Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M504489200 on February 6, 2006

J. Biol. Chem., Vol. 281, Issue 14, 9482-9489, April 7, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/14/9482    most recent
M504489200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jiang, J.
Right arrow Articles by Gu, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jiang, J.
Right arrow Articles by Gu, J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

beta1,4-Galactosyltransferase V Functions as a Positive Growth Regulator in Glioma*

Jianhai Jiang, Xiaoning Chen, Jialin Shen, Yuanyan Wei, Tao Wu, Yanzhong Yang, Hanzhou Wang, Hongliang Zong, Junwu Yang, Si Zhang, Jianhui Xie, Xiangfei Kong, Weicheng Liu, and Jianxin Gu1

From the Key Laboratory of Medical Molecular Virology Ministry of Education and Health, Gene Research Center, Shanghai Medical College of Fudan University (former Shanghai Medical University), Shanghai 200032, China

Received for publication, April 25, 2005 , and in revised form, February 3, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
beta1,4-galactosyltransferase V (GalT V; EC 2.4.1.38 [EC] ) can effectively galactosylate the GlcNAcbeta1->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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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). beta1,4-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 beta1,4-galactosyltransferase (GalT) family, could effectively galactosylate the GlcNAcbeta1->6 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 PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—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. [{gamma}-32P]dATP and the ECL assay kit were from Amersham Biosciences. Sialidase was from Roche Applied Science. The following antibodies were purchased from Santa Cruz Biotechnology: anti-JNK1/2, anti-phospho-JNK, anti-ERK1/2, anti-phospho-ERK and anti-glyceraldehyde-3-phosphate dehydrogenase. Anti-Cyclin D1 Ab, anti-Cyclin D2 Ab, anti-Cyclin D3 Ab, and anti-E2F1 Ab were from Pharmingen. Biotinylated PHA-L or RCA-I was purchased from Vector Laboratories. Anti-AKT Ab, anti-phospho AKT (Ser473) Ab, and anti-phospho-AKT (Thr308) Ab were purchased from Cell Signaling. FITC-conjugated streptavidin and horseradish peroxidase-conjugated streptavidin were purchased from Southern Biotechnonlogy Associates. Anti-HA Ab was purchased from Roche Applied Science. Normal human brain tissues and glioma tissues were obtained from Huashan Hospital, Shanghai, China. Other reagents were commercially available in China.

Cell Culture and Transfection—Human glioma cell lines SHG44 (10), U87, and U251 were cultured in RPMI 1640 medium or DMEM containing 10% bovine calf serum, 100 units/ml penicillin, and 50 µg/ml streptomycin at 37 °C in a humidified CO2 incubator (5% CO2, 95% air). Transformed astrocytes C8-D30 (American Type Culture Collection) were cultured in DMEM containing 10% fetal bovine serum, 1.5 g/liter sodium bicarbonate and 4.5 g/liter glucose. SHG44 cells stably transfected with pcDNA3.0 or GalT V antisense cDNA have been described previously (11). HA-GalT V- and HA-tagged mutant format of GalT V- or mock-transfected stable cells were generated by transfection with pcDNA3.0 or HA-GalT V or the HA-tagged mutant format of GalT V, followed by selection in G418. Individual clones were picked and analyzed. Cell transfection was performed with Lipofectamine (Invitrogen) according to the manufacturer's instructions. Cells were harvested and measured by flow cytometry 24–48 h after transfection, as reported previously (12).

Plasmids—Expression constructs for HA-pcDNA3.0, Ras DA, Ras DN, ERK1, JNK1, pGL3-SV40, pGL3-Basic, constitutively active AKT (Gag-AKT), HA-GalT V, pSilencer-2.0, and pRL-CMV have been described previously (11, 13, 14). GalT V site-directed mutagenesis constructs were derived from HA-GalT V by PCR amplification using TakaRa MutanBEST mutagenesis kit and specific primers (Y268G forward, 5'-TATCTGCTTCCTGGCACCGAGTT-3'; reverse 5'-ACATATACTTATCCAATTTGGTT-3'; W294G forward, 5'-TAATGCTTTCGGCGGTTGGGGTGG-3'; reverse, 5'-AGGAAAGCCATTGATTTTCCGA-3'). Mutated constructs were sequenced, and the correct ones were selected for further experiments. A 320-bp fragment (containing nucleotides from -200 to +120 relative to the transcription start) of the GalT V promoter was prepared by PCR amplification of genomic DNA using a sense primer containing a XhoI restriction site and an antisense primer containing a HindIII restriction site (sense, 5'-TACTCGAGAGGGTCGGCGGCGAGC-3'; antisense, 5'-ATAAGCTTCAGGCGGCCGCTAGAGA-3') as reported previously (15). Following digestion with restriction enzymes, the GalT V promoter fragment was directionally cloned into the pGL3-Basic (Promega). To prepare site-mutated promoter, the putative Sp1-binding site CCCCGCC between nucleotide positions -78 and -71 was changed to CCCCAAA and named M (Sp1). The mutation was created from pGL3 (-200/+120) by PCR using a TakaRa MutanBEST mutagenesis kit. Mutated constructs were sequenced, and the correct ones were selected for further experiments.


