as selectin ligands (43) . A phage harboring the IELLQAR peptide was also shown to inhibit the binding of sialyl Lewis A oligosaccharides to E-selectin, and the phage competes with sialyl Lewis A similar to the competition with sialyl Lewis X (Fig. 2BCitation ). These combined results demonstrate that the IELLQAR peptide closely mimics E-selectin carbohydrate ligands.

The Peptide Can Also Bind to P- and L-Selectin.
The above results indicate that IELLQAR peptide inhibits the binding of E-selectin to sialyl Lewis X oligosaccharide. The next question we raised was how far this peptide mimic can be extended to block the interaction involving other selectins.

As shown in Fig. 3Citation , P- and L-selectin bound to multivalent sialyl Lewis X oligosaccharides albeit with lower affinity, consistent with the previous report (50) . These adhesions could be inhibited by the phage harboring IELLQAR (Fig. 3ACitation ). In contrast, we did not detect significant binding to the C-type lectin domain of brevican fused with IgG (Fig. 3BCitation ). Brevican is a proteoglycan of which the lectin domain binds to HNK-1 glycan, sulfo->3GlcAß1->3Galß->4GlcNAcß->R (37) . The above results were obtained most likely because the homology between the lectin domain of a selectin and brevican is lower (~31% identity) compared with the homology between different selectins (~52% identity; Ref. 51 ).



View larger version (27K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Effect of IELLQAR phage on the binding of E-, P-, and L-selectins to sialyl Lewis X oligosaccharides or the binding of IELLQAR phage to brevican lectin. A, the binding of sialyl Lewis X oligosaccharides to selectins was measured in the presence (+) or absence (-) of IELLQAR phage, as determined in Fig. 2Citation . B, the binding of IELLQAR phage to the brevican lectin chimera or the plate containing a control protein A was measured in the presence of 1 mM CaCl2. Experiments were carried out in duplicates and repeated twice. Representative results are shown; bars, SD.

 
E-Selectin-mediated Adhesion Can Be Inhibited by Phage Harboring the IELLQAR Peptide.
We then tested whether phage harboring the IELLQAR peptide can inhibit the adhesion of HL-60 cells to E-selectin. HL-60 cells are enriched with sialyl Lewis X oligosaccharides (5) and have been shown to bind to E-selectin through sialyl Lewis X oligosaccharides (6, 7, 8, 9) . The results shown in Fig. 4ACitation demonstrate that the adhesion of HL-60 cells to E-selectin coated on plates can be inhibited efficiently by the phage harboring the IELLQAR peptide. This efficiency is similar to that observed in the inhibition for the binding of sialyl Lewis X oligosaccharides to E-selectin, as shown in Fig. 2ACitation .



View larger version (27K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. Effect of IELLQAR phage on HL-60 or B16-FTIII·M cell adhesion to E-selectin. The adhesion of HL-60 (A) or B16-FTIII·M (B) cells to E-selectin was measured in the presence of IELLQAR phage ({blacksquare}) or control FAQLDWH phage (). {square}, cell bindings in the absence of phage. Experiments were carried out in triplicates; bars, SD. The results were repeated three times, and representative results are shown.

 
To determine whether this inhibition can be observed in the adhesion of other cells to E-selectin, B16 melanoma cells, transfected with Fuc-TIII, B16-FTIII, were tested. As shown previously (22) , B16-FTIII·M cells, which moderately express sialyl Lewis X oligosaccharides, bind to E-selectin whereas the parent B16 cells do not. The adhesion of B16-FTIII·M cells to E-selectin was efficiently inhibited by phage harboring the IELLQAR peptide (Fig. 4BCitation , ). In contrast, the phage harboring the control FAQLDWH peptide barely had an effect even at its highest concentration (Fig. 4BCitation , [GRAPHIC]). These results clearly demonstrate that IELLQAR can effectively compete with sialyl Lewis X oligosaccharide for binding to E-selectin.

IELLQAR Peptide Can Inhibit Sialyl Lewis X-dependent Lung Colonization of Tumor Cells.
Recently, we have shown that B16-FTIII·M cells expressing moderate amounts of sialyl Lewis X produce lung tumor nodules to a greater degree than those expressing negligible amounts of sialyl Lewis X (22) . This tumor formation was inhibited by preincubation of antisialyl Lewis X antibody, which indicates that sialyl Lewis X on tumor cells mediate tumor formation (22) . To test whether IELLQAR peptides can inhibit this lung colonization of tumor cells, B16-FTIII·M cells were injected i.v. after preinjection of the octavalent IELLQAR or control octavalent FAQLDWH peptide.

