MAGE-E1, a New Member of the Melanoma-Associated Antigen Gene Family and Its Expression in Human Glioma

[CANCER RESEARCH 61, 4809–4814, June 15, 2001] MAGE-E1, a New Member of the Melanoma-associated Antigen Gene Family and its Expression in Human Glioma

Manabu Sasaki, Kensuke Nakahira,1 Yozo Kawano, Hiromichi Katakura, Toshiki Yoshimine, Keiji Shimizu,2 Seung U. Kim, and Kazuhiro Ikenaka National Institute for Physiological Sciences, Okazaki, 444-8585 Japan [M. S., K. N., Y. K., H. K., K. I.]; Department of Physiological Sciences, Graduate University for Advanced Studies, Okazaki, 444-8585 Japan [K. I.]; Department of Neurosurgery, Osaka University Graduate School of Medicine, Suita, 565-0871 Japan [M. S., T. Y., K. S.]; Division of Neurology, Department of Medicine, University of British Columbia, Vancouver, Canada [S. U. K.]; and Brain Disease Center, Ajou University, Suwon, 442-721 Korea [S. U. K.]

ABSTRACT level of each gene (1). Thus, it is possible to pick up specifically up-regulated genes from an entire gene pool by comparing SAGE To unearth glioma-specific genes in human glioblastoma, the serial profiles between various cancers and normal tissues (2). In SAGE, analysis of gene expression technique was applied to a primary glioblas- each mRNA is distinguished by a short nucleotide fragment (ϳ14 toma, using cultured human astrocytes as a normal control. Among the top 147 most-expressed tags in glioblastoma, we found a tag, TTTTGGG- base, called a “tag”), which is cleaved from a single particular position TAT, that originated from an unidentified gene and which was not of the mRNA. As tags are collectively subcloned and sequenced, detected in human astrocyte cultures. Real-time quantitative reverse tran- thousands of mRNAs can be analyzed in a relatively short time. The scription-PCR showed that MAGE-E1 expression was 2.6–15-fold en- original gene of each tag can be identified by searching databases, and riched in glioblastoma relative to human astrocytes. Expressed sequence the level of the expression can be quantified as the frequency of the tags containing this tag were homologous to the melanoma-associated appearance of the tag. The great advantage of the technique is that tags antigen gene (MAGE) family, and this new cDNA, named MAGE-E1, was from unidentified genes can also be quantified, which was impossible ,cloned by the 5؅-rapid amplification of cDNA ends technique. Three using other techniques such as Northern blotting. In the present study alternatively spliced variants (MAGE-E1a–c) were found, and deduced we performed SAGE experiments to search glioblastoma-specific amino acid sequence showed that MAGE-E1a and -E1b shared the MAGE- genes in a glioblastoma sample and discovered expression of a novel conserved region, whereas -E1c did not. This suggests that although MAGE-E1c is expressed from one of the MAGE family, it has distinct gene, a new member of MAGE family, which is up-regulated in functions from other members. Tissue distribution analysis showed that glioblastoma as compared with normal tissue. MAGE-E1 was distinct from other MAGEs. MAGE-E1 expression was MAGE is a gene family that encodes melanoma antigens. detected only in brain and ovary among normal tissues. Interestingly, MAGE-A1 was originally isolated from the melanoma genomic DNA MAGE-E1a and/or -E1b were specifically expressed in glioma cells among coding an antigen recognized by CTLs (3). Many genes homologous cancer cells. These results indicate that MAGE-E1 is a novel and glioma- to MAGE-A1 have been determined on Xq 28, Xp21.3, Xq26, and specific member of MAGE family. Xp11.23 (4–8) and are classified as MAGE-A,-B,-C, and -D, respectively. Previous studies have described the characteristics of