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[Cancer Research 61, 2162-2168, March 1, 2001]
© 2001 American Association for Cancer Research

Tumor Biology

The Gene for the Axonal Cell Adhesion Molecule TAX-1 Is Amplified and Aberrantly Expressed in Malignant Gliomas

David S. Rickman, Rachana Tyagi, Xiao-Xiang Zhu, Miroslav P. Bobek, Suzan Song, Mila Blaivas, David E. Misek, Mark A. Israel, David M. Kurnit, Donald A. Ross, Phillip E. Kish and Samir M. Hanash1

Departments of Pediatrics [D. S. R., R. T., X-X. Z., S. S., D. E. M., D. M. K., S. M. H.], Surgery [M. P. B., D. A. R., P. E. K.], Pathology [M. B.], Human Genetics [D. M. K.], and The Comprehensive Cancer Center [M. B., D. A. R., P. E. K., S. M. H.], University of Michigan Medical School, Ann Arbor, Michigan 48109, and Brain Tumor Research Center, Department of Neurological Surgery, University of California, San Francisco, California 94143-0520 [M. A. I.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The human TAX-1 gene encodes a Mr 135,000 glycoprotein that is transiently expressed on the surface of a subset of neurons during development and is involved in neurite outgrowth. The TAX-1 gene has been mapped to a region on chromosome 1 that has been implicated in microcephaly and the Van der Woude syndrome. Using restriction landmark genome scanning to search for amplified genes in gliomas, we found TAX-1 to be amplified in 2 high-grade gliomas among a group of 26 gliomas investigated. Real-time reverse transcription-quantitative PCR analysis detected high levels of TAX-1 mRNA in glial tumors, even in the absence of TAX-1 gene amplification. Immunohistochemical analysis revealed abundant levels of TAX-1 in neoplastic glial cells of glioblastoma multiforme tumors. Because glial tumors are highly invasive and in view of the role of TAX-1 in neurite outgrowth, we investigated the potential role of TAX-1 in glioma cell migration. Using an in vitro assay, we found that the migration of glioma tumor cells is profoundly reduced in the presence of either an anti-TAX-1 antibody or a TAX-1 antisense oligonucleotide. Our findings suggest that TAX-1 plays a role in glial tumorigenesis and may provide a potential target for therapeutic intervention.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Malignant gliomas are the most common primary tumors of the adult central nervous system (1) . The molecular and pathogenic mechanisms leading to malignant gliomas are poorly understood. Chromosome abnormalities including double minute chromosomes, which contain amplified genes, are frequently found in high-grade gliomas (2) . The technique of restriction landmark genome scanning provides a means to search for genomic alterations in tumors throughout the genome (3 , 4) . By using different combinations of enzymes and/or electrophoresis conditions for the separation of restriction digests in two dimensions, the number of individual fragments analyzed can reach several thousand. The use of the rare cleaving restriction enzyme NotI to digest genomic DNA allows visualization of DNA fragments that occur preferentially in CpG islands of the genome. Because of the localization of the CpG islands in proximity to transcribed sequences, the two-dimensional patterns obtained with this enzyme are highly targeted to a functional component of the genome (5) . We have used the two-dimensional approach to identify genes amplified in gliomas (6) . Here we provide evidence that the TAX-1 gene (also known as CNTN2), which encodes a cell adhesion protein known to be transiently expressed in developing neurons and involved in axonal guidance, is amplified at the genomic level and/or overexpressed in gliomas.

TAX-1 is a member of the immunoglobulin superfamily, more specifically, a part of the subgroup whose members are GPI-anchored to the membrane, contain four fibronectin III-like domains, six C2 immunoglobulin-like domains, and are expressed mainly in axons or regenerating neurons (7, 8, 9) . TAX-1 encodes a Mr 135,000 glycoprotein that is transiently expressed in a subset of developing neurons and is the human homologue of the rat TAG-1 and the chick axonin-1 (10 , 11) . On the basis of in situ hybridization and immunocytochemical data, TAG-1 and axonin-1 are exclusively expressed in neurons (12, 13, 14, 15, 16) . Likewise, the TAX-1 protein is specifically expressed in the neuronal lineage of cells in the developing human nervous system (17) .2