As shown in Fig. 5ACitation , numerous metastatic foci developed in the lungs of mice that had received the control phage (upper row), whereas only a few foci were detected in the lungs of the mice receiving the phage harboring IELLQAR peptide (lower row). The number of lung nodules pretreated with the phage harboring IELLQAR peptide was almost completely diminished (Fig. 5BCitation ). Almost identical results were obtained when the IELLQAR peptide or control peptide was used (Fig. 5, C and DCitation ).



View larger version (96K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 5. Lung tumor nodules formed by mouse B16-FTIII·M cells and human lung adenocarcinoma HAL-8Luc cells. A, after IELLQAR phage (lower row) or the control phage (upper row) were injected into C57BL/6 mice, B16-FTIII·M cells were injected, and the lungs from these mice were examined. C, B16-FTIII·M cells were injected into C57BL/6 mice after octameric IELLQAR peptide (lower row) or octameric control FAQLDWH peptide (upper row) was injected. B and D, the tabulated results derived from A and C, respectively. E and F, HAL-8Luc cells were injected into nude mice with preinjection of the octameric IELLQAR (lower row) and the control peptide (upper row) peptide, and derived lungs were examined. Each experiment was carried out using six mice; bars, SD.

 
To determine whether the IELLQAR peptide can inhibit metastasis of other tumor cells, human lung HAL-8Luc cells were injected i.v. into nude mice after preinjection of the IELLQAR peptide or a control peptide. The results, shown in Fig. 5, E and FCitation , demonstrated that the IELLQAR peptide inhibited lung colonization of HAL-8Luc cells, and that the weight of lung pretreated with the IELLQAR peptide was similar to that of control mice (see ref. 22 ).

These results, combined, demonstrate that sialyl Lewis X-dependent experimental tumor metastasis is significantly inhibited by pretreatment of the peptide mimicking sialyl Lewis X oligosaccharide.

Finally, to determine whether the peptide inhibits the adhesion of tumor cells to endothelial cells in vivo, mice were killed 1 h after B16-FTIII·M tumor cells were injected and the lungs were examined. As shown in Fig. 6, A and CCitation , tissue sections of the lungs from mice treated with control phage or a control peptide showed that the melanoma cells had already penetrated the vascular endothelium into the mesenchyme, consistent with the previous results (22) . In contrast, the B16-FTIII·M cells were nonadhesive and appeared apoptotic in lungs pretreated with the IELLQAR peptide (Fig. 6, B and DCitation ). Less than 5% of tumor cells adhered to endothelial cells among 100 cells examined in the latter experiments. These results, taken together, indicate that the IELLQAR peptide competes with sialyl Lewis X on tumor cell surfaces, which results in the impairment of the initial attachment of tumor cells to the endothelium and prevents tumor metastasis in vivo.



View larger version (114K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 6. Immunostaining of tumor cells in C57BL/6 mice. B16-FTIII·M cells were injected into C57BL/6 mice after preinjection of the control FAQLDWH phage (A) or IELLQAR phage (B) or the FAQLDWH control peptide (C) or IELLQAR peptide (D). One h after injection of the tumor cells, lungs were removed and immunocytochemically examined using antimelanoma antibody. Arrows, tumor cells; bar, 20 µm. Experiments were repeated two more times, and almost identical results were obtained.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study demonstrates that it is possible to identify a functional peptide mimicking a selectin ligand using appropriate anticarbohydrate antibodies. The IELLQAR peptide binds to E-selectin in a calcium-dependent manner and directly competes with sialyl Lewis X oligosaccharides for binding to E-selectin (Figs. 1Citation and 2Citation ). This is a remarkable improvement over a previous report showing that peptides identified by a phage-displaying library, using E-selectin as a target molecule, did not compete with sialyl Lewis X or exhibit calcium-dependent binding (41) . It is possible that low affinity of E-selectin to its ligands may be a reason for this difficulty. On the other hand, carbohydrate-mimicking peptides were successfully isolated using anticarbohydrate antibodies as a target molecule (30 , 31 , 44, 45, 46) . In particular, a peptide reacting with anti-GD1{alpha} antibody was found to inhibit lung colonization of murine RAW117-H10 lymphoma (31) Our studies extended these previous studies and demonstrated that isolated peptides can compete with carbohydrate ligands for carbohydrate-binding proteins that are not identical to, but overlap with, an epitope recognized by an anticarbohydrate antibody. In this regard, it is noteworthy that the peptide identified in the present study may not be a perfect mimic of sialyl Lewis X, although it competes well with sialyl Lewis X. It is expected that this kind of approach will be useful for identifying peptides mimicking carbohydrate ligands for other carbohydrate-binding proteins.