the MAGE-A subfamily as follows: (a) they were not expressed in normal INTRODUCTION cells except for testis and placenta; and (b) some antigens coded by The prognosis of malignant glioma treatment in human patients is the gene family are presented by the human leukocyte antigen (9). distressingly bad, and the 5-year survival rate of patients after surgery These findings suggest that immunoreaction against MAGE proteins is only 8% in Japan. New therapeutic means to cure glioma, including are expected to affect only the tumor cells but not normal cells and/or immunotherapy and gene therapy, are being developed; glioma- tissues, because testis and placenta lack human leukocyte antigen specific genes to be used for these new therapies have been sought but expression. Immunotherapy with MAGE-A peptides showed tumor without any success. Although genes for GFAP3 and vimentin are regression without significant side effects in melanoma patients who known to be expressed in glioma cells and used for cell typing, they were vaccinated with the MAGE peptide (10). are also expressed in normal astrocytes and are not exclusive for In the present study, we demonstrate that the novel MAGE-related glioma. Glioma-specific genes, like ␣-fetoprotein for hepatocellular gene, MAGE-E1, is up-regulated in malignant glioma. Distribution carcinoma or MAGE genes for melanoma, should provide us with a within normal tissues indicates that MAGE-E1 is a unique member of tool for diagnosis and a target molecule for treatment of malignant MAGE gene family that is specifically expressed in brain and ovary. gliomas. Such genes should also be useful for understanding the pathogenesis and progression of gliomas. MATERIALS AND METHODS Recent progress in molecular biology provides us with tools to uncover tissue- or cell type-specific genes. SAGE is one such tech- Materials. Primary glioblastoma (GB-1–8) tissue block was obtained sur- niques, and it enables us to detect all expressed genes, including gically from adult patients. A human glioma cell line, U87 MG, was cultured unknown ones, in a given tissue and to quantify the relative expression with DMEM (Sigma Chemical Co., St. Louis, MO) containing 10% fetal bovine serum (Life Technologies, Inc., Rockville, MD). Total RNA was extracted by the guanidinium isothiocyanate-CsCl gradient method (11). Received 5/12/00; accepted 4/11/01. Poly(A) RNA fractions of GB-1 and U87 MG were obtained with an mRNA 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 Isolation Kit (Stratagene, La Jolla, CA). Cultured human astrocytes (HA-1) 18 U.S.C. Section 1734 solely to indicate this fact. were prepared as reported previously (12). Poly(A) RNA fraction was obtained 1 To whom requests for reprints should be addressed, at National Institute for Physi- from HA-1 with a QuickPrep mRNA Purification Kit (Amersham Pharmacia ological Sciences, Okazaki, 444-8585 Japan. Phone: 81-564-55-7841; Fax: 81-564-55- Biotech, Uppsala, Sweden). Total RNAs of lung cancer (non-small cell lung 7843; E-mail: [email protected]. 2 Present address: Department of Neurosurgery, Kochi Medical School, Nankoku, cancer), stomach cancer, colon cancer, and hepatocellular carcinoma were 783-8505 Japan. provided by Dr. Shunichiro Okumura (Kansai Medical University, Osaka, 3 The abbreviations used are: GFAP, glial fibrillary acidic protein; MAGE, melanoma- Japan). associated antigen gene; SAGE, serial analysis of gene expression; RT-PCR, reverse SAGE. SAGE was performed with 2.5 ␮g of the poly(A) RNA according transcription-PCR; EST, expressed sequence tag; RACE, rapid amplification of cDNA ends; CGAP, Cancer Genome Anatomy Project; VEGF, vascular endothelial growth to the original method (1). Tags were analyzed by SAGE software provided by factor; HSP-70, heat shock protein 70. Dr. Kenneth W. Kinzler (Johns Hopkins University, Baltimore, MD). The 4809

