Enhancement of metastatic ability by ectopic expression of ST6GalNAcI on a gastric cancer cell line in a mouse model.

ST6GalNAcI is a sialyltransferase responsible for the synthesis of sialyl Tn (sTn) antigen which is expressed in a variety of adenocarcinomas including gastric cancer especially in advanced cases, but the roles of ST6GalNAcI and sTn in cancer progression are largely unknown. We generated sTn-expressing human gastric cancer cells by ectopic expression of ST6GalNAcI to evaluate metastatic ability of these cells and prognostic effect of ST6GalNAcI and sTn in a mouse model, and identified sTn carrier proteins to gain insight into the function of ST6GalNAcI and sTn in gastric cancer progression. A green fluorescent protein-tagged human gastric cancer cell line was transfected with ST6GalNAcI to produce sTn-expressing cells, which were transplanted into nude mice. STn-positive gastric cancer cells showed higher intraperitoneal metastatic ability in comparison with sTn-negative control, resulting in shortened survival time of the mice, which was mitigated by anti-sTn antibody administration. Then, sTn-carrying proteins were immunoprecipitated from culture supernatants and lysates of these cells, and identified MUC1 and CD44 as major sTn carriers. It was confirmed that MUC1 carries sTn also in human advanced gastric cancer tissues. Identification of sTn carrier proteins will help understand mechanisms of metastatic phenotype acquisition of gastric cancer cells by ST6GalNAcI and sTn.

CD43, but not P-selectin glycoprotein ligand-1, functions as an E-selectin counter-receptor in human pre-B-cell leukemia NALL-1.

B-cell precursor acute lymphoblastic leukemia (BCP-ALL/B-precursor ALL) is characterized by a high rate of tissue infiltration. The mechanism of BCP-ALL cell extravasation is not fully understood. In the present study, we have investigated the major carrier of carbohydrate selectin ligands in the BCP-ALL cell line NALL-1 and its possible role in the extravascular infiltration of the leukemic cells. B-precursor ALL cell lines and clinical samples from patients with BCP-ALL essentially exhibited positive flow cytometric reactivity with E-selectin, and the reactivity was significantly diminished by O-sialoglycoprotein endopeptidase treatment in NALL-1 cells. B-precursor ALL cell lines adhered well to E-selectin but only very weakly to P-selectin with low-shear-force cell adhesion assay. Although BCP-ALL cell lines did not express the well-known core protein P-selectin glycoprotein ligand-1 (PSGL-1), a major proportion of the carbohydrate selectin ligand was carried by a sialomucin, CD43, in NALL-1 cells. Most clinical samples from patients with BCP-ALL exhibited a PSGL-1(neg/low)/CD43(high) phenotype. NALL-1 cells rolled well on E-selectin, but knockdown of CD43 on NALL-1 cells resulted in reduced rolling activity on E-selectin. In addition, the CD43 knockdown NALL-1 cells showed decreased tissue engraftment compared with the control cells when introduced into gamma-irradiated immunodeficient mice. These results strongly suggest that CD43 but not PSGL-1 plays an important role in the extravascular infiltration of NALL-1 cells and that the degree of tissue engraftment of B-precursor ALL cells may be controlled by manipulating CD43 expression.

E2F-6 suppresses growth-associated apoptosis of human hematopoietic progenitor cells by counteracting proapoptotic activity of E2F-1.

E2F-6 is a dominant-negative transcriptional repressor against other members of the E2F family. In this study, we investigated the expression and function of E2F-6 in human hematopoietic progenitor cells to clarify its role in hematopoiesis. We found that among E2F subunits, E2F-1, E2F-2, E2F-4, and E2F-6 were expressed in CD34(+) human hematopoietic progenitor cells. The expression of E2F-6 increased along with proliferation and decreased during differentiation of hematopoietic progenitors, whereas the other three species were upregulated in CD34(-) bone marrow mononuclear cells. Overexpression of E2F-6 did not affect the growth of immature hematopoietic cell line K562 but suppressed E2F-1-induced apoptosis, whereas it failed to inhibit apoptosis induced by differentiation inducers and anticancer drugs. Among E2F-1-dependent apoptosis-related molecules, E2F-6 specifically inhibited upregulation of Apaf-1 by competing with E2F-1 for promoter binding. E2F-6 similarly suppressed apoptosis and Apaf-1 upregulation in primary hematopoietic progenitor cells during cytokine-induced proliferation but had no effect when they were differentiated. As a result, E2F-6 enhanced the clonogenic growth of colony-forming unit-granulocyte, erythroid, macrophage, and megakaryocyte. These results suggest that E2F-6 provides a failsafe mechanism against loss of hematopoietic progenitor cells during proliferation. Disclosure of potential conflicts of interest is found at the end of this article.

Not core 2 beta 1,6-N-acetylglucosaminyltransferase-2 or -3 but -1 regulates sialyl-Lewis x expression in human precursor B cells.

Sialyl-Lewis x (sLeX), one of the major selectin ligands, is expressed on T and B cells in a differentiation or activation stage-specific manner. We have demonstrated before that sLeX expression and core 2 beta 1,6-N-acetylglucosaminyltransferase (C2GnT) were simultaneously regulated during precursor B (pre-B) cell differentiation. Three C2GnT family genes, designated C2GnT-1, -2, and -3, were previously identified, but their roles have not been fully examined. In this study, we have investigated the roles of C2GnTs in the regulation of sLeX expression level during pre-B cell differentiation comparing with alpha 1,3fucosyltransferase-VII (FucT-VII) and alpha 2,3sialyltransferase-IV (ST3Gal-IV). Overexpression of not FucT-VII and ST3Gal-IV but C2GnT-1 blocked the down-regulation of sLeX expression by differentiation induction. Neither C2GnT-2 nor -3 but C2GnT-1 transcript was mainly expressed in B lineage cell lines and bone marrow-derived B lineage cells. Significant down-regulation of C2GnT-1 of the three C2GnTs was observed in KM3 cells during differentiation. The expression of C2GnT-1 correlated well to sLeX expression and differentiation stage. Furthermore, introduction of short interfering RNA against C2GnT-1 markedly reduced C2GnT-1 expression and resulted in down-regulation of sLeX expression. These results suggest that not the other glycosyltransferases but C2GnT-1 regulates sLeX expression level during differentiation of pre-B cells, providing the cells with substrate of sLeX structure biosynthesis.