Our results demonstrated that IELLQAR peptide bind to all members of the selectin family (Fig. 3Citation ). The results indicate that the peptide probably represents a common carbohydrate structure recognized by all of the selectins. Our results are thus in contrast to the previous report that peptides mimicking streptococcal group A antigen recognize only each antibody for which the peptide was screened (44) . On the other hand, the IELLQAR peptide apparently represents a specific ligand for selectins inasmuch as it did not bind to the lectin domain of versican (Fig. 3Citation ). Taken together, the present results indicate that it is possible to isolate a versatile peptide commonly recognized by a particular family of carbohydrate-binding proteins.

We have also demonstrated that sialyl Lewis X-mediated adhesion of HL-60 and B16-FTIII·M cells can be inhibited by the IELLQAR peptide (Fig. 4Citation ). More importantly, sialyl Lewis X-dependent lung colonization of B16-FTIII·M cells and human lung adenocarcinoma HAL-8Luc cells were significantly suppressed by a single preinjection of IELLQAR peptide (Fig. 5Citation ). Moreover, B16-FTIII·M tumor cells could not adhere to endothelial cells in the presence of an IELLQAR peptide (Fig. 6Citation ). In contrast, the same tumor cells penetrated endothelial cells in the presence of a control peptide, reaching the lung mesenchyme, as seen in previous studies (22) . As far as we know, this is the first report on the in vivo inhibition of tumor cells to endothelial cells by a carbohydrate-mimicking peptide, thereby inhibiting experimental tumor metastasis.

These results reinforced the previous conclusions that these tumor cells lodge in metastatic sites using sialyl Lewis X- or sialyl Lewis A-mediated adhesion to selectin or its related molecule on endothelial cells (21, 22, 23) . The findings are consistent with the previous studies showing a correlation between the presence of sialyl Lewis X and tumor metastasis (see "Introduction"). In one of those studies, liver metastasis of human colon cancer cells occurred more frequently in sublines that strongly expressed sialyl Lewis X (52) . A clinicopathological study using immunohistochemistry provided evidence that the greater amount of sialyl Lewis X and sialyl Lewis A in cancer cells is closely correlated to poor prognosis (18 , 19) . Our present study thus supports these findings and further demonstrates that sialyl Lewis X, and possibly sialyl Lewis A, play a primary role in the adhesion of cancer cells to endothelial cells at metastatic sites.

One of the major remaining questions is which molecule(s) on endothelial cells actually binds to sialyl Lewis X-bearing (and sialyl Lewis A-bearing) tumor cells. In the above colonic and lung carcinoma cells lines, it was shown that highly metastatic cells bind more strongly to E-selectin than their poorly metastatic counterparts (20 , 21) . In patients with breast carcinomas, the expression of sialyl Lewis X and sialyl Lewis A was associated with the expression of E-selectin (53) . However, it is not known whether E-selectin is the one that initiates tumor cell adhesion.

As shown here and in the published work (22) , the tumor cells tested in the present study rapidly adhere to endothelial cells, most likely within a few minutes after injection. Tumor cells were found in extracellular matrix and mesenchymal cells 1 h after the injection (Fig. 6Citation ). In contrast, P- and E-selectin can be significantly expressed only when endothelial cells are activated. It is not clear how tumor cells can activate endothelial cells in such a short time (6, 7, 8, 9, 10) . Even after activation, expression of P- and E-selectin is only seen after 10 and 60 min, respectively. This is consistent with recent reports that E-selectin expression was at a maximum at 6 h after the injection of tumor cells (54) . Our preliminary results also indicate that lung endothelial cells of host mice were negative for selectins as assessed by immunohistochemical detection (data not shown). It was shown that P-selectin binding requires a specific counter-receptor, PSGL-1, which contains a sulfated tyrosine in addition to sialyl Lewis X oligosaccharides. PSGL-1 was not detected in tumor cells tested in the present study (data not shown; see also Ref. 55 ).

Our results suggest that a novel C-type lectin or carbohydrate-binding protein may be involved in tumor-endothelial cell interactions. Previous clinicopathological studies demonstrate that the expression of core-branched sialyl Lewis X and sialyl Lewis A is highly correlated to a poor prognosis of colonic tumors in patients (19) . The results suggest that the lectin(s), which bind to tumor cells, may preferentially bind to mucin-type O-glycans. Recent studies suggest that a C-type lectin present on natural killer cells can recognize highly dense sialyl Lewis X oligosaccharides (22 , 56) . Similarly, a mannose-binding C-type lectin apparently binds to sulfo->4GalNAcß1->4GlcNAcß1->4 Man->R structure when it is expressed on liver endothelial cells (57) . It is possible that one of the already known or unidentified C-type lectins on the surface of endothelial cells plays a key role in the initial attachment of tumor cells. Identification of this potentially new lectin may lead to novel approaches to inhibit metastasis.