By using real-time RT-QPCR and immunohistochemical staining, we found TAX-1 to be highly expressed in gliomas with TAX-1 gene amplification as well as in other high-grade gliomas. We have demonstrated that either interfering with the availability of TAX-1 or down-regulating its expression inhibits glioma cell migration in vitro.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissue Samples and Cultured Cells.
Thirty-six primary gliomas were obtained, after informed consent, from patients undergoing tumor resection at the University of Michigan Medical Center and the University of California, San Francisco. Portions of the tumors were fixed in 10% formaldehyde and embedded in paraffin for sectioning, whereas the remainder of the tissue samples were snap frozen in liquid nitrogen and stored at -80°C for DNA and/or RNA extraction. In addition, paraffin sections from an additional 17 gliomas were provided by the Department of Pathology, University of Michigan Medical Center. Primary tumor cell cultures were prepared from a GBM by mechanical disaggregation. Viable cells (BTL105) were separated from RBCs and cellular debris by centrifugation on a layer of Ficoll-Paque (Pharmacia Biotech AB) and maintained in tissue culture flasks in DMEM supplemented with 10% FBS (Life Technologies, Inc.), 10 units/ml penicillin, and 0.1 mg/ml streptomycin.

Two-Dimensional DNA Gel Preparation and Cloning of TAX-1.
Whole genomic DNA was purified from 26 gliomas of varying grades and normal adult brain and either used for two-dimensional analysis as described previously (4) or Southern blotting. Briefly, purified DNA was digested with the restriction enzymes NotI and EcoRV, two 6-base cutting enzymes. The protruding ends at the NotI site were end-labeled with [{alpha}-32P]dCTP and [{alpha}-32P]dGTP, and the resulting fragments were size-fractionated (1.0–5.0 kb) for the first dimension in 32-cm, 0.9% disc-agarose gels. The fragments were digested in situ with frequent cutting restriction enzymes (HinfI or DpnII), and the resulting fragments were further fractionated in the second dimension (0.3–2.0 kb) in 25 x 43-cm 5.25% polyacrylamide slab gels. The gels were either dried and exposed to a PhosphoImaging screen (Molecular Dynamics) and digitized by imaging software (ImageQuant) or left hydrated and placed on Hyperfilm (Amersham) for preparative gels. The copy number was estimated by comparing the integrated intensity values obtained for the multicopy fragment corresponding to TAX-1 to 10 neighboring two-copy fragments (4) .

The amplified fragment with first-dimension NotI-EcoRV size of 2300 bp and a second-dimension NotI-HinfI size of 1800 bp was cloned directly from a preparative gel. To this end, radiolabeled or unlabeled DNA digests were fractionated as described above. The radiolabeled-fragment gel was exposed to autoradiographic film and used as a template for locating the amplified spot in the unlabeled-fragment gel. The area of the gel that corresponded to the amplified fragment was excised, and the DNA was eluted with a high salt buffer [50 mM Tris-HCl (pH 8.0), 1 M NaCl, and 10 mM EDTA]. The DNA was ligated to a NotI-HinfI-digested pBluescript vector (Stratagene) at 16°C for 40 h. The resulting plasmid insert was sequenced using T3 and T7 primers and an automated sequencer (DNA sequencing core; University of Michigan). The 475-bp sequence obtained matched to GenBank accession number X92681, which corresponds to the cDNA for TAX-1, the human homologue for the TAG-1 found in rat (10) .

Southern Blot Analysis.
Ten µg of genomic DNA (see above) were digested with BamHI and fractionated in a 1% agarose gel. The DNA was first depurinated in situ in 0.2 N HCl for 1 h, followed by denaturation for 1 h in 1.5 M NaCl, 0.5 M NaOH. The DNA was neutralized in 1.0 M Tris (pH 7.4), 1.5 M NaCl and vacuum transferred to a positively charged nylon membrane (Hybond-N+) in the presence of 20x SSC. To cross-link the DNA, the membrane was UV irradiated and left overnight to dry. The membrane was incubated at 42°C for at least 3 h in hybridization buffer consisting of 50% formamide, 3x SSPE, 5x Denhardt’s solution, 0.5% SDS, and 0.1 mg/ml denatured salmon sperm DNA. A 641-bp PCR fragment of the last exon of TAX-1 was radiolabeled using [{alpha}-32P]dCTP and a random priming kit (Boehringer Mannheim), purified using a G-50 Sephadex column (Quick Spin; Boehringer Mannheim), denatured and added to the hybridization solution. The membrane was incubated overnight at 42°C, washed at a final stringency of 0.1x SSC and 0.5% SDS at 65°C and exposed to a PhosphoImaging screen for analysis using imaging software (ImageQuant). The membrane was then stripped of any remaining probe and rehybridized as described above with a radiolabeled 740-bp KpnI-BamHI fragment of the OP18 cDNA or probes for REN, GAC1, or MDM4. The PCR-generated probes for REN, GAC1, and MDM4 have been described previously (18) . The level of TAX-1 amplification was estimated based on the relative intensity to OP18, another gene also located on chromosome 1 (19) .