original gene of each tag was identified by searching the GenBank database was derived from the cDNA containing the EST sequence. For this purpose, (version 107.0). SAGE profiles reported in the Cancer Genome Anatomy sequences from the tag to the 3Ј-end were amplified from the intermediate Project (CGAP) were obtained through Internet from the web site at http:// product of the SAGE experiment as follows. U87 MG cDNA was synthesized www.ncbi.nlm.nih.gov/SAGE/. from 2.5 ␮g of mRNA with biotinylated oligo(dT) primer. The cDNA was Real-time Quantitative RT-PCR. Real-time quantitative RT-PCR was cleaved by NlaIII. The fragment containing the 3Ј-end was purified with performed with total RNA from four primary glioblastomas (GB-2, -3, -4, -6) avidin-coated magnetic beads (Dynal, Oslo, Norway) and ligated with Link- and human astrocyte cultures (HA-1) with TaqMan Reverse Transcription er-1, which was used in the SAGE experiment. The product was amplified with Reagents and the TaqMan PCR Core Reagent Kit (Perkin-Elmer, Brunchburg, a specific primer containing the tag sequence 5Ј-TAGGGACATGTTTTGGG- Ј Ј NJ) following the instruction manual. 5 -CCAGCTTCTTCTCCTGGATC-3 TAT-3Ј (Tag primer) and a primer, 5Ј-CGGAGCTCTTTTTTTTTTTTTT- Ј Ј (F1 primer) and 5 -GTAACACTGATACCCAAAACATG-3 (R1 primer) TTTT-3Ј (PCR-dT primer) by the following cycling parameters: 30 s at 94°C; Ј were designed to amplify the 3 -half region of an EST, AI082447, containing 30 s at 55°C; 1 min at 72°C for 25 cycles; and a final extension at 72°C for 3 the tag sequence of MAGE-E1. TaqMan probe for detection of MAGE-E1, min. The product was subcloned into the pCR-2.1 vector (Invitrogen, Gron- 5Ј-CGGCTTCTTCCTGTCAGTCGGAGGT-3Ј (MAGE-E1 probe) was de- ingen, Netherlands). The sequence was analyzed by the dideoxy sequence signed to detect a sequence within the F1 primer and the R1 primer (Fig. 1A). method. Human ␤-actin primers and probe were purchased from Perkin-Elmer. The The 3Ј-half region of the EST was also confirmed by RT-PCR experiment reverse-transcribed sample from GB-6 was diluted (1:2, 1:4, 1:8, and 1:16) to with F1 primer and R1 primer, which were described above (Fig. 1A). The R1 construct the standard curve. Experiments were run in duplicate, and the primer sequence was chosen from the region between the tag and the 3Ј-end. average value of the threshold cycle (C ) was calculated for MAGE-E1 and T The PCR product was subcloned into the pCR-2.1 vector and sequenced. ␤-actin, respectively. PCR reaction and the resulting relative increases in Ј reporter fluorescent dye emission were analyzed by the 7700 sequence detector The upstream region of the cDNA was cloned by the 5 -RACE method. The (Perkin-Elmer, Brunchburg, NJ). Analysis of the signal and the construction of Linker-1 was blunted with T4 DNA polymerase (Takara, Tokyo, Japan). U87 the standard curve was performed by the Sequence Detector v1.6.3 software MG cDNA synthesized with biotinylated-oligo(dT) primer was immobilized to (Perkin-Elmer). 1 mg of avidin-coated magnetic beads and connected with 100 pmol of the ␮ Cloning of MAGE-E1 cDNA. An EST, AI082447, was found to contain a blunted Linker-1 with T4 DNA ligase (5 units/ l; Boehringer-Mannheim, tag sequence, CATGTTTTGGGTAT. To use the sequence information of this Mannheim, Germany). The product was amplified with the Linker-1-specific EST for the cloning of corresponding cDNA, we first confirmed that the tag primer used in SAGE and the R2 primer (Fig. 1A), 5Ј-CATCTTGCATGC- CACCTCCG-3Ј, with the LA-PCR Kit (Takara, Tokyo, Japan) by the following cycling parameters: 30 s at 94°C; 30 s at 55°C; 10 min at 72°C for 25 cycles; and a final extension at 72°C for 10 min. The product was subcloned into the pCR-2.1 vector. A clone containing a 1.3-kb fragment was obtained and named G-1. The second 5Ј-RACE was done with 5Ј-TTCCAGGGTG- TACGTTGCT-3Ј (R3 primer). The entire open reading frame was obtained by the third round of 5Ј-RACE with 5Ј-CTACTAGGGCCCTCAGTAGCAG-3Ј (R4 primer). To ensure the continuity of the coding sequence obtained by 5Ј-RACE, a RT-PCR experiment was performed with 1 ␮g of U87 MG total RNA with 5Ј-CTGCGGGGTTCCTGGTGC-3Ј (F2 primer) and 5Ј-GGGT- GCTGTGTCGAGAAGGA-3Ј (R5 primer). Because of the GϩC-rich region, PCR was performed with the LA-PCR Kit following the cycling parameters: 30 s at 94°C; 30 s at 58°C; 4 min at 72°C for 24 cycles; and a final extension at 72°C for 5 min. The amplified products were subcloned into pCR-2.1, and analyzed by sequencing. Three independent clones were obtained. Sequences were registered with the DNA Data Bank of Japan database as accession nos. AB040527, AB040528, and AB040529 for MAGE-E1a,-E1b, and -E1c, respectively. Northern Blot Analysis. Northern blot experiments were done according to standard method (11). Fifty ␮g of total RNA from U87 MG cell line and a primary glioblastoma (GB-6) were separated on a formaldehyde gel to make a filter. RNA ladder (New England Biolabs, Inc. Beverly, MA) was used as a size marker. Human Multiple Tissue Northern blot (MTN; Clontech, Palo Alto, CA) was purchased to investigate the normal tissue distribution of MAGE-E1. A 1.3-kb BstXI-fragment of the MAGE-E1 cDNA (G-1) was labeled with [␣-32P]-dCTP using a High Prime labeling kit (Boehringer Mannheim) and used as a probe. For the positive control, a human ␤-actin cDNA supplied in the MTN was used. Signals were visualized using a BAS 2000 Bio Imaging Analyzer (Fujix, Tokyo, Japan). RT-PCR. One ␮g of total RNA from five glioblastoma (GB-1, -5, -6, -7, Fig. 1. A, the 3Ј-terminal fragment of MAGE-E1 cDNA was amplified from the intermediate product of SAGE by PCR with a specific primer containing the tag sequence and -8) and other cancers was reverse-transcribed with oligo(dT) primer (Tag primer) and oligo(dT)-primer. This fragment contained the region from the tag to the following the method described previously (11). Samples without reverse Ј 3 -end of MAGE-E1. The region of the tag is shown in an open box. The upstream region transcriptase were prepared for negative control. 5Ј-CCTGTGCTTTCTCT- of MAGE-E1 cDNA was amplified with F1 primer and R1 primer designed according to Ј Ј Ј the sequence information of AI082447 shown in this figure. The R1 primer sequence was CAGGCT-3 (F3 primer) and 5 -TCTCTCTCTCCTCCTCGCTGC-3 (R6 chosen from the region between the tag and the 3Ј-end (open box). The tag sequence is primer) were designed to amplify an 882-bp fragment of MAGE-E1c and a represented by a bold character. TaqMan probe for MAGE-E1 (underlined) was designed 473-bp fragment of MAGE-E1a and -E1b. The reverse-transcribed product was to detect a sequence within the F1 and R1 primers. The R2 primer for 5Ј-RACE was designed according to the nucleotide sequence in the region confirmed by the second amplified with F3 primer and R6 primer with the LA-PCR Kit following the experiment. B, structure of MAGE-E1 cDNA. Three variants, MAGE-E1a, -E1b, and -E1c, cycling parameters: 30 s at 94°C; 30 s at 58°C; 1 min at 72°C for 30 cycles; were obtained by RT-PCR cloning covering the entire coding region. Their length was and a final extension at 72°C for 3 min. Fragments were subcloned and 2484 bp, 2496 bp, and 2997 bp, respectively. MAGE-E1c and -E1b had an additional 12 Ј sequenced to confirm their identity. A positive-control experiment was per- bp in the 3 -terminal region of -E1a, and -E1c also possessed another 411 bp in the ␤ Ј middle. They used the same ATG (arrow), but different stop codons (arrowhead). The formed with a set of primers for human -actin, 5 -CAAGAGATGGCCACG- primers used in 5Ј-RACE and RT-PCR are demonstrated below the schematic drawing. GCTGCT-3Ј and 5Ј-TCCTTCTGCATCCTGTCGGCA-3Ј. 4810