In conclusion, we demonstrate that a peptide mimicking a carbohydrate ligand shared by all of the three selectins can be isolated using anti-Lewis A antibody as a target molecule. The identified IELLQAR peptide inhibited sialyl Lewis X-dependent experimental tumor metastasis, which provided direct evidence for the roles of sialyl Lewis X and possibly sialyl Lewis A in tumor metastasis. These exciting results suggest that tumor metastasis may be inhibited by the injection of peptides mimicking tumor-associated carbohydrate antigens such as sialyl Lewis X.

ACKNOWLEDGMENTS
We thank Dr. John Lowe, University of Michigan Medical Center, Ann Arbor, MI for supplying pcDNAI-Fuc-TIII; Drs. Kei Kishimoto, Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT, and Shigeru Tsuboi, The Burnham Institute, La Jolla, CA for vectors encoding chimeric P- and L-selectin, respectively; and Dr. Yu Yamaguchi for brevican-chimeric protein. We also thank Drs. Edgar Ong and Atsushi Suzuki for their critical reading of the manuscript and Susan Fanno and Susan Wynant for organizing the manuscript.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by National Cancer Institute Grants P01 CA71932 (to M. N. F, M. F,), R37 CA33000 (to M. F.), CA74238 and CA28891 (to E. R.), and National Cancer Institute Cancer Center Support Grant CA30199 (to R. P.). Back

2 To whom requests for reprints should be addressed, at The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037. Phone: (858) 646-3143; Fax: (858) 646-3193; E-mail: michiko{at}burnham-inst.org Back

3 Present address: Tohoku University School of Medicine, Aoba-ku, Sendai 980, Japan. Back

4 The abbreviations used are: GD1{alpha}, NeuAc{alpha}2–3Galß1–3(NeuAc{alpha}2–6)GalNAcß1–4 Glcß1–1 Cer; LB, Luria broth; TBS, Tris-buffered saline; Fuc-TIII, {alpha}1,3-fucosyltransferase III. Back