Western Blot Analysis.
BTL105 or U87 cells were cultured in DMEM supplemented with 10% FBS, as described above, for 72 h in standard culture conditions. The medium was removed and concentrated 10-fold (Centricon 10; Amicon), and the proteins were solubilized in loading buffer [final concentration: 62 mM Tris (pH 6.8), 0.5% DTT, and 1.5% SDS] containing a protease inhibitor cocktail (Boehringer Mannheim) and stored at -80°C. The cells were scraped off the tissue culture dish in the presence of loading buffer, and the resulting cell-associated proteins were stored at -80°C until further use. Proteins were separated by SDS-PAGE in a 7.5% acrylamide/piperazine diacrylamide gel. The proteins were transferred to a polyvinylidene fluoride membrane (Immobilon-P; Millipore) in a semidry blotting apparatus (Semi-Phor TE70; Hoffer Scientific Instruments) in 5% methanol, 50 mM sodium borate, pH 9.0 (anode), and 20% methanol, 50 mM sodium borate, pH 9.0 (cathode). The membranes were incubated in a blocking solution containing 5% milk for at least 3 h at room temperature and then in a solution containing 1% milk with either a rabbit polyclonal antibody to the TAX-1 protein (Transduction Laboratories, Nashville, TN) or a mouse monoclonal antibody against ß-tubulin (Sigma). The proteins were visualized using a chemiluminescence kit (Amersham) and exposure to autoradiographic film. The cells treated with antisense or scrambled oligonucleotide (see below) were analyzed in the same manner. Autoradiographic film was scanned, and the levels of TAX-1 and ß-tubulin proteins were quantitated with Visage System (BioImage, Ann Arbor, MI).

RT-QPCR.
Total RNA was extracted from BTL105 and U87 cells with TRIzol reagent (Life Technologies, Inc.), following the protocol that was provided. For each sample, 2 µg of RNA were treated with DNase I (Boehringer Mannheim) at 37°C for 30 min to remove contaminating DNA and then denatured in the presence of random hexamer primers (Promega). The samples were divided in half and incubated with Superscript II (Life Technologies, Inc.) reverse transcriptase in the presence of 1.0 mM DTT and 1.0 mM each of dTTP, dGTP, dCTP, and cATP at 42°C for 40 min. The resulting cDNA was treated with RNase H (Boehringer Mannheim) and subjected to PCR amplification. Forward primers for the human GAPDH (Amplimer set 5406-1; Clontech) and TAX-1 genes (5'-CACACCTCACCATCCTTCAGTC) were end-labeled with [{gamma}-32P]dATP using T4 polynucleotide kinase (New England Biolabs) and used with unlabeled reverse primers (TAX-1 reverse primer, 5'-CACATTTATCCTCTGCCCTTCC) for PCR. Initially, the cDNA was denatured in the presence of the labeled forward and unlabeled reverse primers for TAX-1 in the PCR mixture containing Expand High Fidelity enzyme blend (Boehringer Mannheim) at 94°C for 3 min. The template was amplified for 10 cycles of 60°C for 30 s, 72°C for 2 min, and 93°C for 30 s and for 5 cycles of 60°C for 30 s, 72°C for 2 min, and 93°C for 30 s. Labeled forward and unlabeled reverse primers for GAPDH and fresh Expand High Fidelity enzyme were added, and the template was denatured at 94°C for 3 min. The templates were amplified for an additional 20 cycles at 60°C for 30 s, 72°C for 2 min, and 93°C for 30 s. We determined that 35 and 20 cycles were within the linear range of amplification for TAX-1 and GAPDH cDNA from treated cells, respectively (data not shown). The products were resolved on a 5% urea-polyacrylamide denaturing gel. Vacuum-dried gels were exposed to a PhosphorImaging screen (Molecular Dynamics) and analyzed using imaging software (ImageQuant). Background-subtracted values obtained for the level of TAX-1 expression were based on the relative intensity of the TAX-1 PCR product to that obtained for GAPDH.

