Glycobiology Advance Access originally published online on May 4, 2005
Glycobiology 2005 15(9):887-894; doi:10.1093/glycob/cwi071
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Polysialic acid facilitates tumor invasion by glioma cells
4 Glycobiology, Cancer Research Center, The Burnham Institute, La Jolla, CA 92037; 5 Department of Pathology, Shinshu University School of Medicine, Matsumoto 390-8621, Japan; 6 Department of Neurosurgery, Shinshu University School of Medicine, Matsumoto 390-8621, Japan; 7 Developmental Neurobiology Programs, Cancer Research Center, The Burnham Institute, La Jolla, CA 92037
1 These authors contributed equally to this work.
2 To whom correspondence should be addressed; e-mail: minoru{at}burnham.org
3 Present address: Department of Obstetrics and Gynecology, Keio University School of Medicine, Tokyo 160-8582, Japan
Received on March 15, 2005; revised on April 26, 2005; accepted on April 27, 2005
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Abstract |
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Polysialic acid (PSA) is thought to attenuate neural cell adhesion molecule (NCAM) adhesion, thereby facilitating neural cell migration and regeneration. Although the expression of PSA has been shown to correlate with the progression of certain tumors such as small cell lung carcinoma, there have been no studies to determine the roles of PSA in gliomas, the most common type of primary brain tumor in humans. In this study, we first revealed that among patients with glioma, PSA was detected more frequently in diffuse astrocytoma cells, which spread extensively. To determine directly the role of PSA in glioma cell invasion, we transfected C6 glioma cells with polysialyltransferases to express PSA. In those transfected cells, PSA is attached mainly to NCAM-140, whereas the mock-transfected C6 cells express equivalent amounts of PSA-free NCAM-140. Both PSA negative and positive C6 cell lines exhibited almost identical growth rates measured in vitro. However, PSA positive C6 cells exhibited increased invasion to the corpus callosum, where the mock-transfected C6 glioma cells rarely invaded when inoculated into the brain. By contrast, the invasion to the corpus callosum by both the mock-transfected and PSA positive C6 cells was observed in NCAM-deficient mice. These results combined indicate that PSA facilitates tumor invasion of glioma in the brain, and that NCAM–NCAM interaction is likely attenuated in the PSA-mediated tumor invasion.
Key words: polysialic acid / glioma / tumor invasion / NCAM / polysialyltransferases
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Introduction |
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Polysialic acid (PSA) is a unique carbohydrate of a linear homopolymer of
2,8-linked sialic acid (Finne, 1982
). PSA is primarily attached to N-glycans of the neural cell adhesion molecule (NCAM) in neural cells (Finne, 1982; Edelman, 1984; Rutishauser and Landmesser, 1996; Kiss and Rougon, 1997; Kleene and Schachner, 2004), whereas it is attached to mucin-type glycoproteins in human breast carcinoma and leukemic cells (Martersteck et al., 1996). Polysialylated NCAM is abundant in the embryonic brain. Most NCAM in the adult brain does not contain PSA, but polysialylated NCAM is continuously present in the hippocampus and olfactory bulbs, where neuronal generation persists in the adult (Edelman, 1984; Rutishauser and Landmesser, 1996; Kiss and Rougon, 1997; Kleene and Schachner, 2004).
The cDNAs-encoding polysialyltransferases have been cloned, and these enzymes are called ST8Sia IV (also called PST) and ST8Sia II (also called STX) (Livingston and Paulson, 1993; Eckhardt et al., 1995; Nakayama et al., 1995; Scheidegger et al., 1995; Yoshida et al., 1995). Both ST8Sia II and ST8Sia IV catalyze the transfer of multiple 2,8-linked sialic acid residues to a glycan containing NeuNAc2
3/6Galß14GlcNAcR (Angata et al., 2000). During development, the expression of ST8Sia II and ST8Sia IV genes is specifically regulated (Hildebrandt et al., 1998a; Ong et al., 1998). The amount of ST8Sia II is more significantly reduced postnatally compared with ST8Sia IV (Hildebrandt et al., 1998a; Ong et al., 1998). Mutant mice with ST8Sia II deficiency exhibited misguidance of infrapyramidal mossy fibers and the formation of ectopic synapses in the hippocampus. This altered hippocampus development was associated with higher exploratory drive (Angata et al., 2004). Mutant mice with ST8Sia IV deficiency, on the other hand, bore a restricted phenotype involving an impairment of long-term potentiation in the hippocampal CA1 region (Eckhardt et al., 2000). These results suggest that ST8Sia II and ST8Sia IV may differentially direct the synthesis of PSA in temporal and spatial-specific manners.