Received 8/ 5/99. Accepted 11/11/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Feizi T. Demonstration by monoclonal antibodies that carbohydrate structures of glycoproteins and glycolipids are onco-developmental antigens. Nature (Lond.), 314: 53-57, 1985.[Medline]
  2. Hakomori S. Aberrant glycosylation in tumors and tumor-associated carbohydrate antigens. Adv. Cancer Res., 52: 257-331, 1989.[Medline]
  3. Fukuda M. Possible roles of tumor-associated carbohydrate antigens. Cancer Res., 56: 2237-2244, 1996.[Abstract/Free Full Text]
  4. Fukuda M., Spooncer E., Oates J. E., Dell A., Klock J. C. Structure of sialylated fucosyl lactosaminoglycan isolated from human granulocytes. J. Biol. Chem., 259: 10925-10935, 1984.[Abstract/Free Full Text]
  5. Mizoguchi A., Takasaki S., Maeda S., Kobata A. Changes in asparagine-linked sugar chains of human promyelocytic leukemic cells (HL-60) during monocytoid differentiation and myeloid differentiation. Decrease of high-molecular-weight oligosaccharides in acidic fraction. J. Biol. Chem., 259: 11949-11957, 1984.[Abstract/Free Full Text]
  6. Lowe J. B., Stoolman L. M., Nair R. P., Larsen R. D., Berhend T. L., Marks R. M. ELAM-1-dependent cell adhesion to vascular endothelium determined by a transfected human fucosyltransferase cDNA. Cell, 93: 10925-10935, 1990.
  7. Phillips M. L., Nudelman E., Gaeta F. C. A., Perez M., Singhal A. K., Hakomori S-I., Paulson J. C. ELAM-1 mediates cell adhesion by recognition of a carbohydrate ligand, sialyl-Lex. Science (Washington DC), 250: 1130-1132, 1990.[Abstract/Free Full Text]
  8. Walz G., Aruffo A., Kolanus W., Bevilacqua M., Seed B. Recognition by ELAM-1 of the sialyl-Lex determinant on myeloid and tumor cells. Science (Washington DC), 250: 1132-1135, 1990.[Abstract/Free Full Text]
  9. Polley M. J., Phillips M. L., Wayner E., Nudelman E., Singhal A. K., Hakomori S-I., Paulson J. C. CD62 and endothelial cell-leukocyte adhesion molecule 1 (ELAM-1) recognize the same carbohydrate ligand, sialyl-Lewisx. Proc. Natl. Acad. Sci. USA, 88: 6224-6228, 1991.[Abstract/Free Full Text]
  10. Rosen S. D., Bertozzi C. R. Leukocyte adhesion: two selectins converge on sulphate. Curr. Biol., 6: 261-264, 1996.[Medline]
  11. Hiraoka N., Petryniak B., Nakayama J., Tsuboi S., Suzuki M., Yeh J-C., Izawa D., Tanaka T., Miyasaka M., Lowe J. B., Fukuda M. A novel, high endothelial venule-specific sulfotransferase directs expresion of 6-sulfo sialyl Lewisx, an L-selectin ligand displayed by CD34. Immunity, 11: 79-89, 1999.[Medline]
  12. Bistrup A., Bhakta S., Lee J. K., Belov Y. Y., Gunn M. D., Zuo F-R., Huang C-C., Kannagi R., Rosen S. D., Hemmerich S. Sulfotransferase of two specificities function in the reconstitution of high endothelial cell ligands for L-selectin. J. Cell Biol., 145: 899-910, 1999.[Abstract/Free Full Text]
  13. Itzkowitz S. H., Yuan M., Fukushi Y., Lee H., Shi Z., Zurawski V., Jr., Hakomori S., Kim Y. S. Immunohistochemical comparison of Lea, monosialosyl Lea (CA 19–9) and disialosyl Lea antigens in human colorectal and pancreatic tissues. Cancer Res., 48: 3834-3842, 1988.[Abstract/Free Full Text]
  14. Fukushima K., Hirota M., Terasaki P. I., Wakasaka A., Togashi H., Chia D., Suyama N., Fukushi Y., Nudelman E., Hakomori S-I. Characterization of sialylated Lewis X as a new tumor-associated antigen. Cancer Res., 44: 5279-5285, 1984.[Abstract/Free Full Text]
  15. Takada A., Ohmori K., Yoneda T., Tsuyuoka K., Hasegawa A., Kiso M., Kannagi R. Contribution of carbohydrate antigens sialyl Lewis A and sialyl Lewis X to adhesion of human cancer cells to vascular endothelium. Cancer Res., 53: 354-361, 1993.[Abstract/Free Full Text]
  16. Fukushi Y., Nudelman E., Levery S. B., Hakomori S., Rauvala H. Novel fucolipids accumulating in human adenocarcinoma. III. A hybridoma antibody (FH6) defining a human cancer-associated difucoganglioside (VI3NeuAcV3III3 Fuc2nLc6). J. Biol. Chem., 259: 10511-10517, 1984.[Abstract/Free Full Text]
  17. Berg E. L., Robinson M. K., Mansson O., Butcher E. C., Magnani J. L. A carbohydrate domain common to both sialyl Lea and sialyl LeX is recognized by the endothelial cell leukocyte adhesion molecule ELAM-1. J. Biol. Chem., 266: 14869-14872, 1991.[Abstract/Free Full Text]
  18. Nakamori S., Kameyama M., Imaoka S., Furukawa H., Ishikawa O., Sasaki Y., Kabuto T., Iwanaga T., Matsushita Y., Irimura T. Increased expression of sialyl Lewis X antigen correlates with poor survival in patients with colorectal carcinoma: clinicopathological and immunohistochemical study. Cancer Res., 53: 3632-3637, 1993.[Abstract/Free Full Text]
  19. Shimodaira K., Nakayama J., Nakamura N., Hasebe O., Katsuyama T., Fukuda M. Carcinoma-associated expression of core 2 ß-1,6-N-acetylglucosaminyltransferase gene in human colorectal cancer: role of O-glycans in tumor progression. Cancer Res., 57: 5201-5206, 1997.[Abstract/Free Full Text]
  20. Sawada R., Tsuboi S., Fukuda M. Differential E-selectin dependent adhesion efficiency in sublines of a human colon cancer exhibiting distinct metastatic potentials. J. Biol. Chem., 269: 1425-1431, 1994.[Abstract/Free Full Text]
  21. Martin-Satué M., Marrugat R., Cancelas J. A., Blanco J. Enhanced expression of {alpha}(1,3)-fucosyltransferase genes correlates with E-selectin-mediated adhesion and metastatic potential of human lung adenocarcinoma cells. Cancer Res., 58: 1544-1550, 1998.[Abstract/Free Full Text]
  22. Ohyama C., Tsuboi S., Fukuda M. Dual roles of sialyl Lewis X oligosaccharides in tumor metastasis and rejection by natural killer cells. EMBO J., 18: 1516-1525, 1999.[Medline]
  23. Martin-Satué M., de Castellarnau C., Blanco J. Overexpression of {alpha}(1,3)-fucosyltransferase VII is sufficient for the acquisition of lung colonization phenotype in human lung adenocarcinoma HAL-24Luc cells. Br. J. Cancer, 80: 1169-1174, 1999.[Medline]
  24. Mulligan M. S., Lowe J. B., Larsen R. D., Paulson J., Zheng Z. L., DeFrees S., Maemura K., Fukuda M., Ward P. A. Protective effects of sialylated oligosaccharides in immune complex-induced acute lung injury. J. Exp. Med., 178: 623-631, 1993.[Abstract/Free Full Text]
  25. Mulligan M. S., Paulson J. C., De Frees S., Zheng Z. L., Lowe J. B., Ward P. A. Protective effects of oligosaccharides in P-selectin-dependent lung injury. Nature (Lond.), 364: 149-151, 1993.[Medline]
  26. Buerke M., Weyrich A. S., Zheng Z., Gaeta F. C., Forrest M. J., Lefer A. M. Sialyl Lewis X-containing oligosaccharide attenuates myocardial reperfusion injury in cats. J. Clin. Invest., 93: 1140-1148, 1994.
  27. Koivunen E., Wang B., Ruoslahti E. Isolation of a highly specific ligand for the {alpha}5ß1 integrin from a phage display library. J. Cell Biol., 124: 373-380, 1994.[Abstract/Free Full Text]
  28. Healy J. M., Murayama O., Maeda T., Yoshino K., Sekiguchi K., Kikuchi M. Peptide ligands for integrin {alpha}vß3 selected from random phage display libraries. Biochemistry, 34: 3948-3955, 1995.[Medline]
  29. Pasqualini R., Ruoslahti E. Organ targeting in vivo using phage display peptide libraries. Nature (Lond.), 380: 364-366, 1996.[Medline]