Figure 1
View larger version (46K):
[in this window]
[in a new window]
  		FIGURE 1.
Effects of reduction in the GalT V expression on glioma cell SHG44 invasiveness and growth. A, RT-PCR analysis of GalT V mRNA expression level in normal brain and multiple grade I–IV brain tumors. B, transfection with GalT V antisense cDNA reduced the expression of GalT V in SHG44 cells using RT-PCR analysis. C, cell morphology of mock- (Control) or antisense-transfected SHG44 cells. D, when cells were grown to confluence in RPMI 1640 medium containing 10% FBS, photographs were taken. E, cell migration assay of mock- (Control) or antisense-transfected SHG44 cells. Wound healing assay was prepared as described under "Experimental Procedures" (left panel). And, the wound-induced migration of cells was measured after 24 h (right panel). F, decreasing the GalT V expression in SHG44 cells inhibited the invasive ability assayed in a modified Boyden chamber (p < 0.05, n = 3). G, nude mice were injected with either mock- (Control) or antisense-transfected SHG44 cells. 3 weeks later, photographs were taken (right panel, middle panel). Tumors were removed and weighted. Results were shown as mean ± S.D. of tumor weights (left panel).

 


Figure 2
View larger version (38K):
[in this window]
[in a new window]
  		FIGURE 2.
Effects of GalT V overexpression on glioma cells and transformed astrocytes invasiveness and survival. A, Western blot assay demonstrated HA-GalT V expression in the indicated cells stably transfected with HA-GalT V construct using anti-HA antibody. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Western blot served as a loading control. B, cell migration assay of mock- (Control) or HA-GalT V-transfected cells. C, HA-GalT V-transfected cells were more invasive than the control cells assayed in a modified Boyden chamber (p < 0.05, n = 3). D, 100,000 cells were plated into individual wells of 6-well tissue culture plates in sextuplicate in same condition, grown overnight in DMEM with 10% FBS, and serum-starved for 10 days. Fixed and stained with toluidine blue O (0.1%) in 4% paraformaldehyde diluted in PBS, viable cells were counted. E, the ectopic expression of GalT V promoted glioma growth in vivo. At 3 weeks after injection with the indicated cells, tumors were removed and weighted. Results were shown as mean ± S.D. of tumor weights.

 
Reverse Transcriptase (RT)-PCR—Total RNA (1 µg) extracted was used as a template for cDNA synthesis, with a TaKaRa RNA PCR Kit and specific primers (GalT V forward, 5'-TGAGAACAATCGGTGCATCAG-3'; reverse, 5'-CTCAATCCGCCAAATAACTC-3'; beta-actin forward, 5'-ATGGGTCAGAAGGATTCCTAT-3'; reverse, 5'-GCGCTCGGTGAGGATCTTCAT-3') as reported previously (9). Amplification was carried out for 22–27 cycles under saturation, each at 94 °C, 45 s; 60 °C, 45 s; 72 °C, 1 min in a 50-µl reaction mixture containing 2 µl of each cDNA, 0.2 µM concentration of each primer, 0.2 mM dNTP, and 2.5 units of TaqDNA polymerase. After amplification, 10 µl of each reaction mixture was analyzed by 1% agarose gel electrophoresis, and the bands were then visualized by ethidium bromide staining. The PCR products for GalT V were 657 bp.

Invasion and Migration Assay—Boyden chamber invasion assay was performed basically as described previously by Albini et al. (16). Cells were added to the upper compartment of the chamber, and 800 µl of medium (containing 0.1% bovine serum albumin) was added into the lower chamber. Cells were incubated and allowed to migrate for 24 h. After removal of non-migrated cells, cells that had migrated through the filter were counted under a microscope in five fields at a magnification of x400. Wound healing assay was performed as described previously (17). Briefly, subconfluent cells in 6-well plates were serum-starved overnight. Over 20 wounds were made on the cell monolayer by scratching with a 200-µl sterile tip. After being rinsed with phosphate-buffered saline (PBS) three times, the medium was replaced with complete growth medium. Cells were photographed at 0 and 24 h after scraping, and the wound-induced migration of cells was measured after 24 h.

Survival Assay—Cells were plated onto 6-well dishes. After 24 h, the medium was removed, cells were washed twice with Dulbecco's phosphate-buffered saline (DPBS), and the serum-free medium was added. Cultures were visually inspected daily. Cell numbers were determined after trypan blue staining of viable cells in parallel plates (18).

Clonogenic Survival Assay—To determine IR sensitivity, proliferating cells were x-irradiated at the indicated doses using x-ray generator operating at 320 kV/12 mA and allowed to recover for 4 h before trypsinization. The trypsinized cells (5 x 103) were replanted in duplicate 100-mm dishes and undisturbed for 12 days. The plates were then stained with crystal violet, and the numbers of colonies that were >1 mm in diameter were counted.