Table 1 Known and unknown genes up regulated in primary glioblastoma (GB-1) showing Ͼ5-fold increase in GB-1 were selected from the top-147 relative to cultured human astrocytes (HA-1) most abundantly expressed genes in GB-1. Ninety-seven genes were Tag sequence GB-1 Gene Accession no. obtained, which included 76 known and 21 unknown genes. Table 1 A. Unknown genes shows a portion of these genes, which also showed a 5-fold increase GCAACAGCAA 0.42% sec61 ␥ AF054184 in GB-1 over another human astrocytes profile reported in the CGAP. CAGAGATGAA 0.17% HSP-70 NM_005345 By this selection, 34 known and 18 unknown genes were determined. CTGACTTGTG 0.14% MHC class I HLA-B44 M15470 All of these genes were undetectable in HA-1. In the list of known AGACCAAAGT 0.13% Heat shock 40kD protein 1 D49547 GTGCGCTGAG 0.13% HLA class I locus C heavy chain X58536 genes (Table 1A), previously reported glioma-related genes such as GACTCTTCAG 0.12% ␣-1-antichymotrypsin J05176 VEGF and brain-specific angiogenesis inhibitor 2 were also included. ATGAAACCCT 0.11% Homo sapiens leukemia AF006199 susceptibility mRNA GFAP and vimentin, which have been used as a glioma-specific AAGACCCAGA 0.11% Pleiotrophin M57399 marker by earlier investigators, are absent from the list because of the TGAAAGTGTG 0.09% Heat shock 105kD D86956 high-level expression in normal astrocytes (see Table 2). Some other ACTCCCATAT 0.08% Human transcriptional activator U49857 mRNA glioma-related genes such as cyclin-dependent kinase 4 and epidermal TGCCTGCACC 0.08% Cystatin C X05607 growth factor receptor were also found in the profile of GB-1; GGAGTGGACA 0.07% Ribosomal protein L18 L11566 however, the levels were too low (0.001%) to be selected. Genes TGCACTTCAA 0.07% Hevin X82157 ACCGCCGTGG 0.06% Cytochrome b-245, ␣ M21186 which had not been focused on in previous glioma studies are also polypeptide included in Table 1A. For example, expression of heparin-binding CAATGTGTTA 0.06% NADH dehydrogenase 1 ␣ X81900 subcomplex, 1 neurite-promoting factor 1 in glioma was not reported previously, and CGACCCCACG 0.06% Apolipoprotein E M12529 HSP-70 has been reported for other cancers but not for glioma. CTGACCTGTG 0.06% HLA-B M28205 The 18 tags which could not be identified by the database search are GCAAGAAAGT 0.06% ␤-globin V00497 GAGAGTGTCT 0.06% Fibroblast collagenase inhibitor M12670 listed in Table 1B. Many of them, 15 out 18, were found in EST GTGAAACCTG 0.06% Dihydrolipoamide branched chain J03208 sequences; thus the rest, 3 tags, were totally unknown. Among the transacylase ESTs found by the search, ESTs containing the tag TTTTGGGTAT GTATGGGCCC 0.06% Cartilage glycoprotein-39 M80927 TAGTTTACAC 0.06% KIAA0282 gene D87458 showed high homology with MAGE-D1. We decided to focus on the TCACCGGTCA 0.06% Gelsolin X04412 original gene of this tag and named it MAGE-E1. Our SAGE profiles GTTGTCTTTG 0.06% Complement component 3 K02765 TGACTGTGCT 0.06% Neurogranin U89165 (Table 2, above the dotted line) showed that MAGE-E1 was expressed TTTCCAATCT 0.06% VEGF AF022375 abundantly in glioblastoma (0.052%) but not detected in human TTTGAAATGA 0.06% Spermidine M55580 astrocytes. Compared with GFAP and vimentin, MAGE-E1 expression GAAACTGAAC 0.05% Methionine aminopeptidase U29607 GAAATGTTTC 0.05% Nuclear pore complex interacting NM_006985 was less abundant but highly specific to glioblastoma. protein Our findings are supported by the