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␤1,4-galactosyltransferase V (GalT V; EC 2.4.1.38) can effectively galactosylate the GlcNAc␤13 6Man arm of the highly branched N-glycans that are characteristic of glioma. Previously, we have reported that the expression of GalT V is increased in the process of glioma. However, currently little is known about the role of GalT V in this process. In this study, the ectopic expression of GalT V could promote the invasion and survival of glioma cells and transformed astrocytes. Furthermore, decreasing the expression of GalT V in glioma cells promoted apoptosis, inhibited the invasion and migration and the ability of tumor formation in vivo, and reduced the activation of AKT. In addition, the activity of GalT V promoter could be induced by epidermal growth factor, dominant active Ras, ERK1, JNK1, and constitutively active AKT. Taken together, our results suggest that GalT V functioned as a novel glioma growth activator and might represent a novel target in glioma therapy.The carbohydrate moieties of cell surface glycoconjugates play an important role in cell adhesion and metastasis (1). One of the most prominent transformation-associated changes in the sugar chains of glycoproteins is an increase in the large N-glycans of cell surface glycoprotein (2). ␤1,4-galactosyltransferase (GalT) 2 family are the enzymes responsible for the biosynthesis of N-acetyllactosamine on N-glycans by transferring UDP-galactose to the terminal N-acetylglusamine (N-GlcNAc) residues, and this family consist of seven members, from GalT I to GalT VII (3,4).GalT V, a member of ␤1,4-galactosyltransferase (GalT) family, could effectively galactosylate the GlcNAc␤136 branch (5), which is a marker of glioma (6). The expression change of GalT V has been investigated using NIH3T3 and the highly malignant transformed cell line MTAg. Northern blot analysis has revealed that the transcript of GalT V gene increases by 2-3-fold in the transformed cells (7). Similar results have been obtained in several human cancer cell lines (8). Consistently, our previous study has shown that the expression of GalT V is increased in the process of glioma development, with the highest level in grade IV glioma (9). Despite this knowledge, currently little is known about the role of GalT V in the process of glioma formation.The experiments reported here were undertaken to further study the role of GalT V in glioma malignancy, including cell migration, invasion, growth, and survival. Our results indicate that GalT V functioned as a novel glioma growth activator and could represent a novel target in glioma therapy. EXPERIMENTAL PROCEDURESMaterials-Restriction enzymes, bovine calf serum, fetal bovine serum (FBS), DMEM, RPMI 1640 medium, and TRIzol reagent were from Invitrogen. G418, phenylmethylsulfonyl fluoride, aprotinin, pepstatin, epidermal growth factor (EGF), etoposide (VP16), and toluidine blue O were from Sigma. [␥-32 P]dATP and the ECL assay kit were from Amersham Biosciences. Sialidase was from Roche Applied Science. The following antibodies were purchased from Sa...