Figure 3
View larger version (42K):
[in this window]
[in a new window]
  		FIGURE 3.
GalT V acts as a catalytic enzyme in the promotion of its tumorigenic effects on glioma. A, a schematic diagram of HA-tagged GalT V construct (WT) and its point mutation formats (Y268G/W294G). B, proteins from U87 cells transfected with vector or full-length of GalT V (WT), or point mutant constructs (Y268G/W294G) were separated by SDS-PAGE and the binding to RCA-I was analyzed by RCA-I-lectin. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Western blot served as a loading control. C, Western blot assay demonstrated W294G expression in the indicated cells stably transfected with the GalT V mutant construct W294G using anti-HA antibody. D, migration assay of mock- (Control), HA-GalT V-(WT), or W294G-transfected cells. Wound healing assay was prepared as described under "Experimental Procedures," and the wound-induced migration of cells was measured after 24 h. E, Matrigel invasion assay was performed with the cells stably transfected with mock (Control), HA-GalT V (WT), or W294G. Values were shown as mean ± S.D. of triplicates from two independent experiments.

 
Flow Cytometry of Glycan Level of Membrane Protein and Lectin Blotting—After grown to subconfluence, cells were treated with sialidase for 5 h. Endogenous peroxidase activity was blocked with 0.3% H2O2 for 30 min. After being washed with PBS, cells were harvested after treatment with EGTA (2 mM) and incubated with biotinylated PHA-L or RCA-I (2 µg/µl) for 45 min at 4 °C. Then, the cells were washed with PBS and probed with FITC-conjugated streptavidin (1:128) for 30 min at 4 °C. Next, the cell samples were subjected to flow cytometry. Lectin blotting assay was performed as reported previously (13).

Analysis of Apoptosis by Flow Cytometry—Adherent and non-adherent cells were collected, washed twice in PBS, and fixed with ice-cold 70% ethanol for at least 1 h. The fixed cells were washed and stained with propidium iodide. After incubation for 45 min at 37 °C, the DNA content was determined by quantitative flow cytometry with standard optics of FACScan flow cytometer (BD Biosciences FACStar). The percentage of apoptotic cell was quantified from sub-G1 events.

Implantation of Tumor Cells in Mice—Tumorigenicity assay was performed as described previously by Dong Xie et al. (19). At confluence, the cells were harvested, centrifuged, and then resuspended in a sterile solution of PBS at a final concentration of about 1.0 x 107 cells/ml. A 100-µl aliquot of resuspended cells (about 1.0 x 106 cells) was injected subcutaneously between the shoulder blade ~3 cm from the tail. After 3 weeks, photographs were taken, and tumors were harvested and individually weighed after mice were anesthetized. Data were presented as tumor weight (mean ± S.D.). Statistical analysis was performed by computer program software using the Student's t test.

Statistics and Presentation of Data—All experiments were repeated three times. All numerical data were expressed as mean ± S.D. Data were analyzed using the two-tailed t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
We adopted a semiquantitative RT-PCR to analyze the expression of GalT V mRNA in normal brain tissues and glioma tissues. Consistent with our previous study (9), GalT V mRNA expression was correlated with staging to the glioma tumor (Fig. 1A). To elucidate the biological significance of GalT V in glioma, the GalT V antisense cDNA construct was stably transfected into glioma cell line SHG44 (Fig. 1B) (11). In culture, the control SHG44 cells showed invasive growth with spindle-shaped morphology (Fig. 1C, left panel) and grew in an actinomorphic manner (Fig. 1D, left panel). However, antisense-transfected SHG44 cells exhibited a round morphology (Fig. 1C, right panel) and grew in a ramble way (Fig. 1D, right panel). To examine the property of GalT V in glioma, we studied the effects of reduction in GalT V expression on the motility of SHG44 cells, assayed by wound healing and Boyden chamber assays. As depicted in Fig. 1, E and F, reduction in GalT V expression resulted in a significant decrease in cell migration in vitro and the ability to migrate through Matrigel-coated 8-µm pore size membranes. Similar results were obtained in agarose drop explant assay (data not shown). Moreover, reduction in the expression of GalT V resulted in a total suppression of glioma growth in vivo, compared with the controls (Fig. 1G).

To evaluate the relationship between GalT V overexpression and tumor behavior, transformed astrocytes C8-D30 and glioma cell lines U87 and U251 were stably transfected with HA-GalT V (Fig. 2A). GalT V overexpression in glioma cells U87 and U251 and transformed astrocytes C8-D30 resulted in a striking increase of cell migration (Fig. 2B), an almost 3-fold increase in vitro invasiveness through a reconstituted Matrigel basement membrane (Fig. 2C), a great increase in colony number (data not shown) and greater numbers of viable cells in serum-free conditions relative to the control cell lines (Fig. 2D). To examine the effect of GalT V overexpression on the ability of tumor formation in vivo, HA-GalT V- or vector-transfected glioma cells were injected subcutaneously into nude mice. As depicted in Fig. 2E, the HA-GalT V-transfected cells developed tumors with a markedly large size during the 3 weeks of observation compared with the control cells. Collectively, these experiments demonstrated that GalT V could promote glioma cell invasiveness and survival.