other SAGE profiles obtained CCTTGAGTAC 0.05% Aldolase C AF054987 from the CGAP (Table 2, below the dotted line). MAGE-E1 was more GTGAAACCTT 0.05% Carnitine palmitoyltransferase II M58581 GCTTAACCTG 0.05% Glutamate dehydrogenase 1 M20867 abundantly expressed in glioblastoma (GB, 0.041%) than in normal GCTTGGCCTG 0.05% Brain-specific angiogenesis AB005298 adult brain (NB, 0.006%) or human astrocytes (HA, 0.010%). In brain inhibitor 2 ACGAGTCATT 0.05% KIAA0927 protein AB023144 tumor cells, the expression level was high in medulloblastoma (MB, 0.026%) but low in fibrillary astrocytoma (FA, 0.006%). These results B. Known genes indicate that MAGE-E1 is up-regulated in undifferentiated brain tu- TAGAAAGGCA 0.11% ESTs AA594490 mors. CTCGTTAAGA 0.09% ESTs AA122179 Because the CGAP profile shows a 4-fold increase of MAGE-E1 CTAAATCTTT 0.09% ESTs AI420977 ACGAGGGGTG 0.08% ESTs AA873072 expression in glioblastoma in comparison with human astrocyte cul- CCTGTGGTCC 0.08% ESTs T63193 tures, real-time quantitative RT-PCR was performed to quantify AAGAGCGCCG 0.07% ESTs W28593 AGGGTCGCAG 0.07% No match MAGE-E1 expression in the other four glioblastomas (GB-2, -3, -4, TAACAAAGGA 0.06% ESTs AA083277 and -6) relative to cultured human astrocyte (HA-1). MAGE-E1 TTTTGGGTATa 0.05% ESTs AI082447 expression was up-regulated in all four tumors, showing 2.6–15-fold CTCCTTAAGA 0.05% ESTs AI760380 GCAGGGTGGG 0.05% ESTs AI083856 enrichment (Table 3). CT negative control without reverse tran- CCTGTAGTCT 0.05% No match scriptase was 40 (data not shown). GGCTCTGGGA 0.05% ESTs AW245985 Cloning of MAGE-E1 cDNA. To use sequence information of the TCACTGCACT 0.05% No match TGGAGAAGAG 0.05% ESTs AA521075 ESTs found by the search, first we confirmed that the MAGE-E1 tag GCAAGCCAAC 0.05% ESTs AI669642 TACTGTGGAT 0.05% ESTs AA844056 TCGCAAGAAT 0.05% ESTs AI922855 Table 2 Expression of MAGE-E1, GFAP, and Vimentin observed in SAGE profiles a We focused on ESTs containing a tag, TTTTGGGTAT, showing high homology with Our SAGE profiles appear above the dotted line and the other profiles, obtained from MAGE-D1. the CGAP, appear below the dotted line. TTTTGGGTAT ACTTTGTCCC TCCAAATCGA Total tags (MAGE-E1) (GFAP) (Vimentin) GB-1 9556 0.052% 0.303% 0.115% RESULTS HA-1 12353 Not detected 0.113% 0.105% ...... Identification of the Genes Specifically Up-Regulated in Gli- GBa 70669 0.041% 0.200% 0.127% oma. The SAGE experiment was performed with a primary glioblas- NBb 95254 0.006% 0.126% 0.005% HAc 52533 0.010% 0.004% 0.187% toma (GB-1) tissue fragment obtained surgically from a patient. For FAd 17622 0.006% 0.153% 0.023% comparison, cultured human astrocytes (HA-1) obtained from an MBe 19181 0.026% 0.005% 0.261% embryo brain of 14 weeks’ gestation was analyzed in parallel because a GB, glioblastoma multiforme. glioblastoma is defined as an astrocytic tumor according to the WHO b NB, normal brain. c HA, human astrocytes. classification. Numbers of the tags analyzed for GB-1 and HA-1 were d FA, fibrillary astrocytoma. 9,556 and 12,353, respectively. By comparing obtained profiles, genes e MB, medulloblastoma. 4811

Table 3 MAGE-E1 expression in glioblastoma relative to cultured human astrocytes detected by real-time quantitative RT-PCR experiment MAGE-E1 MAGE-E1 MAGE-E1 Relative to ␤-actin ␤-actin MAGE-E1/ a b ␤ c CT Average CT HA Average CT Relative to HA -actin HA-1 25.20 25.06 25.13 1.0 19.54 1.0 1.0 GB-2d 23.11 23.37 23.24 3.1 21.26 0.39 7.9 GB-3 22.35 22.83 22.59 4.7 20.45 0.61 7.7 GB-4 23.32 23.20 23.26 3.1 19.23 1.2 2.6 GB-6 23.36 22.81 23.09 3.3 22.30 0.22 15 a CT, threshold cycle in real-time quantitative RT-PCR experiment. b HA, human astrocytes. c MAGE-E1 expression level normalized against ␤-actin. d GB, primary glioblastoma.