PSA has been found in various tumors including small cell and nonsmall cell lung carcinomas, multiple myeloma, neuroblastoma, and Wilms’ tumor (Roth et al., 1988; Van Camp et al., 1990; Fukuda, 1996; Smith et al., 1996; Hildebrandt et al., 1998b; Seidenfaden et al., 2000; Tanaka et al., 2000). In both small cell and nonsmall cell lung carcinomas and multiple myeloma, the expression of PSA is correlated with tumor progression (Scheidegger et al., 1994; Smith et al., 1996; Hildebrandt et al., 1998b; Tanaka et al., 2000). In one particular study, small cell lung carcinoma cells expressing different amounts of PSA were isolated from H69 cell line by clonal dilution of cells. After subcutaneous inoculation of these tumor cells, tumor cells expressing PSA produced more intracutaneous metastasis than tumor cells poorly expressing PSA, although a comparable amount of NCAM was expressed in these variants (Scheidegger et al., 1994).
Gliomas are the most common type of primary brain tumors in humans. It is highly invasive in nature, but tumor metastasis to other organs is rare (Thorsen and Tysnes, 1997). To elucidate the mechanisms of glioma invasion and migration, transfections of various genes to glioma cell lines have been tested for tumor invasion and migration (Kaye et al., 1986; Edvardsen et al., 1994; Chicoine and Silbergeld, 1995; Thorsen and Tysnes, 1997; Owens et al., 1998). In particular, the forced expression of NCAM-140 in glioma cell lines resulted in reduced migration when the glioma cells were inoculated into the brain (Edvardsen et al., 1994) or assayed for migration through a reconstituted basal lamina, Matrigel (Chicoine and Silbergeld, 1995). However, no studies have addressed the direct roles of PSA in glioma invasion in the brain.
In this study, we first found that among the 44 patients with astrocytoma examined, PSA was detected in nine cases of the 30 NCAM-positive astrocytoma, in particular, a diffuse astrocytoma subtype which spreads extensively. ST8Sia IV and ST8Sia II transcripts were also detected in a recurred case of diffuse astrocytoma, which expressed PSA. We then assayed experimental tumor formation of C6 rat glioma cell line in the brain after transfection with ST8Sia II or ST8Sia IV cDNA to express PSA. Mock-transfected and PSA-positive C6 cell lines were inoculated into the brain of wild type and NCAM-deficient mice. The results obtained indicate that PSA expressed on glioma cells facilitates tumor invasion, most likely due to the attenuation of NCAM–NCAM interaction.
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Results |
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Expression of PSA, ST8Sia II mRNA, and ST8Sia IV mRNA in human astrocytoma
Immunohistochemistry by using anti-NCAM monoclonal antibody demonstrated that NCAM was expressed along cytoplasmic processes of the tumor cells in 30 (68.2%) of 44 patients examined, irrespective of the histological grade of the tumor, that is, five cases (83.3%) of six pilocytic astrocytoma, 10 (66.7%) of 15 diffuse astrocytoma, 11 (68.8%) of 16 anaplastic astrocytoma, and four (57.1%) of seven glioblastoma multiforme. On the other hand, PSA was immunohistochemically detected in nine (30%) of 30 patients, which also expressed NCAM in the tumor cells; one case (20%) of pilocytic astrocytoma, four cases (40%) of diffuse astrocytoma, three cases (27.3%) of anaplastic astrocytoma, and one case (25%) of glioblastoma multiforme (Figure 1). Clinical records of the patients at the surgical operation revealed that four (44.4%) of nine PSA-positive patients were recurred cases, whereas five (23.8%) of 21 patients expressing NCAM alone were recurred cases. This result suggests that PSA expressed on the tumor cells was associated with recurrence of the disease. These results combined with the morphological examination of tumor cells suggest that PSA is expressed more frequently in those gliomas that spread extensively, which makes it difficult to remove all of the glioma cells in the first operation.
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Indeed in one recurred case of diffuse astrocytoma, significant amounts of PSA (Figure 2C) as well as NCAM (Figure 2B) were detected in the cell surface and cytoplasmic processes of tumor cells, and the magnetic resonance imaging of this patient revealed that the recurred tumor mainly occupied the right frontal lobe of cerebrum and invaded the left frontal lobe through the corpus callosum. To determine whether two polysialyltransferases, ST8Sia II and ST8Sia IV, play a major role in the biosynthesis of PSA in the glioma cells, we analyzed serial tissue sections of the above case by in situ hybridization using specific RNA probes for ST8Sia II and ST8Sia IV. The results show that both ST8Sia II (Figure 2G) and ST8Sia IV (Figure 2E) transcripts were detectable in the tumor cells, and ST8SiaIV apparently plays a major role in the polysialylation of this tumor.
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Isolation of C6 glioma cells expressing PSA
To directly demonstrate the roles of PSA in glioma invasion, we measured tumor formation of C6 glioma cells after the cells were transfected with ST8Sia II or ST8Sia IV cDNA. After transfecting with pcDNA3-ST8Sia II or pcDNA3-ST8Sia IV, clonal cell lines expressing PSA were isolated, resulting in C6-ST8Sia II and C6-ST8Sia IV. As shown in Figure 3A, C6-ST8Sia II and C6-ST8Sia IV were positive for both PSA and NCAM, whereas the mock-transfected C6 cell line was positive only for NCAM. The results also demonstrated that all three cell lines express almost identical amounts of NCAM as assessed by fluorescence activated cell sorter (FACS) analysis.
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Western blot analysis of C6, C6-ST8Sia II, and C6-ST8Sia IV illustrates that PSA-containing proteins migrated as a large heterogeneous molecular mass of 170–240 kDa (Figure 3B). After removing PSA by endoneuraminidase-N (endo-N) digestion, NCAM molecules migrated at almost the same position as the NCAM of C6 cells and NCAM-140 expressed on HeLa cells. The results also show that a small amount of NCAM-180 is expressed although this band might also represent a dimer of NCAM-140 (Angata et al., 1997
). These results indicate that most PSA is attached to NCAM-140 in C6-ST8Sia II and C6-ST8Sia IV. NCAM was estimated to contain 20–40 sialic acids and 10–35 sialic acids for C6-ST8Sia II and C6-ST8Sia IV cells, respectively, using the procedure described previously (Angata et al., 1998). Polysialylation by ST8Sia IV was less efficient than that by ST8Sia II, probably because C6 cells that express a large amount of PSA after transfection by ST8Sia IV tend to detach from the substratum.
Expression of PSA does not increase cell growth
To determine whether the expression of PSA facilitates tumor cell growth in vitro, the cells were plated at low density and the growth rate was determined. The results demonstrated that the expression of PSA did not alter the rate of tumor cell growth when assessed by a cell proliferation assay in vitro (Figure 4).
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Tumor invasion of C6 glioma cells expressing PSA
Although many of invasion studies were carried out in vitro using for example, Matrigel, this assay is not suited for studying invasion in the brain because substantial differences exists in the extracellular components between Matrigel and the brain (Yamaguchi, 2000). We thus opted to utilize in vivo invasion assay.
When the mock-transfected C6 glioma cells were inoculated into the caudate putamen, tumor cells gradually spread to surrounding tissue and then mostly to the cerebral cortex (Figure 5A and B), consistent with C6 glioma derived from diffuse fibrillary astrocytoma (Thorsen and Tysnes, 1997). When PSA-expressing C6-ST8Sia II cells were inoculated in the same manner, more tumor invasion was observed, in particular, to the corpus callosum (Figure 5D–F). These tumor cells were positive for PSA, and the positive staining was abolished by pretreatment with endo-N (Figure 5G and H). It is noteworthy that each glioma cell highly extended along myelinated fibers in the corpus callosum (Figure 5E insert), in contrast to spindle shape of the mock-transfected C6 cells (Figure 5B insert). Similar results were obtained for PSA-expressing C6-ST8Sia IV cells (Figure 6). By contrast, C6 cells negative for PSA scarcely exhibited invasion to the corpus callosum under the same conditions (Figures 5B and C and 7). Almost identical results were obtained on more than two independently cloned cells.
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Invasion to corpus callosum in the absence of NCAM
The above results showed that the invasion of PSA-expressing C6 cells to the corpus callosum may be one of the important characteristics in glioma progression. This observation is similar to the fact that axons of the corpus callosum express detectable amounts of PSA on NCAM extending in myelin sheath (Seki and Arai, 1991). To determine whether invasion by C6 cells is attenuated by PSA-free NCAM–NCAM interaction, we inoculated C6 and C6-ST8Sia II cells into the brain of NCAM-deficient mice. Figure 7 shows that both C6 and C6-ST8Sia II cells invaded into the corpus callosum in NCAM-deficient mice. The results indicate that NCAM–NCAM interaction may prevent C6 cells from invading the corpus callosum in wild-type mice brain, and C6 cells lacking PSA can also invade into the corpus callosum in the absence of NCAM in the host animal.
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Discussion |
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This study demonstrated that glioma cells from patients with astrocytoma express PSA. The frequency of PSA expression was highest in diffuse astrocytoma, which spread extensively, and was apparently associated with recurrence of the disease. We have also revealed that in a patient with recurred diffuse astrocytoma associated with the invasion of corpus callosum, both ST8Sia IV and ST8Sia II confer the expression of PSA on the tumor cells. In previous studies, the overexpression of ST8Sia II has been correlated to the progression of nonsmall cell lung carcinoma (Tanaka et al., 2000
). This study expanded these findings to glioma, showing that PSA formed mainly by ST8Sia IV is associated with the invasive character of glioma cells.
This study also provides direct evidence, for the first time, that PSA plays a role in tumor invasion in the brain. Because glioma tumors rarely metastasize extracranially, we assayed tumor formation inside the brain. C6 glioma cells became highly invasive to the corpus callosum by the acquisition of PSA through the transfection of ST8Sia II or ST8Sia IV. The results strongly suggest that polysialylation facilitates tumor migration. PSA in the transfected C6 glioma cells was shown to attach mostly to the 140-kDa transmembrane NCAM isoform (NCAM-140). Similarly, NCAM-140, but not NCAM-120, is polysialylated in differentiated C2C12 cells (Suzuki et al., 2003). It has been shown that the loss of NCAM-induced metastatic dissemination of pancreatic ß cell tumors was observed when ß cell tumor-bearing transgenic mice was crossbred with NCAM knockout mice (Perl et al., 1999). This phenotype was reversed by introducing wild-type NCAM-120. These results indicate that PSA attached to NCAM-140 facilitates cell migration. NCAM-140 contains a transmembrane domain, which is absent in NCAM-120. It is tempting to speculate that enhancing glioma invasion may require signal transduction transmitted directly from the extracellular to cytoplasmic domains of NCAM-140. By using TE671 cells that express significant amount of PSA, it has been shown that intraperitoneal injection of TE671 produced lung and liver metastasis. Repeated injection of endo-N to remove PSA resulted in the diminishment of lung or liver metastasis (Daniel et al., 2001). Moreover, when metastases occurred in endo-N-injected animals, they strongly expressed polysialylated NCAM, which escaped from endo-N treatment. These results combined with the results obtained in this study indicate that the expression of PSA leads to increased migration, resulting in increased metastasis.
C6 glioma cells represent a well-differentiated astrocytoma (Thorsen and Tysnes, 1997). After inoculation to the caudate putamen, C6 glioma cells migrated toward the cerebrum cortex and formed tumors in the caudate putamen and cerebrum. Interestingly, PSA-expressing C6 cells invaded the corpus callosum as found in a recurred patient of diffuse astrocytoma. By contrast, such invasion to the corpus callosum was rarely found in the PSA-negative C6 cells. Similarly, PSA expression was more frequently associated with diffuse astrocytoma in the patients examined, suggesting PSA might facilitate cell migration of astrocytoma. The corpus callosum consists of myelinated fibers crossing two hemispheres of the brain. This study also suggests that polysialylated NCAM may weakly interact with adhesive molecules in the corpus callosum (Seki and Arai, 1991), thus allowing cells to migrate in the corpus callosum as shown in previous studies for other systems (Ono et al., 1994; Chazal et al., 2000).
These results, as a whole, indicate that NCAM–NCAM interaction may prevent C6 cells that do not express PSA from migrating into the corpus callosum. It is likely that polysialylation attenuates NCAM–NCAM interaction and facilitates the invasion of polysialylated C6 cells into the corpus callosum. It has also been reported that NCAM may facilitate axonal growth by the stimulation of fibroblast growth factor receptor (Saffell et al., 1997). Similarly, polysialylated NCAM stimulates the signaling by brain-derived neutrophil factor (BDNF), most likely because polysialylated NCAM accumulates BDNF, and presents it to its receptor (Muller et al., 2000; Vutskits et al., 2001; Zhang et al., 2004). Further studies will be necessary to determine if any of those mechanism play a role in glioma invasion facilitated by PSA.
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Materials and methods |
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Tissue collection
Tissue blocks of the primary astrocytomas resected from 44 patients were retrieved from the archives of Shinshu University Hospital, Matsumoto, Japan. According to the World Health Organization (WHO) classification of astrocytic tumors (Kleihuses and Cavenee, 2000
), they were categorized into four subtypes, that is, pilocytic astrocytoma (six cases), diffuse astrocytoma (15 cases), anaplastic astrocytoma (16 cases), and glioblastoma multiforme (seven cases). These tissue specimens were fixed for 48 h in 20% buffered formalin (pH 7.4), embedded in paraffin, and sectioned at 4 and 7 µm thickness for immunohistochemistry and in situ hybridization, respectively, as described previously (Machida et al., 2001). The Ethical Committee of Shinshu University School of Medicine approved the protocols for this study.
Immunohistochemistry and in situ hybridization
Immunohistochemical detection of PSA and NCAM was performed by using mouse monoclonal antibodies, 5A5 (mouse IgM, University of Iowa Hybridoma Bank, Iowa City, IA) and 123C3 (mouse IgG1, Zymed, Carlsbad, CA), respectively. For the NCAM immunostaining, microwave irradiation in a 1.0 mM ethylenediamine tetra-acetic acid (EDTA)–NaOH solution (pH 8.0) was carried out before the incubation with 123C3 antibody, as described previously (Kim et al., 2002). For secondary antibody, Envision+ (DAKO, Glostrup, Denmark), which is dextran polymers conjugating a large number of goat antibodies against mouse immunoglobulins and horseradish peroxidase, was used to increase the sensitivity of immunodetection (Sabattini et al., 1998). The counterstaining was performed with hematoxylin. In control experiments, the primary antibodies were omitted from the staining procedure, and for the PSA staining, pretreatment with endo-N that cleaves PSA (Hallenbeck et al., 1987) was also carried out. In these controls, no specific staining was noted.
To construct RNA probes for in situ hybridization, we amplified the ST8Sia IV-specific region (nucleotides –47 to +113; the first nucleotide of the initiation codon is +1) by polymerase chain reaction (PCR) using a primer set of 5'-GCTCTAGAAGGTGCGGGAGCTGG-3' and 5'-GGGGTACCGATGAGTTGCGTCTCCT-3'. Similarly, the STX-specific region (nucleotides +1 to +138) was amplified by using primers 5'-GCTCTAGATGCAGCTGCAGTTCCGGA-3' and 5'-GGGGTACCGTTCACAGCTGATCTGATTGT-3'. In these primers, the XbaI and Asp718 sites are underlined. These cDNAs were cloned into the XbaI and Asp718 sites of pGEM-3Zf (+) (Promega, Madison, WI), and the resultant vectors were used as a template for the construction of the RNA probes, as described previously (Angata et al., 1997; Yeh et al., 2001).
Transfection of C6 glioma cell line with ST8Sia II or ST8Sia IV cDNA vector
pcDNAI-ST8Sia II and pcDNAI-ST8Sia IV harboring cDNA encoding a full-length human ST8Sia II and ST8Sia IV were cloned, as described previously (Nakayama et al., 1995; Angata et al., 1997). The cDNA inserts of pcDNAI-ST8Sia II and pcDNAI-ST8Sia IV were excised by HindIII–XhoI and HindIII–XbaI, respectively, and cloned into corresponding sites of pcDNA3 (Invitrogen, Carlsbad, CA) resulting in pcDNA3-ST8Sia II and pcDNA3-ST8Sia IV.
A rat C6 glioma cell line was transfected with pcDNA3-ST8Sia II or pcDNA3-ST8Sia IV and selected by G418. Clonal cells expressing PSA were chosen after staining with 12F8 anti-PSA antibody (BD Biosciences, San Diego, CA), as described previously (Angata et al., 1997). These cells were designated as C6-ST8Sia II and C6-ST8Sia IV. C6 cells were also transfected with pcDNA3 that lacks cDNA insert, and a cell line isolated after selection with G418 was named mock-transfected C6 cells, C6. These cells were subjected to FACS analysis, as described before (Ohyama et al., 1999).
Western blot analysis
C6, C6-ST8Sia II, and C6-ST8Sia IV cells were subjected to western blot analysis, as described previously (Angata et al., 1997). A portion of cell pellet was digested with endo-N. The proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) (6%) and subjected to western blotting using anti-PSA (12F8) or anti-NCAM (5B8) antibody followed by peroxidase-conjugated goat IgG specific to rat IgM or mouse IgG and ECL Plus kit (Amersham Biotech, Piscatany, NJ). HeLa cells expressing NCAM-140 (Nakayama et al., 1995) were used as a positive control.