  30. Hoess R., Brinkmann U., Handel T., Pastan I. Identification of a peptide which binds to the carbohydrate-specific monoclonal antibody B3. Gene, 128: 43-49, 1993.[Medline]
  31. Ishikawa D., Kikkawa H., Ogino K., Hirabayashi Y., Oku N., Taki T. GD1{alpha}-replica peptides functionally mimic GD1{alpha}, an adhesion molecule of metastic tumor cells, and suppress the tumor metastasis. FEBS Lett., 441: 20-24, 1998.[Medline]
  32. Scott J. K., Smith G. P. Searching for peptide ligands with an epitope library. Science (Washington DC), 249: 386-390, 1990.[Abstract/Free Full Text]
  33. Aruffo A., Kolanus W., Walz G., Fredman P., Seed B. CD62/P-selection recognition of myeloid and tumor cell sulfatides. Cell, 67: 35-44, 1991.[Medline]
  34. Sueyoshi S., Tsuboi S., Sawada-Hirai R., Dang U. N., Lowe J. B., Fukuda M. Expression of distinct fucosylated oligosaccharides and carbohydrate-mediated adhesion efficiency directed by two different {alpha}-1,3-fucosyltransferases. Comparison of E- and L-selectin-mediated adhesion. J. Biol. Chem., 269: 32342-32350, 1994.[Abstract/Free Full Text]
  35. Tsuboi S., Isogai Y., Hada N., King J. K., Hindsgaul O., Fukuda M. 6'-Sulfo sialyl Lex but not 6-sulfo sialyl Lex expressed on the cell surface supports L-selectin-mediated adhesion. J. Biol. Chem., 271: 27213-27216, 1996.[Abstract/Free Full Text]
  36. Tsuboi S., Fukuda M. Branched O-linked oligosaccharides ectopically expressed in transgenic mice reduce primary T-cell immune responses. EMBO J., 16: 6364-6373, 1997.[Medline]
  37. Miura R., Aspberg A., Ethell I. M., Hagihara K., Schnaar R. L., Ruoslahti E., Yamaguchi Y. The proteoglycan lectin domain binds sulfated cell surface glycolipids and promotes cell adhesion. J. Biol. Chem., 274: 11431-11438, 1999.[Abstract/Free Full Text]
  38. Weitz-Schmidt G., Stokmaier D., Scheel G., Nifant’ev N. E., Tuzikov A. B., Bovin N. V. An E-selectin binding assay based on a polyacrylamide-type glycoconjugate. Anal. Biochem., 238: 184-190, 1996.[Medline]
  39. Kukowska-Latallo J. F., Larsen R. D., Nair R. P., Lowe J. B. A cloned human cDNA determines expression of a mouse stage-specific embryonic antigen and the Lewis blood group {alpha}(1,3/1,4)fucosyltransferase. Genes Dev., 4: 1288-1303, 1990.[Abstract/Free Full Text]
  40. Inufusa H., Kojima N., Yasutomi M., Hakomori S. Human lung adenocarcinoma cells with different lung colonization potential (LCP), and a correlation between expression of sialosyl dimeric Lex (defined by Mab FH6) and LCP. Clin. Exp. Metastasis, 9: 245-257, 1991.[Medline]
  41. Martens C. L., Cwirla S. E., Lee R. Y., Whitehorn E., Chen E. Y-F., Bakker A., Martin E. L., Wangstrom C., Gopalan P., Smith C. W., Tate E., Koller K. J., Schatz P. J., Dower W. J., Barrett R. W. Peptides which bind to E-selectin and block neutrophil adhesion. J. Biol. Chem., 270: 21129-21136, 1995.[Abstract/Free Full Text]
  42. Nelson R. M., Dolich S., Aruffo A., Cecconi O., Bevilacqua M. P. Higher-affinity oligosaccharide ligands for E-selectin. J. Clin. Invest., 91: 1157-1166, 1993.
  43. Welply J. K., Abbas S. Z., Scudder P., Keene J. L., Broschat K., Casnocha S., Gorka C., Steininger C., Howard S. C., Schmuke J. J., Graneto M., Rotsaert J. M., Manger I. D., Jacob G. S. Multivalent sialyl-LeX: potent inhibitors of E-selectin-mediated cell adhesion; reagent for staining activated endothelial cells. Glycobiology, 4: 259-265, 1994.[Abstract/Free Full Text]
  44. Harris S. L., Craig L., Methroke J. S., Rashed M., Zwick M. B., Kenar K., Toone E. J., Greenspan N., Auzanneau F. I., Marino-Albernas J-R., Pinto M. B., Scoot J. K. Exploring the basis of peptide-carbohydrate crossreactivity: evidence for discrimination by peptides between closely related anti-carbohydrate antibodies. Proc. Natl. Acad. Sci. USA, 94: 2454-2459, 1997.[Abstract/Free Full Text]
  45. Taki T., Ishikawa D., Hamasaki H., Handa S. Preparation of peptides which mimic glycosphingolipids by using phage petide library and their modulation on ß-galactosidase activity. FEBS Lett., 418: 219-223, 1997.[Medline]
  46. Lou Q., Pastan I. A LewisY epitope mimicking peptide induces anti-LewisY immune responses in rabbits and mice. J. Pept. Res., 53: 252-260, 1999.[Medline]
  47. Hanjan S. N., Kearney J. F., Cooper M. D. A monoclonal antibody (MMA) that identifies a differentiation antigen on human myelomonocytic cells. Clin. Immunol. Immunopathol., 23: 172-188, 1982.[Medline]
  48. Magnani J. L., Ball E. D., Fanger M. W., Hakomori S. I., Ginsburg V. Monoclonal antibodies PMN 6, PMN 29, and PM-81 bind differently to glycolipids containing a sugar sequence occurring in lacto-N-fucopentaose III. Arch. Biochem. Biophys., 233: 501-506, 1984.[Medline]
  49. Gooi H. C., Feizi T., Kapadia A., Knowles B. B., Solter D., Evans M. J. Stage-specific embryonic antigen involves {alpha}1 goes to 3 fucosylated type 2 blood group chains. Nature (Lond.), 292: 156-158, 1981.[Medline]
  50. Foxall C., Watson S. R., Dowbenko D., Fennie C., Lasky L. A., Kiso M., Hasegawa A., Asa D., Brandley B. K. The three members of the selectin receptor family recognize a common carbohydrate epitope, the sialyl Lewisx oligosaccharide. J. Cell Biol., 117: 895-902, 1992.[Abstract/Free Full Text]
  51. Kansas G. S. Selectins and their ligands: current concepts and controversies. Blood, 88: 3259-3287, 1996.[Free Full Text]
  52. Izumi Y., Taniuchi Y., Tsuji T., Smith C. W., Nakamori S., Fidler I. J., Irimura T. Characterization of human colon carcinoma variant cells selected for sialyl LeX carbohydrate antigen: liver colonization and adhesion to vascular endothelial cells. Exp. Cell Res., 216: 215-221, 1995.[Medline]
  53. Renkonen J., Paavonen T., Renkonen R. Endothelial and epithealial expression of sialyl LeX and sialyl Lea in lesions of breast carcinoma. Int. J. Cancer, 74: 296-300, 1997.[Medline]
  54. Khatib A-M., Kontogiannea M., Fallavollita L., Jamison B., Meterissian S., Brodt P. Rapid induction of cytokine and E-selectin expression in the liver in response to metastatic tumor cells. Cancer Res., 59: 1356-1361, 1999.[Abstract/Free Full Text]
  55. Handa K., White T., Ito K., Fang H., Wang S., Hakomori S. 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: 773-781, 1995.
  56. Matsumoto N., Ribaudo R. K., Abastado J-P., Margulies D. H., Yokoyama W. M. The lectin-like NK cell receptor Ly-49A recognizes a carbohydrate-independentepitpoe on its MHC class I ligand. Immunity, 8: 245-254, 1998.[Medline]
  57. Fiete D., Beranek M. C., Baenziger J. U. The macrophage/endothelial cell mannose receptor cDNA encode a protein that binds oligosaccharides terminating with SO4-4-GalNAcß1,4GlcNAcß or Man at independent sites. Proc. Natl. Acad. Sci. USA, 94: 11256-11261, 1997.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
S. Hatakeyama, K. Sugihara, J. Nakayama, T. O. Akama, S.-M. A. Wong, H. Kawashima, J. Zhang, D. F. Smith, C. Ohyama, M. Fukuda, et al.
Identification of mRNA splicing factors as the endothelial receptor for carbohydrate-dependent lung colonization of cancer cells
PNAS, March 3, 2009; 106(9): 3095 - 3100.
[Abstract] [Full Text] [PDF]