MAGE-E1 Expression in Normal and Various Cancer Tissues. Northern blot analysis showed that MAGE-E1 mRNA was detected as a single band of about 3 kb in U87 MG cell line and a primary glioblastoma (GB-5; Fig. 3A). The size was consistent with that of cDNAs cloned and sequenced (Fig. 1B). Signals of similar size were observed in human brain and faintly in ovary (Fig. 3B) but not in other tissue, including testis and placenta, in which all other MAGE genes are expressed. A set of primers for RT-PCR was designed to cover the additional 411 bases of MAGE-E1c. Using these primers, an 882-bp fragment of MAGE-E1c could be distinguished from a 473-bp fragment of MAGE-E1a and -E1b. RT-PCR showed that MAGE-E1a and/or -E1b (Fig. 4, lower bands) were expressed abundantly in glioma, whereas -E1c (Fig. 4, upper bands) was expressed in all tumor samples examined. The expression pattern in cultured human astrocytes Fig. 2. A, structure of the deduced polypeptides coded by MAGE-E1 mRNAs. The (HA-1) was similar to that of glioblastomas (data not shown). Non- deduced polypeptides encoded by MAGE-E1 mRNAs are schematically demonstrated. specific signals were not observed in the control experiment that was All of them possess the NH2-terminal 348 amino acids. MAGE-E1b is identical to MAGE-E1a except for 2 amino acids missing in the COOH terminus. MAGE-E1c lacks performed without reverse transcriptase (data not shown). a large portion of COOH-terminal half and has a distinct 66-amino acid tail. Ⅲ, MAGE- conserved domain. B, homology of deduced MAGE-E1a polypeptides with other MAGEs. Homology search with the database showed that deduced MAGE-E1a polypeptides were DISCUSSION highly homologous with other MAGE family members at a region spanning 421–600 amino acids: 78.3% identical to MAGE-D1; 41.1% to MAGE-A1; 43.5% to MAGE-B1; In the present study, we focused on the genes up-regulated in and 41.4% to MAGE-C1. glioblastoma (Table 1A). The list of these genes illustrated the character of glioblastoma as a cancer. As previously reported, VEGF elicits angiogenesis in various tumors (13). Up-regulation of this gene had actually originated from the same gene that gave us the ESTs. An is consistent with the character of glioblastoma as a hypervascular intermediate product of the SAGE experiment was amplified with a tumor. Finding of the genes associated with protein synthesis in the tag-specific primer and the oligo(dT)-primer by PCR and sequenced list, such as sec61␥ and ribosomal protein L18, may reflect the active (see “Materials and Methods”). The resulting sequence matched the growth of glioma cells. Several glioma-related genes were found in Ј ESTs. Then, 5 -RACE was performed with specific primers designed the SAGE profile as we expected, however, our results indicate that according to the EST. Finally, RT-PCR cloning covering the entire coding region was performed. Three variants, possibly originated by an alternative splice, were obtained (MAGE-E1a,-E1b, and -E1c; Fig. 1B). The length of MAGE-E1a,-E1b, and -E1c cDNAs was 2485 bp, 2497 bp and 2997 bp, respectively. MAGE-E1c and -E1b had an additional 12 bp in the 3Ј-terminal region of -E1a, and -E1c also possessed another 411 bp in the middle. Structure of the Predicted Polypeptides and Homology with MAGE Proteins. The three species of transcripts used the same ATG but different stop codons, resulting in different open reading frames

(Fig. 2A). All three products share the NH2-terminal 348 amino acids. MAGE-E1b is identical to MAGE-E1a except for 2 amino acids missing in the COOH terminus. MAGE-E1c lacks a large portion of the COOH-terminal half and has a distinct 66-amino acid tail. The homology search with on database showed that MAGE-E1a and -E1b were highly homologous with other MAGE family members at a region spanning amino acid 421–600: 78.3% identical and 96.1% similar to MAGE-D1; 41.1% and 76.1% to MAGE-A1; 43.5% and Fig. 3. MAGE-E1 expression in glioma and normal tissues. A, Northern blot analysis 76.7% to MAGE-B1; and 41.4% and 76.1% to MAGE-C1 (Fig. 2B). showed that MAGE-E1 mRNA was detected as a single band of about 3 kb in the U87 MG cell line and a primary glioblastoma (GB-6). The size was consistent with that of MAGE-E1c lacked the conserved domain, suggesting that it has dif- MAGE-E1 cDNAs. B, a single band ϳ3 kb long was observed in brain and ovary but not ferent functions from the members of MAGE gene family. in other tissues, including testis and placenta, in which all other MAGEs are expressed. 4812