Transcription factor E1AF is widely known to play critical roles in tumor metastasis via directly binding to the promoters of genes involved in tumor migration and invasion. Here, we report for the first time E1AF as a novel binding partner for ubiquitously expressed Sp1 transcription factor. E1AF forms a complex with Sp1, contributes to Sp1 phosphorylation and transcriptional activity, and functions as a mediator between epidermal growth factor and Sp1 phosphorylation and activity. Sp1 functions as a carrier bringing E1AF to the promoter region, thus activating transcription of glioma-related gene for ␤1,4-galactosyltransferase V (GalT V; EC 2.4.1.38). Biologically, E1AF functions as a positive invasion regulator in glioma in cooperation with Sp1 partly via up-regulation of GalT V. This report describes a new mechanism of glioma invasion involving a cooperative effort between E1AF and Sp1 transcription factors.E1AF, a member of a subfamily of ETS domain transcription factors, is capable of regulating transcription by binding to the Ets-binding site (EBS) in the promoter of its target genes (39) and is involved in a number of processes, including neuronal pathfinding (23) and mammary gland development and male sexual function (22,25). Pathologically, E1AF plays an important role in HER2/Neu-mediated mammary oncogenesis and hepatocyte growth factor-induced cancer invasiveness and metastasis via directly binding to the promoters of genes involved in tumor migration and invasion (17,18,22,29,30,38,39,41), suggesting the contribution of E1AF to various malignant phenotypes of cancer cells. However, the mechanisms of E1AF-induced tumor metastasis remain to be discovered.Sp1 is a well-known DNA-binding nuclear protein that is widely expressed in tissues (2). It binds to GC box motifs in promoters of numerous genes involved in cell growth regulation and cancer (7), including p21 (14), caspase-8 (28), cyclin D1, and GalT V (35, 47), which effectively galactosylates the GlcNAc␤1,6 branch of N-glycans and functions as a positive regulator in glioma invasion (9,16,20). Biologically, Sp1 plays important roles in a wide variety of physiological processes, including the cell cycle, hormonal activation, apoptosis, angiogenesis, oncogenesis, etc. (10). Sp1 phosphorylation is tied to functional changes in DNA binding and promoter activation, contributes to the regulation of cell physiology, and functions as a link between various pathophysiological signals and transcription of their target genes (6).Here, we found that E1AF physically and functionally interacted with Sp1 through a glutamine-rich (Gln-rich) domain and contributed to Sp1 phosphorylation and transcriptional activity. Sp1 functioned as a carrier bringing E1AF to the region of the glioma-related gene GalT V promoter, thus activating its transcription. Furthermore, E1AF functioned as a positive invasion regulator in glioma in cooperation with Sp1. This report describes new mechanisms of glioma invasion involving cooperative efforts of E1AF and Sp1 transcription factor...

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beta1,4-Galactosyltransferase V (beta1,4GalT V; EC 2.4.1.38) is considered to be very important in glioma for expressing transformation-related highly branched N-glycans. Recently, we have characterized beta1,4GalT V as a positive growth regulator in several glioma cell lines. However, the role of beta1,4GalT V in glioma therapy has not been clearly reported. In this study, interfering with the expression of beta1,4GalT V by its antisense cDNA in SHG44 human glioma cells markedly promoted apoptosis induced by etoposide and the activation of caspases as well as processing of Bid and expression of Bax and Bak. Conversely, the ectopic expression of beta1,4GalT V attenuated the apoptotic effect of etoposide on SHG44 cells. In addition, both the beta1,4GalT V transcription and the binding of total or membrane glycoprotein with Ricinus communis agglutinin-I (RCA-I) were partially reduced in etoposide-treated SHG44 cells, correlated well with a decreased level of Sp1 that has been identified as an activator of beta1,4GalT V transcription. Collectively, our results suggest that the down-regulation of beta1,4GalT V expression plays an important role in etoposide-induced apoptosis and could be mediated by a decreasing level of Sp1 in SHG44 cells, indicating that inhibitors of beta1,4GalT V may enhance the therapeutic efficiency of etoposide for malignant glioma.

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Previous study indicated that beta1,4-galactosyltransferase I (beta1,4GT1) was up-regulated by cycloheximide (CHX) and thus enhanced apoptosis induced by CHX in SMMC-7721 cells. In this study, we reported that constitutively active Akt protein (myr-Akt) inhibited CHX-induced apoptosis in SMMC-7721 cells through down-regulating beta1,4GT1. However, the two PI3K inhibitors LY294002 and wortmannin treatment up-regulated beta1,4GT1 through enhancing Sp1 protein expression and consequently increased CHX-induced SMMC-7721 cells apoptosis. Besides, our results suggested that beta1,4GT1 and cell surface galactose residues synthesized by elevated beta1,4GT1 played an important role in SMMC-7721 cells apoptosis treated with CHX and PI3K inhibitor together. Moreover, we found that CHX accentuated beta1,4GT1 through down-regulating Akt expression to mediate SMMC-7721 cells apoptosis. Taken together, PI3K inhibitors LY294002 and wortmannin up-regulated beta1,4GT1 and enhanced CHX-induced apoptosis in SMMC-7721 cells, which suggested that PI3K inhibitors might have therapeutic potential when combined with CHX in the treatment of hepatoma.