Cell proliferation assay
C6, C6-ST8Sia II, and C6-ST8Sia IV were seeded in 96-well plates at 105 cells/mL in -MEM Earle’s Salts (Irvine Scientific, Santa Ana, CA) containing 10% fetal bovine serum and cultured for various periods. The number of living cells was measured each day by using CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega), as described previously (Ohyama et al., 1999).
Implantation of C6 cells in mice brain
C6 tumor cells were inoculated into the caudate putamen of adult C57Bl/6 mice, as described previously (Kaye et al., 1986; Chicoine and Silbergeld, 1995). After anesthetizing with tribromoethanol (0.015 mL/g body weight by intraperitoneal injection), C57Bl/6 mice (7- to 9-week-old males, 8 mice for each group of experiments) were set on a stereotaxis frame, and a 1 cm incision was made in the left frontal region of the head. A craniotomy was performed by using a 2-mm bit on a dental drill, and the dura was punctured with a 25-gauge needle. C6 glioma cell suspension [4.8 x 104 cells in 4 µL phosphate-buffered saline (PBS)] was injected by using a Hamilton syringe with a cone-tipped needle attached to the stereotactic frame. Injection was made into the left caudate putamen of the animal at 0.7 mm anterior to the bregma, at 1.8 mm lateral to the midline, and at a depth of 3.0 mm into the brain.
After surgery, the animals were allowed to recover under observation and then returned to their cage. Twenty-one days after surgery, the animals were sacrificed, and brain specimens were prepared for histological analysis. Under the same conditions, C6 and C6-ST8Sia II were inoculated into the brain of mutant mice deficient in NCAM (Cremer et al., 1994) obtained from the Jackson Laboratory (Bar Harbor, ME). NCAM-deficient mice were backcrossed with C57BL/6 mice three generations before use.
Examination of the brain tissue from mice
The mice were deeply anesthetized with tribromoethanol, followed by perfusion with PBS, pH 7.3, and then with 50 mL of 4% paraformaldehyde, 0.2% glutaraldehyde, and 1 mM MgCl2 in 0.1 M sodium phosphate buffer (pH 7.3). Each mouse brain was postfixed in 2% paraformaldehyde, 0.2% glutaraldehyde, and 1 mM MgCl2 in 0.1 M sodium phosphate buffer (pH 7.3) at 4°C overnight. Fixed specimens were embedded in paraffin and cut at 4 µm thickness. Because our preliminary experiments revealed that an intermediate filament, vimentin, which could be detected in astrocytoma (Cosgrove et al., 1989), was strongly expressed in C6 glioma cells (data not shown), the mouse tissue sections were stained with each of two mouse monoclonal antibodies, 5A5 for PSA and V9 mouse IgG1 (DAKO) for vimentin, followed by treatment with Envision+ (DAKO), as described above, or Ventana ES automated DAB immunohistochemical system (Ventana Medical Systems, Tucson, AZ) (Hayama et al., 2002). The V9 antibody was developed by immunizing swine vimentin and cross-reacts with human, rat, and chicken vimentins. For vimentin staining, microwave irradiation for 25 min in 0.05 M Tris buffer (pH 8.8) containing 1.0 mM EDTA was carried out. In control experiments, primary antibodies were omitted from the staining procedure, and only Envision+ or Ventana system was applied onto the tissue sections. Counterstaining was performed with hematoxylin.
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Acknowledgments |
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We thank Dr. Nobuyoshi Hiraoka for useful discussion, Dr. Edgar Ong for critical reading of the article, and Ms. Aleli Morse for organizing the article. The work was supported by grants R01 CA33895 (to M.F.) and R01 NS41332 (to Y.Y.) awarded by the National Institutes of Health and by grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Priority Area 14082201) and the Ministry of Health, Labor and Welfare of Japan (3rd Term Comprehensive Control Research for Cancer) (to J.N.).
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Abbreviations |
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Endo-N, endoneuraminidase-N; NCAM, neural cell adhesion molecule; PBS, phosphate-buffered saline; PSA, polysialic acid; ST8Sia II,
2,8-sialyltransferase II; ST8Sia IV, 2,8-sialyltransferase IV |
References |
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