Home page
 RadiologyHome page
J. B. Kruskal, A. Azouz, H. Korideck, M. El-Hallak, S. C. Robson, P. Thomas, and S. N. Goldberg
Hepatic Colorectal Cancer Metastases: Imaging Initial Steps of Formation in Mice
Radiology, June 1, 2007; 243(3): 703 - 711.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
K. Sugihara, D. Sugiyama, J. Byrne, D. P. Wolf, K. P. Lowitz, Y. Kobayashi, M. Kabir-Salmani, D. Nadano, D. Aoki, S. Nozawa, et al.
Trophoblast cell activation by trophinin ligation is implicated in human embryo implantation
PNAS, March 6, 2007; 104(10): 3799 - 3804.
[Abstract] [Full Text] [PDF]

Home page
 GlycobiologyHome page
W. Chen, J. Tang, and P. Stanley
Suppressors of {alpha}(1,3)fucosylation identified by expression cloning in the LEC11B gain-of-function CHO mutant
Glycobiology, March 1, 2005; 15(3): 259 - 269.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
J. Zou, V. V. Glinsky, L. A. Landon, L. Matthews, and S. L. Deutscher
Peptides specific to the galectin-3 carbohydrate recognition domain inhibit metastasis-associated cancer cell adhesion
Carcinogenesis, February 1, 2005; 26(2): 309 - 318.
[Abstract] [Full Text] [PDF]

