sLeX Expression Research Articles

Expression of nm23-H1 Gene and Sialyl Lewis X Antigen in Breast Cancer

The nm23 gene has been proposed as a candidate tumor metastasis suppressor in some human cancers. Sialyl Lewis X (sLex) has been demonstrated to play an important role in the adhesion of human cancer cells to human vascular endothelium, inducing metastasis. Little information has been reported about the correlation between the expression of nm23 and sialylated carbohydrate antigens. In the present study, 102 surgically resected primary breast cancer tissues were sectioned and stained with antibody against nm23-H1 and sLex. Of the 102 cases, 39 (38.2%) cases with a reduced expression of nm23-H1 were observed, and the numbers of sLex-positive cases were 61 (59.8%), respectively. The reduced expression of nm23-H1 and the positive expression of sLex were significantly associated with lymph node involvement. Among the 100 patients who underwent curative surgery, the disease-free survival rate was significantly correlated to both the nm23-H1 and sLex expressions. No interrelated expressions were found between nm23-H1 and sLex. In multivariate analysis using Cox regression model, combination assay of nm23-H1 and sLex expression emerged as independent significant prognostic factors. These results suggest that nm23-H1 gene and sLex may be involved in different steps of the metastatic process in human breast cancer, and immunohistochemical detection of the combination of sLex and nm23-H1 may be a biologic marker of prognostic significance.

Correlation between nm23 protein and several cell adhesion molecules in human gastric carcinoma.

The correlation between nm23 protein (nm23) expression and the expression of several cell adhesion molecules was studied immunohistochemically in 110 resected gastric carcinomas. Formalin‐fixed and paraffin‐embedded samples were serially sectioned and stained with antibodies against nm23, integrin β1 subfamily members (α2β1, α3β1 and α4β1), LFA‐1, ICAM‐1, sialyl Lewisx (sLex) and CD44h, ‐V3, and ‐V6. Primary carcinomas presenting with either lymph node involvement or liver metastasis expressed significantly reduced levels of nm23 compared to tumors without metastasis. The percent of tumors expressing each adhesion molecule was as follows: α2β1, 27.3%; α3β1, 20.0%; α4β1, 14.5%; LFA‐1, 14.5%; ICAM‐1, 12.7%; sLex, 67.3%; CD44h, 55.5%; CD44V3, 20.0%; and CD44V6, 4.5%. Expression of α2β1 integrin and high levels of sLex were significantly correlated with lymph node metastasis, and expression of α3β1 integrin and high levels of sLex were correlated with liver metastasis. Expression of ICAM‐1 was inversely correlated with liver metastasis. Comparing the expression of each cell adhesion molecule with nm23 immunoreactivity, expression of sLex was significantly associated with nm23 expression. Of tumors expressing high levels of sLex, 75% showed reduced nm23 expression, compared to 52% of tumors with weak or no sLex expression (P < 0.05). A similar tendency was also observed in the metastasized secondary tumors. These results suggest that reduced nm23 expression may promote the metastatic properties of cancer cells in concert with increased sLex expression.

S27.4. Tumor cell transmigration across cultured endothelium and invasion through reconstituted basement membrane in vitro

Sialyl Lewis antigens (sLex/a) are cancer-associated carbohydrate determinants, serve as ligands of the selectin family and are associated with hematogenous metastasis of cancer. So far, the clinicopathologic values of sialyl Lewis x (sLex) and sialyl Lewis a (sLea) in lung cancer have remained controversial. Using immunohistochemistry, the expressions of sLex and sLea antigens, and an airway mucin (MUC5AC) protein, which was supposed to be the major carrying protein of sialyl Lewis moieties, were studied in surgically resected tumor tissues of 61 patients with stages I or II NSCLC. Thirty-two (52.5%) of the 61 studied subjects were found to be positive for expression of sLea, 40 (65.6%) were positive for expression of sLex, and 16 (26.2%) were positive for MUC5AC protein. Both the expression of sLex and MUC5AC were associated with adenocarcinoma subtype. Patients bearing tumors with MUC5AC and/or sLex expression had a higher probability of post-operative distant metastasis. Survival analysis demonstrated that patients bearing tumors with expression of sLex antigen or MUC5AC had shorter overall survival. The multivariate logistic regression showed that age >65 years old (OR = 0.207, 95% CI = 0.075–0.569, P = 0.002), nodal status (OR = 6.575, 95% CI = 2.459–17.583, P < 0.001), and MUC5AC (OR = 5.545, 95% CI = 1.998–15.386, P = 0.001) were independent factors affecting survival. We concluded that the expression of sLex was related to MUC5AC protein, while patients with tumors co-expressing both MUC5AC and sLex antigen had the worst survival.