The GalT V protein consists of a short NH2-terminal cytoplasmic domain, a stem region, and a catalytic domain, which contains two conserved residues (Tyr268/Trp294) which are important for the galactosylation activity of GalT I (5, 20–22). To investigate the contribution of these residues in the galactosylation activity of GalT V, HA-tagged point mutants of GalT V (Y268G/W294G) were constructed and transiently transfected into U87 cells (Fig. 3A). As depicted in Fig. 3B, Trp294 was involved in the galactosyltransferase activity of GalT V. To investigate the contribution of the GalT V galactosylation activity in its tumorigenic effects, the mutation construct W294G was stably transfected into transformed astrocytes C8-D30 and glioma cell lines U87 and U251 (Fig. 3C). As shown in Fig. 3, D and E, the point mutation (W294G) abolished the ability of GalT V to promote the migration ability and invasive potential of glioma cells, indicating that an intact catalytic domain might be essential for GalT V tumorigenic function in glioma. This conclusion was further supported by clonogenic assay and tumorigenicity assay in vivo (data not shown).

The expression of beta1,6-linked GlcNAc-bearing N-glycans has been reported as a marker of tumor progress in glioma (6). To assess the contribution of GalT V in the expression of N-glycan of cell membrane protein, flow cytometry analysis was performed using FITC-conjugated RCA-I which interacts with oligosaccharides terminating with the Galbeta1->4GlcNAc group or FITC-conjugated PHA-L, which interacts with highly branched N-Glycans with the Galbeta1->4GlcNAcbeta1->6-(Galbeta1->4GlcNAcbeta1->2)Man branch (15). As expected, reduction in the expression of GalT V decreased the binding with RCA-I or PHA-L on the cell surface (Fig. 4A). Consistent with this, a significant decrease of the binding of total glycoprotein with RCA-I (Fig. 4B, left panel) or PHA-L (Fig. 4B, right panel) was observed for 50–80-kDa and 80–100-kDa protein bands in the antisense-transfected cells compared with the mock-transfected cells.

Furthermore, reduction in the expression of GalT V inhibited cell cycle progression (Fig. 5A, left panel) and reduced the endogenous expression of Cyclin D1, Cyclin D3, and E2F1 (Fig. 5A, right panel), which are important regulators of cellular proliferation and highly expressed in glioma (23–25). In addition, suppression of GalT V expression promoted apoptosis induced by chemotherapeutic agent etoposide (VP16), which was widely used for treatment of malignant gliomas (26) (Fig. 5B, left panel), and inhibited the ability to form invisible clones with x-irradiated treatments (Fig. 5B, right panel). We next examined the contribution of GalT V in the activation of AKT and MAPK kinase, which have shown to associate with glioma invasiveness (18, 27–29). As shown in Fig. 5C, reduction in the expression of GalT V led to a reduction of the levels of phospho-AKT (Ser473/Thr308) and phospho-JNK1/2 (Thr183/Tyr185) status. In summary, these data strongly suggest that GalT V could represent a novel target in glioma therapy.


Figure 4
View larger version (42K):
[in this window]
[in a new window]
  		FIGURE 4.
Reduction in the GalT V expression could reduce the expression of N-Glycans. A, both mock- (Control) and antisense-transfected SHG44 cells were incubated with biotinylated RCA-I or L-PHA followed by incubation with FITC-conjugated streptavidin. Analysis was performed using FACScan. The dotted lines represented fluorescence of the secondary antibody alone. The numbers on the left inside of the top panel gave the mean fluorescence intensity of the secondary antibody alone, whereas those in the right inside gave the mean fluorescence intensity of the indicated antibody staining done. B, proteins were separated by SDS-PAGE, and the binding to RCA-I or PHA-L was analyzed by RCA-I-lectin (left panel) or PHA-L-lectin (right panel). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Western blot served as a loading control.