For the 30 tumor samples, we relied on the TaqMan assay (Perkin-Elmer model 7700, Foster City, CA) to quantitate the amount of TAX-1 mRNA. The forward and reverse primers for the TAX-1 gene used in the assay were 5'-GTGGTCCCTTTCCGAAATGA and 5'-GGTGGAGCGTGGGAGTCA, respectively. The fluorescently tagged probe used for the TAX-1 gene spans exons 21 and 22 (5'-6FAM-TCATTCTGGTACAGCATCTTATAGCCGGTGAC-TAMRA). The forward and reverse primers used for the GAPDH gene were 5'-TGTGAACCATGAGAAGTATGACAACA and 5'-CACGATACCAAAGTTGTCATGGAT, respectively. We used 5'-6FAM-CATCAGCAATGCCTCCTGCACCAC-TAMRA for the GAPDH probe, which spans two exons. The primers and probe for a given sequence were added to the reaction mixture containing RNA from the sample of interest in the presence of Multi-Scribe reverse transcriptase, RNase inhibitor, and AmpliTaq Gold as per the suggestions of Perkin-Elmer, except that we scaled our total reaction volume down to 10 µl. Reverse transcription was accomplished by a 30-min incubation at 60°C. The reaction was stopped by a 10-min incubation at 95°C that denatures the cDNA-RNA hybrid and activates the AmpliTaq Gold enzyme. The synthesized cDNA is PCR amplified using AmpliTaq Gold in a two-step amplification (15 s at 95°C to denature the DNA and 1 min at 60°C to permit annealing and elongation). To assay the initial concentration of the reactants (the sequence abundance), the number of cycles at which the reaction crosses a threshold value is measured. This number, the CT value, varies directly with the initial sequence abundance. To measure the relative abundance of two genes in a given RNA sample, the amplification value derived using the first sequence (TAX-1) was divided by the amplification value using the second sequence (GAPDH). Derivation of this fraction is independent of RNA sample concentration, eliminating the requirement to measure RNA concentration accurately.

Immunostaining.
Because of the limited availability of anti-TAX-1 antibodies, paraffin sections from 29 different gliomas were immunostained with an antibody made against the TAG-1 protein (3.1C12 at a 1:2 dilution of a hybridoma supernatant). This antibody does not recognize TAX-1 in Western blot analysis (data not shown). This mouse monoclonal antibody, developed by Thomas M. Jessell and Jane Dodd, was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of The National Institute of Child Health and Human Development and maintained by The University of Iowa (Department of Biological Sciences, Iowa City, IA). Serial sections were stained with H&E, with an antibody against GFAP (Dako, at a 1:6400 dilution), or isotype-specific mouse immunoglobulins (Sigma) using a Ventana ES automated immunostainer and peroxidase-based visualization (Ventana Medical Systems, Tucson, AZ.).

Migration Assay.
BTL105 or U87 cells were trypsinized from the culture dishes and centrifuged at 1200 x g for 5 min. The cell pellet was resuspended in DMEM supplemented with 10% FBS and plated into 96-well Ultralow-Cluster culture dishes (Costar) at a density of 4 x 103 cells/well and incubated under standard culture conditions for 24 h on a rotating shaker. Under these conditions, the cells aggregate and form a single multicellular spheroid. The spheroids were then treated with either a phosphorothiate-modified antisense (AS1) oligonucleotide to TAX-1 (5'-TCCTGGTGGCTGTCCCCATGGTGG), a phosphorothiate-modified oligonucleotide that is a scrambled version of the compliment of AS1 (Scr1, 5'-CCAGCGCCAAGAATGACGCGGACC), at a final concentration of 0.01, 0.1, or 1.0 µM. Cells were also incubated at the same concentrations as stated above with another TAX-1-antisense oligonucleotide (AS2, 5'-GAACTCCAAGCTGAAGAGGAGACA), a scrambled version of the compliment of AS2, Scr2 (5'-CTCTGAGTGTTGGCTACCTTCTCT), the rabbit polyclonal antibody against TAX-1 (see above), or rabbit immunoglobulins (Sigma) at a final concentration of 15 µg/ml. Forty-eight h after constant agitation, individual spheroids were selected and plated into a well of a 12-well tissue culture dish (Falcon) in the presence of fresh medium replenished with the appropriate oligonucleotide or antibody. Cell migration away from an attached spheroid was monitored at 15, 30, 42, and 65 h after plating by imaging the tissue culture well with a digital camera (Pixera Visual Communication System; Pixera Corp.). The migration distance, calibrated with a stage micrometer, was measured using the resulting image.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TAX-1 Gene Amplification in Gliomas.
Two-dimensional analysis of 26 gliomas was undertaken using the NotI/EcoRV/HinfI enzyme combination. One fragment with first-dimension NotI-EcoRV size of ~2300 bp, and a second-dimension NotI-HinfI size of ~1200 bp was found to occur with an increased copy number in two of the tumors (nos. 62 and 307; Fig. 1ACitation ). This fragment was derived from chromosome 1, based on its occurrence in two-dimensional patterns of flow sorted chromosome 1 (20) . This fragment was cloned and sequenced. A database search revealed identity with a sequence in the promoter region of the TAX-1 gene from position -342 to +32 (GenBank accession no. X84419; Ref. 21 ). The TAX-1 gene has been mapped to chromosome band 1q32.1 and implicated in microcephaly and the Van der Woude syndrome (10 , 22) . Sufficient DNA was available for Southern blot analysis of 13 of the 26 tumors, including one tumor (no. 307), which showed amplification of TAX-1 by two-dimensional analysis (Fig. 1B)Citation . TAX-1 gene copy number was estimated at 50 copies/diploid genome in tumor no. 307 by Southern analysis, which was consistent with quantitative analysis of TAX-1 spot intensity in two-dimensional patterns of this tumor. Tumor 62 was estimated at 16 copies/diploid genome based on its quantitative analysis in two-dimensional patterns of this tumor (4) . The GAC1 and REN genes, and more recently, the MDM4 gene, have also been identified to be a part of this amplicon (6 , 18 , 23) . The same Southern blot was screened to determine the status of these genes. We observed coamplification of the GAC1 and REN genes in tumor no. 307. The MDM4 gene was not amplified in any of the tumors analyzed (Fig. 1B)Citation .



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  		Fig. 1. DNA analysis of TAX-1 in gliomas. A, close-up of two-dimensional patterns showing a multicopy fragment (arrow) in tumors 62 and 307 but not in normal brain. Genomic DNA was digested with NotI and EcoRV. The fragments were end-labeled and fractionated in a 0.9% disc-agarose gel. The fragments were digested in situ with HinfI and further fractionated in the second dimension in a 5.25% agarose slab gel. Estimated copy numbers of TAX-1 are 16 and 50 copies of the TAX-1 gene/diploid genome for tumors 62 and 307, respectively (4) . B, Southern analysis of 13 tumors showing TAX-1 gene amplification in tumor 307 (arrow). A 641-bp PCR amplified fragment of the most 3' end exon of the TAX-1 gene was used as a probe. The membrane was subsequently stripped of bound probe and rehybridized with a radiolabeled, 740-bp KpnI-BamHI fragment of the OP-18 cDNA, another gene located on chromosome 1 (19) . The membrane was stripped of bound probe and rehybridized with a 221-bp fragment of the MDM4 gene, a 234-bp fragment of the GAC1 gene, or a 215-bp fragment of the REN gene (18) .

 
TAX-1 mRNA Expression in Gliomas.
Northern analysis of TAX-1 mRNA revealed a low signal in gliomas that could not be reliably quantitated (data not shown). Given this low signal and the limited amount of tumor tissue available, we used a more sensitive fluorescent real-time RT-QPCR to evaluate the expression of TAX-1 in gliomas. The TaqMan RT-QPCR assay has been described previously (24, 25, 26) . The assay is based on the principle that successful PCR yields a fluorescent signal attributable to Taq-mediated exonuclease digestion of a fluorescently labeled oligonucleotide homologous to a sequence between the two primers. The extent of digestion, which depends directly on the amount of PCR product that is produced, can be quantified by measuring the increment in fluorescence that results from decreased energy transfer. This sensitive measurement allows detection in the exponential phase of the PCR reaction that is required for determination of initial sequence abundance. Thirty gliomas of varying grades, using the WHO criteria (1) , were analyzed for the expression of TAX-1 (including 20 of the original 26 tumors, subjected to two-dimensional analysis). As controls, we used samples from fetal brain, known to have high-level expression of TAX-1, and from adult brain, known to have low-level expression of TAX-1. Table 1Citation shows the relative abundance of TAX-1 mRNA in gliomas, normalized to the average of three normal adult brain samples. Listed are 26 high-grade (grades III and IV or GBM) and 4 low-grade (grades I and II) tumors. We detected high levels of TAX-1 mRNA in tumor nos. 62 and 307, both of which contained an amplified TAX-1 gene (Fig. 1)Citation . Increased levels of TAX-1 mRNA (two SDs above the mean for normalized adult brain, i.e., >1.6 in the normalized TAX-1 column) were also detected in 7 tumors without TAX-1 amplification, including one low-grade glioma (no. 327).


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  		Table 1 Expression analysis of TAX-1 in gliomas

 
Expression of TAX-1 Protein in Neoplastic Glial Cells.
The TaqMan results suggested that TAX-1 is expressed in gliomas in the absence of gene amplification. Because tumor tissue samples contain a heterogeneous population of cells, we assessed the specificity of expression of TAX-1 by immunohistochemistry. We used a mouse monoclonal antibody against the rat homologue, TAG-1. This antibody recognizes the human TAX-1 protein in tissue sections and has been used to monitor TAX-1 expression during human spinal cord development (17) . Paraffin sections from 29 gliomas (21 high-grade and 8 low-grade), including 12 that were analyzed by the TaqMan assay, were screened for reactivity with the anti-TAG-1 antibody. In all but two of the high-grade tumors, neoplastic glial cells stained positive for TAX-1, whereas the low-grade tumors showed little or no TAX-1 expression (Fig. 2, A–DCitation ; Table 1Citation ). Immunostaining ratings shown in Table 1Citation indicate the amount of staining for the TAX-1 protein observed in neoplastic cells. Nonneoplastic cells did not show detectable staining. Adjacent paraffin sections from the tumors did not show immunoreactivity using isotype-specific mouse immunoglobulins. The brightest and most extensive staining was observed in GBMs, particularly in neoplastic cells that also expressed GFAP, a marker for cells of glial lineage (27 , 28) . The subcellular localization of TAX-1 varied between tumors as well as between individual cells within tumors. TAX-1 was found associated with the plasma membrane, in the cytoplasm, or occasionally forming a perinuclear rim. In contrast, cellular constituents in normal brain, including glial cells, showed negative immunostaining for TAX-1 (Fig. 2, E and F)Citation . It should be mentioned that we detected low levels of TAX-1 mRNA in many of the GBMs and a high level in 1 low-grade glioma (nos. 327) that were not analyzed by immunohistochemistry. However, we generally observed a high abundance of the TAX-1 protein in high-grade gliomas relative to low-grade gliomas by immunohistochemistry.



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  		Fig. 2. Analysis of TAX-1 expression by immunohistochemistry. Serial paraffin sections from a high-grade tumor [grade IV (Gr. IV)or GBM] stained for the TAX-1 protein (A) and GFAP (B) are shown. A large multinucleated neoplastic cell (arrow) stained positive for both TAX-1 and GFAP. Representative serial paraffin sections of a low-grade tumor [grade I (Gr. I) pilocytic astrocytoma], stained for TAX-1 (C) and GFAP (D), are shown. Although this tumor expressed GFAP abundantly, TAX-1 is barely detectable. A region of normal brain (Nrml. Br.) from a patient with a GBM, in which tumor cells stained positive for TAX-1, shows that normal glial cells stained negative for TAX-1 (E) and positive for GFAP (F).

 
TAX-1 Is Involved in Tumor Cell Migration in Vitro.
The TAX-1 protein has been implicated in the promotion of neurite outgrowth of developing neurons via homophilic and heterophilic interactions with L1, integrins, and other cell adhesion molecules (29, 30, 31) . The TAX-1 protein is attached to the cell membrane by a GPI anchor that may be cleaved, resulting in the release of a secreted form of TAX-1 from the cell surface, which can stimulate neurite outgrowth (11) . Therefore, we examined TAX-1 expression in cultured glioma cells, in view of inhibiting TAX-1. We found expression of TAX-1 in both primary glioma cells that were derived from a GBM explant (BTL105 cells) and in cells from an established cell line derived from a grade III astrocytoma (U87, ATCC HTB14), by RT-QPCR, immunofluorescence (data not shown), and Western analysis. We detected TAX-1 in the cell lysates of BTL105 and U87 but not in the culture media in which the cells were grown (Fig. 3)Citation . Because malignant gliomas are highly invasive, we used an in vitro assay, using U87 and BTL105 cells, to assess the involvement of TAX-1 in cell migration (32 , 33) . Multicellular spheroids were generated with BTL105 cells and incubated either in the presence of an antibody to TAX-1 or with isotype-specific rabbit immunoglobulins as a control. The migration of adherent single cells away from the spheroid was monitored at different time points. In control cultures, migration of single cells was observed at 15 h, and by 40 h, a substantial migration was noted (Fig. 4, A and B)Citation . In contrast, cells incubated in the presence of an anti-TAX-1 antibody were substantially inhibited in their ability to migrate. This result suggested that blocking TAX-1 protein inhibited cell migration. We next determined whether down-regulation of the TAX-1 message would inhibit cell migration. U87 or BTL105 spheroids were cultured in the presence of a 24-base antisense oligonucleotide (AS1) spanning the start codon of the TAX-1 cDNA (GenBank accession X68274) or in the presence of a scrambled oligonucleotide (Scr1) with an identical base composition. Using specific radiolabeled forward and unlabeled reverse primers for the TAX-1 and GAPDH genes, RT-QPCR was performed within the linear range of product amplification. The level of TAX-1 mRNA in treated or untreated cells was determined by its intensity relative to the product obtained for GAPDH. AS1 profoundly reduced the level of the TAX-1 message compared with the level seen in untreated cells or cells treated with Scr1 (Fig. 4, D and E)Citation . A reduced level of the TAX-1 protein was also observed in cells treated with the antisense oligonucleotide relative to cells treated with Scr1 or with a different antisense oligonucleotide (AS2) or a scrambled oligonucleotide (Scr2; Fig. 4, F and GCitation ). Moreover, the magnitude of the TAX-1 protein down-regulation (by ~3-fold) in AS1 treated cells compared with control and Scr1-treated cells corresponds to the fold reduction of mRNA values observed in AS1-treated cells. BTL105 cells treated with AS1 had markedly reduced migration away from the spheroids as compared with untreated cells or cells treated with Scr1 (Fig. 4C)Citation . A similar inhibitory effect of the antisense oligonucleotide was observed with U87 cells (data not shown). Treatment with AS2 and Scr2 did not have an inhibitory effect on BTL105 or U87 cell migration (data not shown). These results suggest that TAX-1 affects tumor cell migration in vitro.



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  		Fig. 3. Analysis of TAX-1 expression in cultured glioma cells by Western blotting. The medium/supernatant from U87 or BTL105 cells was collected after 72 h in culture, concentrated 10-fold, and subjected to SDS-PAGE/Western blot analysis along with the corresponding cell lysate. DMEM supplemented with 10% FCS was concentrated similarly and used as a negative control. Rat brain extract (rat br.) supplied with the anti-TAX-1 antibody (Transduction Laboratories, Nashville, TN) served as positive control. The membrane was probed with a rabbit polyclonal antibody to the TAX-1 protein and then was stripped and reprobed with an antibody against ß-tubulin. Species-specific horseradish peroxidase-conjugated secondary antibodies were used in conjunction with an enhanced chemiluminescence kit (Amersham) to visualize Tax-1 and ß-tubulin. TAX-1 is detected in the cell extracts and not in the culture medium. ext., extract; sup, supernatant.

 


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  		Fig. 4. Effects of inhibiting TAX-1 on the migration of glioma cells in culture. A, three representative BTL105 spheroids cultured for 40 h are shown. One was untreated (Cntl), and the other two were treated with either rabbit immunoglobulins (Rb IgG) or a rabbit anti-TAX-1 antibody (anti-TAX1). Migration distances were obtained by measuring the distance between the peripheral edge of the spheroid and the outermost edge of the ring of migrated cells. B and C, each data point in the graphs is an average of three separate experiments; bars, SD. B, a summary of the migration distances that were measured for antibody-treated spheroids. C, a summary of the measurements taken of cell migration in the presence of 1.0 µM AS1 or Scr1 or untreated over time. Total RNA was extracted from BTL105 cells that were untreated (Cntl) or treated for 65 h with either 1.0 µM of AS1 or Scr1. D, BTL105-related RT-PCR products of the TAX-1 and GAPDH genes. E, the intensity values obtained for TAX-1 relative to those obtained for GAPDH. F and G, Western blot and intensities values obtained for TAX-1 and ß-tubulin extracted from BTL105 cells treated for 65 h with 1.0 µM AS1, Scr1, AS2, Scr2, or not treated (Cntl).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have shown that the gene for the axonal cell adhesion molecule TAX-1 is amplified and aberrantly expressed in high-grade gliomas. TAX-1 maps to position 1q32.1 known to contain an amplicon in some gliomas (34) . Others have reported the amplification of REN, GAC1, and MDM4 and found overexpression of the latter two in a small percentage of tumors (6 , 18 , 23) . In contrast, we have immunohistochemical evidence of expression of TAX-1 in most high-grade gliomas. According to the GB4 radiation hybrid map,3 these genes are ordered as follows (centromeric to telomeric): MDM4, GAC1, REN, and TAX-1 at position 1q32.1. In one of the tumors exhibiting TAX-1 amplification, we showed that REN and GAC1, and not MDM4, are similarly coamplified. It should be noted that we also detected amplification of the TAX-1 gene in one meningioma. REN and GAC1, but not MDM4, were coamplified in this tumor as well. Therefore, MDM4 is not a part of the amplicon in the tumors that we analyzed.

TAX-1 is a member of a subset of proteins in the immunoglobulin superfamily, which are GPI-anchored to the membrane (9 , 35) , contain four fibronectin III-like domains and six C2 immunoglobulin-like domains, and are expressed mainly in axons or regenerating neurons (7) . TAX-1, like its rat and chick homologues TAG-1 and axonin-1, respectively, is exclusively expressed in the neuronal lineage in the human nervous system (11 , 14, 15, 16, 17) . Another member of the immunoglobulin superfamily, L1, has been shown to be expressed in glioma cells in culture and potentially involved in the invasion of tumor cells as a secreted form (36) . However, unlike TAX-1, L1 is expressed in normal glial cells as well as other nonneuronal cells (37, 38, 39) . TAX-1 has been found to be both membrane associated and secreted from the surface of the cell as a soluble factor, which acts as a substratum for the outgrowing neurites (11) . In our studies of glioma cell lines, we did not find evidence that the Tax-1 protein was secreted.

All of the tumors in this study are gliomas and are presumably derived from cells in the glial lineage. Using the TaqMan assay along with immunohistochemistry, we observed TAX-1 to be overexpressed in the tumors containing an amplified TAX-1 gene. The TAX-1 protein was localized in neoplastic, GFAP-positive cells, with highest expression seen in GBMs. Moreover, TAX-1 was found to be expressed in gliomas without TAX-1 gene amplification. The finding of TAX-1 expression in tumors deriving from the glial lineage was unexpected given that in prior studies, TAX-1 and TAG-1 were found to be transiently expressed solely on the surface of fasciculating axons of a restricted subset of neurons during embryonic neural development, with minimal expression in normal adult neural tissue (15, 16, 17) . It should be noted that by in situ hybridization analysis, low levels of TAG-1 mRNA were detected in mouse cerebellar granule cells, in pyramidal cells of areas CA1 and CA3 of the hippocampus, and other areas that are known to maintain their neural plasticity in adult brain (40) . This is consistent with the idea that TAX-1, TAG-1, and axonin-1 are involved in neuronal development. Further investigation is needed to explore the mechanism behind the aberrant expression of TAX-1 in the absence of gene amplification.

During tumorigenesis, newly formed tumor cells expand and infiltrate into surrounding tissue by changing their intracellular/extracellular architecture (41) . There is evidence that altering the expression of adhesion molecules alters the capacity for cell-cell or cell-matrix interaction, which mediates the invasion and growth of neoplastic tissue (35) . Given its structural and functional similarity to L1, we reasoned that TAX-1 may function in the migration of glioma cells. We therefore used an in vitro migration assay to measure the involvement of TAX-1 in cell migration. We showed that either down-regulating the expression level of TAX-1 with an antisense oligonucleotide or masking the protein with an antibody inhibits cell migration. Members of the immunoglobulin superfamily interact with an array of proteins, including extracellular matrix proteins and other cell surface adhesion molecules. Both TAG-1 and axonin-1 form homophilic complexes as well as bind to other cell adhesion molecules, such as L1 and ß1 integrins, and these interactions are essential for their ability to promote neurite outgrowth and fasciculation (29 , 31 , 42) . Therefore, it is of interest to identify the proteins or ligands that interact with TAX-1 to mediate its role in tumor cell migration. Nr-CAM/Bravo, another member of the immunoglobulin subfamily similar to L1, is expressed on the surface of glial cells and interacts with axonin-1, mediating neuron-glia contacts (14) . Nr-CAM/Bravo has been shown to be overexpressed in high-grade gliomas; however, to date, there have been no studies documenting an interaction between TAX-1 and Nr-CAM/Bravo (43) .

Our results suggest that the expression of TAX-1 in neoplastic cells of high-grade gliomas may be related to their ability to migrate and invade surrounding normal tissue. These tumors are typically highly malignant, infiltrative, and aggressive. The restricted expression of TAX-1 makes it a plausible target for intervention in glioma therapy.


   FOOTNOTES
 
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.

1 To whom requests for reprints should be addressed, at The Comprehensive Cancer Center, University of Michigan, A520 MSRB I, Box 0656, Ann Arbor, MI 48109. Phone: (734) 763-9311; Fax: (734) 647-8148; E-mail: shanash{at}umich.edu Back

2 The abbreviations used are: GPI, glycosyl phosphatidylinositol; TAG, transiently expressed axonal glycoprotein; GBM, glioblastoma multiforme; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; 6FAM, 6-carboxy-fluorescein; TAMRA, 6-carboxytetramethylrhodamine; GFAP, glial fibrillary acidic protein; RT-QPCR, reverse transcription-quantitative PCR. Back

3 Internet address: http://www.ncbi.nlm.nih.gov/genemap99. Back

Received 6/19/00. Accepted 12/22/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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