Upregulated β1-6 branch N-glycan marks early gliomagenesis but exhibited biphasic expression in the progression of astrocytic glioma

Glioma is the world’s commonest primary brain malignancy with much of its biology relating to translational and post-translational events still unknown. In this study, we investigated the clinicopathological significance of N-linked β1-6-GlcNAc branches and GnT-V enzyme in the development and progression of astrocytic glioma. Expression of GnT-V and its GlcNAc-β1-6 oligosaccharides by-product together with Con-A binding sugars were assessed immunohistochemically on tissue microarrays of 16 normal brain and 159 tissue samples of astrocytomas of variable grades and histology. Although tissues of both grade I astrocytomas and normal brain showed considerably higher GnT-V expression, GlcNAc-β1-6 expression was significantly high only in tissues of grade I astrocytomas (p < 0.001), which is attributable to elevated levels of the precursor Con-A binding sugar moieties (p < 0.001). The activity of GnT-V enzyme was found to be dependent on the degree of glioma pathogenesis, as the GlcNAc-β1-6 branched expression diminished with every progressive grade of glioma, reaching minimum in glioblastoma (p < 0.001). Having biphasic activity in gliomagenesis, the role of GnT-V in glioma was deciphered by generating different ectopic GnT-V expressions in U-87 cells, which showed the highest GnT-V expression among selected glioma cell lines. Transient GnT-V rescue was achieved in knockdown clones by transfection with GnT-V expression vector. Suppression of GnT-V in U-87 cells slowed cell proliferation with G0/G1 cell cycle phase arrest. Reduced tumorigenicity, invasiveness and cell-ECM interactions were also associated with suppressed in vitro GnT-V activity suggesting GnT-V may act as an oncoprotein. We report for the first time that GnT-V products are involved in early gliomagenesis but their reduced expression, correlating with low Con-A binding sugars level found in high tumor grades predicts the loss of total N-glycosylation in glioma development and may be of potential diagnostic and/or prognostic value in astrocytoma.     

Relationship between metastasis-associated phenotypes and N-glycan structure of surface glycoproteins in human hepatocarcinoma cells

Purpose: To study the relation of N-glycan structure on cell surface glycoproteins to the metastatic phenotypes. Methods: Two human hepatocarcinoma 7721 cell lines transfected with sense or antisense cDNA of GnT-V, named GnT-V/7721 and GnT-V-AS/7721, respectively, were adopted, because GnT-V can change the antennary number and the content of the β1,6 GlcNAc branch in N-glycans. The effects of over- and under-expression of GnT-V on the metastasis-associated phenotype of the transfected cells were investigated and compared with the cells mock-transfected with the plasmid vector. Results: In GnT-V/7721 cells, GnT-V activity was increased by 92% compared with the mock cells. HRP-labeled lectin staining of transfected cells showed elevated intensity with HRP-L-PHA and reduced intensity with HRP-ConA, suggesting the increased antennary number and content of the β1,6 GlcNAc branch in N- glycans. Analysis of the N-glycan structure of [³H]-labeled glycopeptides prepared from cell-surface [³H] glycoproteins using DSA-affinity chromatography also revealed the above change of the N-glycan structure in a more quantitative manner. GnT-V/7721 cells showed a suppressed cell attachment to fibronectin (Fn) or laminin (Ln), and increased cell migration and invasion through matrigel. In contrast, GnT-V-AS/7721 cells showed reduction of both GnT-V activity and content of the β1,6 branch in N-linked glycans, elevation of cell attachment to Fn or Ln, and decline of cell migration and invasion through matrigel. These changes were just the opposite to those in GnT-V/7721 cells. Conclusions: The alteration of N-glycan structure in surface glycoproteins resulting from the activity change of GnT-V contributes to the alterations in metastasis-associated phenotypes. The product of GnT-V, the β1,6 GlcNAc branch in N-linked glycans, is a structural factor of adhesion inhibition and invasion promotion. GnT-V is, therefore, closely related to cancer metastasis and its over-expression is an important molecular mechanism of metastasis. Received: 4 April 2000 / Accepted: 12 July 2000     

白细胞介素1β通过β1整合素糖基化修饰诱导EA.hy926细胞凋亡

目的探讨IL-1β对血管内皮细胞损伤机制。方法以EA.hy926血管内皮细胞为研究对象,利用不同浓度IL-1β刺激24 h,采用Western blot方法检测IL-1β对β1整合素、Bcl-2、Bax、Caspase-3及N-乙酞氨基葡萄糖转移-V(GnT-V)表达的影响;通过Lectin blot方法显示IL-1β对GnT-V所修饰的所有糖蛋白量的影响;免疫共沉淀方法检测IL-1β对β1整合素糖基化修饰量的变化;流式细胞术检测细胞凋亡率的变化。结果 IL-1β可从蛋白翻译水平抑制β1整合素的表达,同时可以下调Bcl-2/Bax的比例,上调Caspase-3的表达。初步揭示IL-1β诱导血管内皮细胞凋亡可能是通过β1整合素N-连接糖基化作用来完成的。结论 IL-1β可通过调控EA.hy926细胞β1整合素的糖基化修饰,增加细胞凋亡率,损伤血管内皮细胞。     

shRNA沉默GnT-V基因表达对前列腺癌PC-3细胞增殖和凋亡的影响

目的构建针对N-乙酰氨基葡萄糖转移酶V（GnT-V）的小片段发夹状RNA（shRNA）表达质粒,研究shRNA表达质粒沉默GnT-V基因后对前列腺癌PC-3细胞增殖和凋亡的影响。方法设计针对GnT-V基因的小于扰RNA（siRNA）靶序列,构建shRNA表达载体并转染PC-3细胞,通过G418筛选,建立稳定表达GnT-V基因的细胞株,采用RT-PCR和蛋白质印迹检测GnT-VmRNA和蛋白的表达,并通过CCK-8增殖实验、流式细胞仪评价GnT-VshRNA对前列腺癌PC-3细胞增殖和凋亡的影响。结果成功构建了GnT-VshRNA表达质粒,且该质粒明显下调GnT-V的表达;PC-3细胞GnT-V／1079的tuRNA和蛋白质水平的抑制率分别为76．5％和67．0％,对PC3细胞呈明显抑制效应;CcK-8增殖实验显示,与对照组相比,PC-3GnT-V／1079的增殖受到明显抑制（P〈0．01）,以48h为著;流式细胞仪检测结果表明,PC-3GnT-V／1079的凋亡率明显增加（P〈0．05）。结论shRNAGnT-V能显著降低GnT-V基因的表达水平,从而有效抑制PC-3细胞增殖。并促进细胞凋亡,该GnT-V的siRNA序列可能成为治疗前列腺癌的有效靶点。     

Reversal effect of GnT-V on the radioresistance of human nasopharyngeal carcinoma cells by alteration β1, 6-GlcNAc branched N-glycans

Radiotherapy is the primary treatment for human nasopharyngeal carcinoma (NPC), yet radioresistance remains a major obstacle to successful treatment in many cases. N-acetylglucosaminyltransferase V (GnT-V), which synthesizes β1, 6-GlcNAc branched N-glycans, is closely related to the radiosensitivity of NPC cells. However, a better understanding of the functional role of GnT-V in NPC radioresistance and the related mechanisms is urgently needed. In the present study, a radioresistant NPC cell line, CNE-2R, was established by repeated γ-irradiation. We found that GnT-V levels, as well as β1, 6-GlcNAc branched N-glycans were significantly increased in the CNE-2R cells as compared with that in the parental cells. Meanwhile, knockdown of GnT-V in the CNE-2R cells enhanced cell radiosensitivity and inhibited the formation of β1, 6-branched N-glycans. In addition, the regulated expression of GnT-V in the CNE-2R cells converted the heterogeneous N-glycosylated forms of CD147. Furthermore, swainsonine, an inhibitor of N-glycan biosynthesis, was also able to reverse the radioresistance of the CNE-2R cells. Taken together, the present study revealed a novel mechanism of GnT-V as a regulator of radioresistance in NPC cells, which may be useful for fully understanding the biological role of N-glycans in NPC radioresistance.     

GnT-V,macrophage and cancer metastasis: a common link

GnT-V generated, β1,6-branched polylactosamines are a common feature shared by normal granulocytes, monocytes, and a variety of malignant cells. Furthermore, activation of GnT-V in oncogenic transformation induces invasiveness and metastatic potential in mice as well as in humans. In view of the common expression of lymphocytic/monocytic trait, motility, and GnT-V by metastatic cancer cells, macrophage fusion hybrids were generated in vitro with Cloudman S91 mouse melanoma cells to test whether the parental traits are co-expressed in hybrids and how those are related to altered phenotypes in relation to metastasis. In fact, the fusion hybrids are highly metastatic in vivo, motile in vitro, and express macrophage-associated traits of increased GnT-V activity, β1,6 branching, and polylactosamine content. A Spontaneously formed lung melanoma metastases have been identified and characterized as host × tumor hybrid containing higher DNA content than parental cells and increased GnT-V activity [1]. The results, taken together, could reflect prior fusion of tumor-associated macrophages with cells of the primary tumor, and therefore establish a possible common link between elevated expression of GnT-V and malignant transformation, a well-known report. Moreover, the fusion hybrids with metastatic potential ranging from high to low offer a genetically matched model system, for identification and characterization of differentially expressed genes in association with metastasis, since the fusion partners are derived from the same species of mouse (DBA/2J). This revised version was published online in July 2006 with corrections to the Cover Date.