 
We next explored the possible relationship between Ras/MAPK and PI3K/AKT signaling pathways and GalT V expression. GalT V mRNA expression levels in serum-starved and serum- or EGF-stimulated SHG44 cells were assessed by RT-PCR analysis. As shown in Fig. 6A, endogenous GalT V mRNA expression was markedly induced by serum (Fig. 6A, upper panel) or EGF (Fig. 6A, lower panel), indicating the contribution of serum or EGF in the transcription regulation of the GalT V gene. To adequately address this question, we constructed the GalT V reporter construct pGL3 (-200/+120), which retained relative strong promoter activity in cancer cells and contained one Sp1-binding site at nucleotide positions -81/-69 (15). As expected, transient transfection of the GalT V reporter plasmid pGL3 (-200/+120) into SHG44 cells showed a dose-dependent reporter gene activity in response to serum (Fig. 6B) or EGF stimulation (Fig. 6C). To determine whether Ras signaling pathway was involved in the GalT V transcription activation, we transiently cotransfected reporter plasmid pGL3 (-200/+120) and either the dominant negative expression construct Ras-DN or the constitutively active expression construct Ras-DA into SHG44 cells. As expected, expression of Ras-DN decreased the GalT V promoter activity in a dose-dependent manner, whereas expression of Ras-DA caused a similarity dependent activation (Fig. 6D), indicating a role of Ras/MAPK signaling pathway in the transcription regulation of GalT V. Consistent with this, transient overexpression of ERK1 or JNK1 into SHG44 cells led to a significant increase in the GalT V promoter activity (Fig. 6E, lane 1–5). As the PI3K-AKT pathway represents one of the most potently pro-survival signaling pathways frequently activated in malignant gliomas (27), we explored the effect of AKT on the GalT V promoter activity. As expected, cotransfection of pGL3 (-200/+120) and constitutively active AKT (Gag-AKT) construct increased the promoter activity in a dose-dependent manner (Fig. 6E, lanes 1, 6, and 7). Sp1, which plays an essential role in the GalT V transcription in cancer cells (15), has been well defined as a nuclear target factor of pro-growth and pro-survival signal transduction pathways in tumor cells, including Ras/MAPK and PI3K/AKT signaling pathways (30, 31). To elucidate the contribution of this Sp1-binding site in the activation of the GalT V promoter by Ras/MAPK and PI3K/AKT signaling pathways, we introduced site-directed mutagenesis into this Sp1-binding site (M (Sp1)). It was found that the mutagenesis of this Sp1-binding site abolished the effects of Ras-DA, Ras-DN, ERK1, JNK1, or Gag-AKT on the GalT V promoter activity (Fig. 6F).


Figure 5
View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 5.
Inhibition of GalT V expression could suppress cell cycle progression and survival. A, mock- (Control) or antisense-transfected SHG44 cells were harvested, and cell cycle parameters were determined as described under "Experimental Procedures" (left panel). Equal amounts of proteins from mock- (Control) or antisense-transfected SHG44 cells were immunoblotted with the indicated antibodies (right panel). B, after a treatment of VP16 in the indicated concentration, the percentage of apoptotic cells was quantified by flow cytometer. The percentage of apoptosis was standardized with that of SHG44 cells with a treatment of VP16 (20 µM). Each value was the mean ± S.D. of at least three independence experiments (left panel). The effect of GalT V on the sensitivity of SHG44 cells after exposure to x-rays is shown. Cells were plated, x-irradiated at the indicated doses, allowed to recover, and then replanted and scored for colony formation, as described under "Experimental Procedures." The colony number was standardized with that of cells without x-ray treatment (right panel). C, equal amounts of proteins from mock- (Control) or antisense-transfected SHG44 cells were immunoblotted with the indicated antibodies. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Malignant gliomas are the most common primary brain tumors and one of the deadliest human cancers (32). They develop as the result of stepwise accumulations of multiple genetic alterations, which result in the activation of oncogenes and/or the inactivation of tumor suppressor genes (33). Our early studies have demonstrated that GalT V is highly expressed in glioma. Moreover, the expression of GalT V increases upon malignant transformation of cells and is correlated with staging to the glioma tumor (9). These findings motivate us to examine the function of GalT V protein in glioma.


Figure 6
View larger version (30K):
[in this window]
[in a new window]
  		FIGURE 6.
Activation of the GalT V promoter by EGF and dominant active Ras and AKT. A, serum-starved SHG44 cells were stimulated with the indicated concentrations of FBS (upper panel) or EGF (lower panel) for 24 h. Relative GalT V mRNA expression levels were determined by RT-PCR analysis. Levels of beta-actin mRNA expression were assessed as loading controls. The GalT V promoter construct pGL3 (-200/+120) was transiently transfected into SHG44 cells. After transfection, cells were treated with the indicated concentration of FBS (B) or EGF (C) for 24 h. The luciferase activity was determined as described above. The values were presented as fold activation over those observed in 1% FBS-treated or non-treated samples. D, SHG44 cells were transiently cotransfected with pGL3 (-200/+120) and increasing amounts of plasmids expressing the constitutively active form of Ras (Ras DA) or dominant negative form of Ras (Ras DN), and the luciferase activity was determined as described above. E, SHG44 cells were transiently cotransfected with pGL3 (-200/+120) and increasing amounts of ERK1, JNK1, or Gag-AKT constructs, and the luciferase activity was determined as described above. F, the promoter constructs pGL3 (-200/+120) (WT) or M (Sp1) were cotransfected into SHG44 cells with the plasmids expressing the constitutively active Ras (Ras DA), dominant negative Ras (Ras DN), ERK1, JNK1, or Gag-AKT. The luciferase activity was determined as described above.