were expressed in mouse blastocysts and embryonic stem cells but not in adult tissues except for testis (19). These findings suggest that products of MAGE genes have some functions in the proliferating phase of the cells. MAGE-E1 might also play an important role in the cell proliferation of malignant glioma. In the present study, we cloned MAGE-E1, the first member of MAGE family expressed predominantly in brain and ovary. Recent studies have also reported novel MAGE genes whose tissue distribution is different from that of MAGE-A,-B, and -C; MAGE-D1 expressed ubiquitously (8) and MAGE-L2 expressed in brain and placenta (20). These observations suggest that MAGE-related genes are Fig. 4. MAGE-E1 expression in various cancers. MAGE-E1a and/or -E1b (lower distributed in various tissues. Radiation hybrid mapping showed that bands) are expressed abundantly in glioma relative to other cancers, whereas E1c (upper the MAGE-E1 gene is located on chromosome X like other MAGEs.4 bands) is expressed in all tumor samples examined. U87, U87 MG cell line; GB, glioblastoma multiforme; LC, lung cancer; SC, stomach cancer; CC, colon cancer; HCC, This result indicates that chromosome X may contain other MAGE- hepatocellular carcinoma. related genes expressed in various tissues. Such genes could be isolated by cDNA cloning from various cancer cells. Indeed, MAGE-D1 was isolated from genes activated in multiple myeloma (8) they are not the best and exclusive markers for gliomas because of the and MAGE-E1, described in the present study, was identified from high-level expression also in normal astrocytes (GFAP and vimentin) genes up-regulated in glioblastoma. Our RT-PCR results show that or relatively low expression in glioblastoma (CDK-4 and EGFR). The MAGE-E1a and/or -E1b are expressed specifically in glioblastomas, list also included genes which has not been focused on in the previous whereas MAGE-E1c is distributed in various tumors. This pattern of studies. A previous study has reported that a complex of HSP-70 and -E1c expression is similar to members of classical MAGE family such antigenic peptides could induce antitumor immunity (14). Our finding as -A,-B, and -C, which are widely found in various tumors. Because of an abundant expression of HSP-70 in glioblastoma suggests that it MAGE-E1c polypeptide lacks the MAGE-conserved domain, it could be a candidate target molecule for the immunotherapy of should play a totally unrelated role in tumor cells, unlike other glioma. As discussed above, our results provide us with new clues to members of MAGE family. MAGE-E1a and -E1b were observed in understand the nature of pathogenesis and progression of gliomas in human astrocytes. This observation suggests that MAGE-E1a and/or vivo. However, we should exercise caution in interpreting our exper- E1b possibly could be originated by a brain-specific alternative splice. imental results. Although freshly resected primary glioblastoma tissue MAGE-E1 products could have dual functions, one of which is unique is the most suitable material to determine the gene expression pattern in glioblastoma, whereas the other is conserved in various other of gliomas, normal cells such as vascular endothelial cells and reactive tumors. Additional studies on the structure and function of MAGE-E1 astrocytes are inevitably included among the tumor cells. Therefore, products should elucidate their biological roles in brain, glioma cells, genes of interest should be identified and analyzed carefully, and their and other cancer cells. expression should be assigned to each cell type. Additional study, such as histochemical staining, is necessary to confirm the expression ACKNOWLEDGMENTS in glioma cells. Our SAGE profiles and that of the CGAP showed that MAGE-E1 We thank Dr. Kenneth W. Kinzler for providing detailed protocol and expression in glioblastoma (GB-1) is Ͼ4-fold higher than that in SAGE software and Dr. Shunichiro Okumura for providing RNA of various cultured human astrocytes (Table. 2). Real-time quantitative RT-PCR cancers. showed that MAGE-E1 was 2.6–15-fold enriched in glioblastoma relative to human astrocytes. This difference of expression levels of REFERENCES MAGE-E1 in glioblastoma could be used for the development of 1. Velculescu, V. E., Zhang, L., Zhou, W., Vogelstein, J., Basrai, M. A., Bassett, D. E., additional modes of diagnosis and treatment of human patients suf- Jr., Hieter, P., Vogelstein, B., and Kinzler, K. W. Characterization of the yeast transcriptome. Cell, 88: 243–251, 1997. fering from malignant gliomas. 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Manabu Sasaki, Kensuke Nakahira, Yozo Kawano, et al.

Cancer Res 2001;61:4809-4814.

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