Expression of sialyl 6-sulfo Lewis X is inversely correlated with conventional sialyl Lewis X expression in human colorectal cancer

Sialyl 6-sulfo Lewis X determinant has been described recently as a major ligand for L-selectin on high endothelial venules of human peripheral lymph nodes. From our investigation of its distribution in human colorectal cancer tissues and cultured colon cancer cells, the sialyl 6-sulfo Lewis X determinant was preferentially expressed in the nonmalignant colonic epithelia rather than cancer cells (P < 0.001; n = 23). This was in contrast to the distribution of conventional sialyl Lewis X, which was preferentially expressed in cancer tissues rather than nonmalignant epithelia (P = 0.007; n = 23), indicating that 6-sulfation predominantly occurs in nonmalignant tissues and is suppressed upon malignant transformation. In confirmation of this, a nonsialylated determinant 6-sulfo Lewis X was also found to be preferentially localized in the nonmalignant epithelia. Significant expression of sialyl 6-sulfo Lewis X was observed in only 2 lines, whereas 8 were positive for conventional sialyl Lewis X, among 13 cultured colon cancer cell lines. Transfection of cells with fucosyltransferase (Fuc-T) VI induced expression of sialyl 6-sulfo Lewis X, whereas transfection of Fuc-T III did not, suggesting that the determinant was synthesized mainly by Fuc-T VI in colonic epithelia. Members of the sialic acid cyclase pathway, the de-N-acetyl sialyl 6-sulfo Lewis X and cyclic sialyl 6-sulfo Lewis X determinants, were also preferentially expressed in the nonmalignant epithelia rather than colonic cancer cells (P < 0.001; n = 23). Stimulation of the sialyl 6-sulfo Lewis X-positive colon cancer cell line with a calcium ionophore ionomycin markedly reduced sialyl 6-sulfo Lewis X and induced cyclic sialyl 6-sulfo Lewis X expression. These results suggested that the metabolic conversion of sialyl 6-sulfo Lewis X into cyclic sialyl 6-sulfo Lewis X by a calcium-dependent enzyme, sialic acid cyclase, as we hypothesized for human leukocytes previously (C. Mitsuoka et al., Proc. Natl. Acad. Sci. USA, 96: 1597-1602, 1999), also occurs in nonmalignant colonic epithelia.     

Attachment of human colon cancer cells to vascular endothelium is enhanced by N-acetylglucosaminyltransferase V

Expression of N-acetylglucosaminyltransferase V (GnT-V) in colon cancer has been shown to be related to hematogenous metastasis and poor prognosis. To investigate the mechanism by which cancer cells expressing GnT-V metastasize to distant organs, we established GnT-V-overexpressing DLD-1 and WiDr cells (human colon cancer cell lines) by transfecting them with a GnT-V expression vector. Attachment to endothelial cells expressing E-selectin was studied, and expression of the E-selectin ligand, sialyl Lewis x, in colon cancer cells was investigated. Both of the cell lines showed reduced adhesion to fibronectin as compared with mock transfectants. In contrast, attachment to human umbilical vein endothelial cells expressing E-selectin was significantly enhanced by GnT-V expression (p < 0.01). Sialyl Lewis x is a ligand for E-selectin and a marker for poor prognosis of colon cancer. Its synthesis in cells has been shown to involve GnT-V. We demonstrated that expression of sialyl Lewis x in colon cancer cells was induced by GnT-V expression. These results suggest that GnT-V induces sialyl Lewis x expression and leads colon cancer cells to metastasize by enhancing their ability to attach to vascular endothelium in distant organs, such as liver or lung. Inhibition of GnT-V activity may prevent metastasis in colon cancer patients with high sialyl Lewis x expression.     

Reduced sialidase expression in highly metastatic variants of mouse colon adenocarcinoma 26 and retardation of their metastatic ability by sialidase overexpression.

Sialidase expression levels are inversely correlated with the metastatic potential of mouse colon adenocarcinoma 26 sublines, as assessed by activity assays and RT-PCR, irrespective of total and cell surface sialic acid contents. Compared with low metastatic NL4 and NL44 cell lines, the highly metastatic NL17 and NL22 cells exhibit low expression of sialidases, accompanied with higher levels of sialylLe(x) and GM3. To investigate whether these properties of NL17 cells can be altered by sialidase overexpression, we transfected a cytosolic sialidase gene into NL17 cells. The result was markedly inhibited lung metastasis, invasion and cell motility with a concomitant decrease in sialylLe(x) and GM3 levels, in line with the case of spontaneously low metastatic sublines having relatively high endogenous sialidase levels, implying that sialidase level is a determining factor affecting metastatic ability. Treatment of the cells with antibodies against sialylLe(x) and GM3 affected cell adhesion and/or cell motility, providing evidence that desialylation of these molecules, as targets of sialidase, is involved in the suppression of metastasis.     

Karyotype evolution in B-cell lymphoid malignancy with an 8;14 translocation

To evaluate the significance of karyotypic evolution of tumor cells with an 8;14 translocation [t(8;14)(q24;q32)], we examined the clinicopathologic features and immunologic phenotypes of nine Japanese patients with various types of B-cell malignancy with the translocation. All these patients had structural rearrangements of the long arm of chromosome No. I (Iq) in a stem line or the subline of tumor cells with a t(8;14). The rearrangements were composed of a translocation involving Iq with other chromosomes and a tandem duplication of Iq, and they were exclusively associated with a partial trisomy for Iq. Two patients with diffuse large-cell lymphoma, whose tumor cells did not express surface immunoglobulins (s-Ig), had the Iq translocation in their highly complex karyotypes. Tumor cells from the other seven patients expressed s-Ig and the karyotypes were relatively simple. Among these patients, the Iq translocation was found in two patients with Burkitt's lymphoma, and the Iq duplication were observed in a stem line or the sublines from four patients with Burkitt's lymphoma-leukemia and one each with small non-cleaved-cell or diffuse large-cell lymphoma. Except for one patient in the stage of IE, these patients had a poor prognosis because of the clinical conversion of extranodal metastases in the earlier disease phase. These findings are compatible with those of Western patients with a t(8;14). Therefore, tumor cells marked primarily with a t(8;14) could have "major routes" in the karyotypic evolution, for which potentials should be recognized as clinical risk factors, and the morphologic presentation and the expression of surface immunoglobulins may be associated with the process of karyotypic evolution.     

Sequential change of carbohydrate antigen associated with differentiation of murine leukemia cells: i-I antigenic conversion and shifting of glycolipid synthesis.

Cell surface carbohydrate antigens and their metabolism were investigated during the course of differentiation of murine cultured leukemia cells (M1) into macrophage-like cells. The major glycolipids in undifferentiated M1 cells were of the ganglio series, with a small amount of lacto-series glycolipids. A novel branched structure was found as a tetraosylceramide of M1- cells. Upon differentiation, synthesis of lacto-series glycolipids was significantly enhanced and synthesis of globo-series glycolipids was newly induced but the ganglio-series synthesis was much reduced. Undifferentiated cells expressed only i antigen (i+I-Pk-); differentiated macrophage-like cells became I-antigen dominant and Pk-antigen positive (i+/-I+Pk+). The changes proceeded in two sequential steps: (i) an enhancement of lacto-series glycolipid synthesis associated with the conversion of i antigen to I antigen, and (ii) subsequent induction of globo-series glycolipid synthesis accompanied by the appearance of Pk antigen. The experimental system offers a clue for studies on the process of branching (i-to-I conversion) as well as the biological significance of three major glycolipids (globo-, lacto-, and ganglio-series) as markers of cell differentiation.     

Accumulation of highly acidic sulfated glycosphingolipids in human hepatocellular carcinoma defined by a series of monoclonal antibodies

An accumulation of sulfated and very complex, highly acidic glycolipids was observed in cultured human hepatocellular carcinoma cells. Among the cells tested, PLC/PRF/5 cells contained a significant amount of very complex sulfated acidic glycolipids, and HepG2 cells were characterized as having a large amount of relatively simple sulfated glycolipids. Several monoclonal antibodies (all IgM) directed to these sulfated and highly acidic glycolipids were established. Among them, 49-D6 and 7-E10 were both directed to SM3 (LacCer-II3-sulfate), a relatively simple sulfated glycolipid, and 34-A4 was directed to SD1a (GgOse4Cer II3,IV3-disulfate) and more complex sulfated glycolipids. The other four antibodies, 26-A10, 34-B9, 79-C8, and 16-E10, reacted with unknown highly acidic glycolipids, which were eluted in 0.9-2.7 M ammonium acetate in DEAE chromatography, indicating that these antigenic glycolipids were far more acidic than the usual glycolipids described until now. Analysis of the glycolipids extracted from the hepatocellular carcinoma tissues and cirrhotic livers of patients and from a normal liver with these monoclonal antibodies revealed that sulfated glycolipids having simple carbohydrate structures such as SM3 accumulated significantly in the cirrhotic liver (2 of 4 cases) as well as hepatocellular carcinoma tissue (15 of 17 cases, 88%), and more complex sulfated glycolipids and highly acidic glycolipids were much more specific to hepatocellular carcinoma tissues (10 of 17 cases, 59%) compared to the cirrhotic liver (0 of 4 cases).