 
Results from previous studies have shown that the expression of GlcNAcbeta1->6Man-branched N-glycans play a major role in glioma malignant (6). This GlcNAcbeta1->6Man structure is synthesized by N-acetylglucosaminyltransferase V, a key enzyme in the processing of asparagine-linked glycans during the synthesis of glycoproteins (34). As the reduction in the expression level of the GalT V gene in SH-SY5Y cells resulted in the decreased galactosylation of highly branched N-glycans, and the expression of GalT V in several human cancer cell lines was highly correlated with that of the N-acetylglucosaminyltransferase V gene (8, 15), the GalT V was considered to be involved in the expression of highly branched N-glycans. Consistently, reduction in the GalT V expression in SHG44 cells decreases the galactosyltransferase of the highly branched N-glycans, which leads to the reduced binding to L-PHA. Similar results are obtained in other cancer cell lines including U87 and HeLa cells (data not shown). Therefore, GalT V might be very important in glioma for expressing the highly branched N-glycans that are involved in glioma growth and metastasis (6).

In this study, the role of GalT V in glioma development has been evaluated in several experimental models. Its forced expression in U87 and U251 cells could markedly stimulate their growth and invasion and migration and significantly enhance their tumorigenicity in vivo. These cells developed larger tumors in nude mice. To further address the physiological functions of GalT V in glioma, we investigated the effect of reduction in the GalT V expression on glioma malignant. Reduction in the expression of GalT V resulted in the changes in cell morphology, and decreasing the GalT V expression in glioma cells promoted apoptosis and decreased the migration potential and glioma growth in vitro and in vivo. These findings indicate that GalT V functioned as a positive growth regulator in glioma.

The regulation of glioma cell growth is a complex and intricately orchestrated process involving multiple interrelated signaling pathways. Studies from a number of laboratories have demonstrated that signaling pathways downstream of Ras are critical for facilitating tumor cell proliferation, inhibiting cell death, and maintaining the tumor phenotype (35, 36). Similarly, activation of the AKT pathway provides a survival advantage by inhibiting programmed cell death or apoptosis and is important for the maintenance of the tumor phenotype by promoting cell motility and cell shape changes for tumorigenesis (37, 38). In an effort to define the mechanism by which the GalT V protein plays a positive growth regulator role in glioma, we analyzed the contribution of GalT V in mitogenic signaling pathways and demonstrated that decreasing the expression of GalT V reduced the AKT activity, indicating that GalT V functioned as a novel glioma growth activator by modulating signaling pathways such as AKT.

Furthermore, Ras/MAPK and PI3K/AKT signaling pathways were involved in the transcription regulation of the GalT V gene. The activity of GalT V promoter could be induced by epidermal growth factor, dominant active Ras, ERK1, JNK1, and constitutively active AKT, suggesting that GalT V might be involved in the pro-survival role of AKT or Ras/MAPK signaling pathways in glioma. However, the mechanism of the activation of the GalT V promoter medicated by AKT or Ras/MAPK signaling pathways remained unknown and should be explored.

Another important finding of this study was the characterization of GalT V as a novel target in glioma therapy, which was suggested by quantitative evidences. (a) The GalT V mRNA expression was correlated with glioma grade. (b) Reduction in the GalT V expression resulted in a decrease in colony number (data not shown) and glioma growth in vivo. (c) The suppression of GalT V expression inhibited cell cycle progression and reduced the expression of Cyclin D1 and E2F1. (d) The impact of GalT V expression in glioma decreased the relative resistance to apoptosis induced by etoposide or x-ray. (e) Reduction in the GalT V expression decreased the AKT activity which has been inversely correlated with survival in patient glioma specimens (39). These results indicate that decreasing the GalT V expression resulted in the changes in the intracellular pro-survival signaling pathways and decreased glioma cell invasion potential, suggesting that manipulating the expression of GalT V might have therapeutic potential for the treatment of glioma.

The finding that GalT V performed a positive growth regulator in glioma and could represent a novel target in glioma therapy expands our understanding of the proteins involved in gliomagenesis. The molecular mechanism of the transcription regulation of GalT V in glioma should be explored next.


   FOOTNOTES
 
* This work was supported by the 863 Program of China (2001AA234031) and the National Natural Scientific Foundation of China (30330320) and a grant from the development of science and technology of Shanghai (02DJ14002). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed. Tel.: 86-21-54237704; Fax: 86-21-64164489; E-mail: jxgu{at}shmu.edu.cn.