P-selectin and its role in the inflammatory process

The limited efficacy of monocyte-derived dendritic cell (mo-DC)-based vaccines is primarily attributed to the reduced mo-DC migratory capacity. One undefined aspect is the initial binding of mo-DCs to endothelial cells and vascular selectins. In this study, we investigated the role and modulation of the selectin binding determinant sialyl Lewisx (sLex) in selectin-dependent mo-DC binding. Our data reveal that sLex is required for maximal binding of mo-DCs to tumor necrosis factor (TNF)-α-activated endothelial cells under static conditions, as evidenced by the use of sialidase. Sialidase treatment also abrogated mo-DC cell tethering to immobilized, purified P-, L-, or E-selectin under flow. The requirement of sLex-dependent binding of mo-DC to selectins was further substantiated by using sLex free sugar and anti-sLex antibody, which significantly suppressed mo-DC-selectin binding. P-selectin glycoprotein ligand-1 is required for mo-DC binding to both P- and L-selectin, but it is dispensable for E-selectin recognition. Interestingly, the extent of mo-DC tethering was maximal on P-selectin, followed by E- and L- selectin. Accordingly, L-selectin mediated faster mo-DC rolling than E- or P-selectin. Interferon (IFN)-γ induces a significant increase in mo-DC surface sLex expression, which is probably due to the enhanced synthesis of C2GnT-I. These findings may contribute to improving mo-DC-based vaccination protocols.

Role of O-linked carbohydrate chains on leukocyte cell membranes in platelet-induced leukocyte activation.

We previously reported that activated platelets stimulated neutrophils and monocytes to produce superoxide anion (O2-) through the interaction between P-selectin and its carbohydrate ligand, sialyl Lewis X (sLeX) (Nagata, K., Tsuji, T., Todoroki, N., Katagiri, Y., Tanoue, K., Yamazaki, H., Hanai N., and Irimura, T. (1993) J. Immunol. 151, 3267-3273). In the present study, we investigated the role of cell surface carbohydrate chains of leukocytes in this process. Glycoconjugate-specific hydrolytic enzymes and inhibitors of glycosylation processing were applied. Granulocyte-like differentiated HL-60 (gHL-60) cells released an increased amount of O2- in response to activated platelets in a P-selectin-dependent manner. When HL-60 cells were differentiated in the presence of benzyl-alpha-N-acetylgalactosaminide (Bzl-alpha-GalNAc), an inhibitor of chain elongation of O-linked carbohydrates, the enhanced generation of O2- was abrogated in parallel with decrease in the expression of sLex structure and in the adhesion capacity to activated platelets. In contrast, treatment with swainsonine or 1-deoxymannojirimycin, inhibitors of processing of N-linked carbohydrate chains, did not show such effects. O-Sialoglycoprotease treatment of gHL-60 cells decreased the activated platelet-induced O2- production with concomitant reduction of cell surface sLex expression. Treatment of these cells with N-glycanase did not affect the O2- production. These results strongly suggested that serine/threonine-linked carbohydrate chains containing sLex played an essential role in the P-selectin-mediated leukocyte activation. By Western blotting analysis of gHL-60 cell lysates, we identified two glycoproteins which carried sLex structures and were sensitive to Bzl-alpha-GalNAc treatment.

SLe(x) expression of normal CD34 positive bone marrow haemopoietic progenitor cells.

We investigated sialylated Lewis x (sLe(x)) antigen expression on CD34 positive (CD34+) haemopoietic progenitors in the bone marrow of eight healthy volunteers using monoclonal antibodies. We found that in all the samples examined, CD34+ bone marrow progenitors strongly expressed the sLe(x) antigen. This contradicts previous publications which reported sLe(x) expression on malignant blast cells but not on normal CD34+ progenitor cells.

Characterization of a specific ligand for P-selectin on myeloid cells. A minor glycoprotein with sialylated O-linked oligosaccharides

Lectin-carbohydrate recognition between the selectins and their ligands are among the earliest events in leukocyte recirculation, leukocyte recruitment into inflamed areas, and abnormal egress of leukocytes in diseases. Previously, we have described a dimeric sialoglycoprotein from myeloid cells with subunits of molecular mass = 120 kDa, which is selectively recognized by P-selectin (Moore, K.L., Stults, N.L., Diaz, S., Smith, D.F., Cummings, R.D., Varki, A., and McEver, R.P. (1992) J. Cell Biol. 188, 445-456). Here, we demonstrate that this P-selectin ligand carries alpha 2-3-linked sialic acids and the sialyl-Lewisx (SLex) tetrasaccharide motif. This glycoprotein contains < 1% of the total membrane-bound sialic acids and a very small fraction of the total SLex on neutrophil membranes. In spite of a relative resistance to sialidase digestion, the predominant form of sialic acid on the ligand is N-acetylneuraminic acid. Selective periodate oxidation of the side chain of sialic acids does not affect P-selectin binding and allows the introduction of tritium label into the truncated sialic acids. beta-Elimination with alkaline borohydride releases labeled O-linked oligosaccharides both from the labeled neutrophil ligand and from the ligand purified from HL-60 cells metabolically labeled with [3H]glucosamine. The ligand from both neutrophils and HL-60 cells is also susceptible to cleavage by the enzyme O-sialoglycoprotease from Pasteurella hemolytica. Analysis of the specificity of this enzyme suggests that the P-selectin ligand carries large numbers of closely spaced sialylated O-linked oligosaccharides. O-Sialoglycoprotease abolishes both direct binding of P-selectin to HL-60 cells and the adhesion of HL-60 cells to immobilized P-selectin, without significantly decreasing overall cell surface SLex expression. This indicates that the 120-kDa ligand may be the major determinant of P-selectin:myeloid cell interaction in vivo. Finally, based on the current and previous data, we hypothesize that the high affinity recognition site(s) of this P-selectin ligand may be derived from a "clustered saccharide patch" of sialylated fucosylated O-linked oligosaccharide sequences.