Home page
 J. Histochem. Cytochem.Home page
J. Pan, H. Yeger, and E. Cutz
Neuronal Developmental Marker FORSE-1 Identifies a Putative Progenitor of the Pulmonary Neuroendocrine Cell Lineage During Lung Development
J. Histochem. Cytochem., December 1, 2002; 50(12): 1567 - 1578.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
C. Ohyama, S. Kanto, K. Kato, O. Nakano, Y. Arai, T. Kato, S. Chen, M. N. Fukuda, and M. Fukuda
Natural killer cells attack tumor cells expressing high levels of sialyl Lewis x oligosaccharides
PNAS, October 15, 2002; 99(21): 13789 - 13794.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
J. Zhang, J. Nakayama, C. Ohyama, M. Suzuki, A. Suzuki, M. Fukuda, and M. N. Fukuda
Sialyl Lewis X-dependent Lung Colonization of B16 Melanoma Cells through a Selectin-like Endothelial Receptor Distinct from E- or P-Selectin
Cancer Res., August 1, 2002; 62(15): 4194 - 4198.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
S.-i. Hakomori
The glycosynapse
PNAS, January 1, 2002; (2002) 12540899.
[Abstract] [Full Text] [PDF]

Home page
 GlycobiologyHome page
T. Jouault, C. Fradin, F. Dzierszinski, M. Borg-Von-Zepelin, S. Tomavo, R. Corman, P.-A. Trinel, J.-P. Kerckaert, and D. Poulain
Peptides that mimic Candida albicans-derived {beta}-1,2-linked mannosides
Glycobiology, August 1, 2001; 11(8): 693 - 701.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
S.-i. Hakomori
Inaugural Article: The glycosynapse
PNAS, January 8, 2002; 99(1): 225 - 232.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fukuda, M. N.
Right arrow Articles by Fukuda, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fukuda, M. N.
Right arrow Articles by Fukuda, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online