2 The abbreviations used are: GalT, beta1,4-galactosyltransferase; GalT V, beta1,4-galactosyltransferase V; RT, reverse transcription; RCA, Ricinus communis agglutinini; PHA, Phaseolus vulgaris leucoagglutinin; FITC, fluorescein isothiocyanate; AS, antisense; DN, dominant negative; DA, dominant active; Ab, antibody; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; EGF, epidermal growth factor; JNK, c-Jun NH2-terminal kinase; ERK, extracellular signal-regulated kinase; HA, hemagglutinin; PBS, phosphate-buffered saline; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hakomori, S. (1996) Cancer Res. 56, 5309-5318[Abstract/Free Full Text]
  2. Couldrey, C., and Green, J. E. (2000) Breast Cancer Res. 2, 321-323[CrossRef][Medline] [Order article via Infotrieve]
  3. Guo, S., Sato, T., Shirane, K., and Furukawa, K. (2001) Glycobiology 11, 813-820[Abstract/Free Full Text]
  4. Sato, T., Guo, S., and Furukawa, K. (2001) Biochimie (Paris) 83, 719-725
  5. Sato, T., Furukawa, K., Bakker, H., Van den Eijnden, D. H., and Van Die, I. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 472-477[Abstract/Free Full Text]
  6. Yamamoto, H., Swoger, J., Greene, S., Saito, T., Hurh, J., Sweeley, C., Leestma, J., Mkrdichian, E., Cerullo, L., Nishikawa, A., Ihara, Y., Taniguchi, N., and Moskal, J. R. (2000) Cancer Res. 60, 134-142[Abstract/Free Full Text]
  7. Shirane, K., Sato, T., Segawa, K., and Furukawa, K. (1999) Biochem. Biophys. Res. Commun. 265, 434-438[CrossRef][Medline] [Order article via Infotrieve]
  8. Sato, T., Shirane, K., Kido, M., and Furukawa, K. (2000) Biochem. Biophys. Res. Commun. 276, 1019-1023[CrossRef][Medline] [Order article via Infotrieve]
  9. Xu, S., Zhu, X., Zhang, S., Yin, S., Zhou, L., Chen, C., and Gu, J. (2001) J. Cancer Res. Clin. Oncol. 127, 502-506[CrossRef][Medline] [Order article via Infotrieve]
  10. Chen, Z. P., Remack, J., Brent, T. P., Mohr, G., and Panasci, L. C. (1999) Clin. Cancer Res. 5, 4186-4190[Abstract/Free Full Text]
  11. Xu, S., Zhang, S., Chen, C., Yan, J., Cai, M., Zhu, X., and Gu, J. (2002) J. Exp. Clin. Cancer Res. 21, 409-414[Medline] [Order article via Infotrieve]
  12. Ma, Y., Yuan, J., Huang, M., Jove, R., and Cress, W. D. (2003) J. Biol. Chem. 278, 16770-16776[Abstract/Free Full Text]
  13. Zhu, X., Jiang, J., Shen, H., Wang, H., Zong, H., Li, Z., Yang, Y., Niu, Z., Liu, W., Chen, X., Hu, Y., and Gu, J. (2005) J. Biol. Chem. 280, 12503-12516[Abstract/Free Full Text]
  14. Zhu, X., Chen, S., Yin, X., Shen, A., Ji, S., Shen, Z., and Gu, J. (2003) Biochem. Biophys. Res. Commun. 309, 279-285[CrossRef][Medline] [Order article via Infotrieve]
  15. Sato, T., and Furukawa, K. (2004) J. Biol. Chem. 279, 39574-39583[Abstract/Free Full Text]
  16. Albini, A., Iwamoto, Y., Kleinman, H. K., Martin, G. R., Aaronson, S. A., Kozlowski, J. M., and McEwan, R. N. (1987) Cancer Res. 47, 3239-3245[Abstract/Free Full Text]
  17. Hauck, C. R., Hsia, D. A., and Schlaepfer, D. D. (2000) J. Biol. Chem. 275, 41092-41099[Abstract/Free Full Text]
  18. Shi, Q., Bao, S., Maxwell, J. A., Reese, E. D., Friedman, H. S., Bigner, D. D., Wang, X. F., and Rich, J. N. (2004) J. Biol. Chem. 279, 52200-52209[Abstract/Free Full Text]
  19. Xie, D., Yin, D., Tong, X., O'Kelly, J., Mori, A., Miller, C., Black, K., Gui, D., Said, J. W., and Koeffler, H. P. (2004) Cancer Res. 64, 1987-1996[Abstract/Free Full Text]
  20. Aoki, D., Appert, H. E., Johnson, D., Wong, S. S., and Fukuda, M. N. (1990) EMBO J. 9, 3171-3178[Medline] [Order article via Infotrieve]
  21. Lo, N. W., Shaper, J. H., Pevsner, J., and Shaper, N. L. (1998) Glycobiology 8, 517-526[Abstract/Free Full Text]
  22. Schwientek, T., Almeida, R., Levery, S. B., Holmes, E. H., Bennett, E., and Clausen, H. (1998) J. Biol. Chem. 273, 29331-29340[Abstract/Free Full Text]
  23. Alemany, R., Gomez-Manzano, C., Balague, C., Yung, W. K., Curiel, D. T., Kyritsis, A. P., and Fueyo, J. (1999) Exp. Cell Res. 252, 1-12[CrossRef][Medline] [Order article via Infotrieve]
  24. Arato-Ohshima, T., and Sawa, H. (1999) Int. J. Cancer 83, 387-392[CrossRef][Medline] [Order article via Infotrieve]
  25. Bacon, C. L., Gallagher, H. C., Haughey, J. C., and Regan, C. M. (2002) J. Neurochem. 83, 12-19[CrossRef][Medline] [Order article via Infotrieve]
  26. Nagane, M., Asai, A., Shibui, S., Oyama, H., Nomura, K., and Kuchino, Y. (1999) Jpn. J. Clin. Oncol. 29, 527-534[Abstract/Free Full Text]
  27. Jetzt, A., Howe, J. A., Horn, M. T., Maxwell, E., Yin, Z., Johnson, D., and Kumar, C. C. (2003) Cancer Res. 63, 6697-6706[Abstract/Free Full Text]
  28. Nakada, M., Niska, J. A., Tran, N. L., McDonough, W. S., and Berens, M. E. (2005) Am. J. Pathol. 167, 565-576[Abstract/Free Full Text]
  29. Perkins, E., Calvert, J., Lancon, J. A., Parent, A. D., and Zhang, J. (2003) Brain Res. Mol. Brain Res. 111, 42-51[Medline] [Order article via Infotrieve]
  30. Pore, N., Liu, S., Shu, H. K., Li, B., Haas-Kogan, D., Stokoe, D., Milanini-Mongiat, J., Pages, G., O'Rourke, D. M., Bernhard, E., and Maity, A. (2004) Mol. Biol. Cell 15, 4841-4853[Abstract/Free Full Text]
  31. Bonello, M. R., and Khachigian, L. M. (2004) J. Biol. Chem. 279, 2377-2382[Abstract/Free Full Text]
  32. DeAngelis, L. M. (2001) N. Engl. J. Med. 344, 114-123[Free Full Text]
  33. Cavenee, W. K. (1992) Cancer 70, 1788-1793[CrossRef][Medline] [Order article via Infotrieve]
  34. Arango, J., and Pierce, M. (1988) J. Cell. Biochem. 37, 225-231[CrossRef][Medline] [Order article via Infotrieve]
  35. Reuveni, H., Geiger, T., Geiger, B., and Levitzki, A. (2000) J. Cell Biol. 151, 1179-1192[Abstract/Free Full Text]
  36. Claes, P., Grobben, B., Van Kolen, K., Roymans, D., and Slegers, H. (2001) Br. J. Pharmacol. 134, 402-408[CrossRef][Medline] [Order article via Infotrieve]
  37. Xiao, A., Wu, H., Pandolfi, P. P., Louis, D. N., and Van Dyke, T. (2002) Cancer Cell 1, 157-168[CrossRef][Medline] [Order article via Infotrieve]
  38. Maier, D., Jones, G., Li, X., Schonthal, A. H., Gratzl, O., Van Meir, E. G., and Merlo, A. (1999) Cancer Res. 59, 5479-5482[Abstract/Free Full Text]
  39. Kurose, K., Zhou, X. P., Araki, T., Cannistra, S. A., Maher, E. R., and Eng, C. (2001) Am. J. Pathol. 158, 2097-2106[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J BiochemHome page
Y. Wei, D. Liu, Y. Ge, F. Zhou, J. Xu, H. Chen, J. Gu, and J. Jiang
Identification of E1AF as a Target Gene of E2F1-induced Apoptosis in Response to DNA Damage
J. Biochem., October 1, 2008; 144(4): 539 - 546.
[Abstract] [Full Text] [PDF]

Home page
 J BiochemHome page
J. Zhou, Y. Wei, D. Liu, X. Ge, F. Zhou, X. Yun, J. Jiang, and J. Gu
Identification of {beta}1,4GalT II as a Target Gene of p53-mediated HeLa Cell Apoptosis
J. Biochem., April 1, 2008; 143(4): 547 - 554.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
J. Jiang, Y. Wei, J. Shen, D. Liu, X. Chen, J. Zhou, H. Zong, X. Yun, X. Kong, S. Zhang, et al.
Functional Interaction of E1AF and Sp1 in Glioma Invasion
Mol. Cell. Biol., December 15, 2007; 27(24): 8770 - 8782.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
T. Sato and K. Furukawa
Sequential Action of Ets-1 and Sp1 in the Activation of the Human beta-1,4-Galactosyltransferase V Gene Involved in Abnormal Glycosylation Characteristic of Cancer Cells
J. Biol. Chem., September 21, 2007; 282(38): 27702 - 27712.
[Abstract] [Full Text] [PDF]

Home page
 GlycobiologyHome page
J. Jiang, J. Shen, T. Wu, Y. Wei, X. Chen, H. Zong, S. Zhang, M. Sun, J. Xie, X. Kong, et al.
Down-regulation of {beta}1,4-galactosyltransferase V is a critical part of etoposide-induced apoptotic process and could be mediated by decreasing Sp1 levels in human glioma cells
Glycobiology, November 1, 2006; 16(11): 1045 - 1051.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/14/9482    most recent
M504489200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jiang, J.
Right arrow Articles by Gu, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jiang, J.
Right arrow Articles by Gu, J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2006 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement