[CANCER RESEARCH 60, 3848–3855, July 15, 2000]

Identification on a Human Sarcoma of Two New Genes withTumor-specific Expression1

Valerie Martelange, Charles De Smet, Etienne De Plaen, Christophe Lurquin, and Thierry Boon2

Ludwig Institute for Cancer Research, Brussels Branch, and Cellular Genetics Unit, Universite Catholique de Louvain, B-1200 Brussels, Belgium

ABSTRACT

GenesMAGE, BAGE, GAGE,and LAGE-1/NY-ESO-1code for antigensthat are recognized on melanoma cells by autologous CTLs. Because thepattern of expression of these genes results in the presence of antigens onmany tumors of various histological types and not on normal tissues, theseantigens qualify for cancer immunotherapy. To identify new genes withtumor-specific expression, we applied a cDNA subtraction approach,i.e.,representational difference analysis, to a human sarcoma cell line. Weobtained two cDNA clones that appeared to be tumor specific. The cor-responding genes were namedSAGE and HAGE because they have thesame pattern of expression as genes of theMAGE family. SAGE encodesa putative protein of 904 amino acids and shows no homology to anyrecorded gene. Like theMAGE-A genes, it is located in the q28 region ofchromosome X. Expression of geneSAGEwas observed mainly in bladdercarcinoma, lung carcinoma, and head and neck carcinoma but not innormal tissues, with the exception of testis. GeneHAGE, which is locatedon chromosome 6, encodes a putative protein of 648 amino acids. Thisprotein is a new member of the DEAD-box family of ATP-dependent RNAhelicases. GeneHAGE is expressed in many tumors of various histologicaltypes at a level that is 100-fold higher than the level observed in normaltissues except testis. Because of this tumor-specific expression, genesSAGE and HAGE ought to encode antigens that could be useful forantitumoral therapeutic vaccination.

INTRODUCTION

Several studies have shown that by cultivating irradiated tumorcells with autologous lymphocytes, it is possible to obtain respondercell populations that display a cytotoxic response against tumor cells(1). The CTL clones derived from such responder populations havebeen found to recognize several antigens (2). The genes coding forthese antigens have been identified by transfection and detection ofthe transfectants by the CTLs. A first important class of tumorantigens recognized by CTLs is encoded by genes that are activated intumors. These genes belong to theMAGE,BAGE,GAGE,andLAGE-1/NY-ESO-1families. They are all expressed in tumors of differenthistological types but not in normal tissues, except for spermatogeniccells, and for some of them, placenta (3–8). A second categorycontains differentiation antigens encoded by genes expressed in nor-mal melanocytes and in melanoma cells, such as tyrosinase, Melan-A/Mart-1, gp100, and gp75 (9–12). A third class constitutes antigensproduced by point mutations in genes that are expressed ubiquitously,e.g., MUM-1, cyclin-dependent kinase 4,b-catenin, and HLA-A2(13–16). Finally, there are antigens derived from genes overexpressed

in tumors relative to normal cells, such as HER-2/neu and PRAME(17, 18).

The antigens encoded by genes that are expressed only in tumors andin germ-line cells appear to be strictly tumor specific, because the sper-matogenic cells that express these genes do not express HLA moleculesand are therefore incapable of presenting antigens to T cells (19, 20).Because these genes are expressed in a large proportion of tumors ofvarious histological types, they appear to be excellent potential sources ofantigens for cancer immunotherapy. Clinical trials involvingMAGEantigens are ongoing, and tumor regressions have been observed (21–24).

For the purpose of finding new genes that present the same patternof expression as theMAGE, BAGE,GAGE,andLAGE-1/NY-ESO-1genes, we have recently applied subtraction of cDNA from a tumorwith cDNA from a panel of normal tissues. This approach has led tothe identification of a new member of theMAGE family, geneMAGE-C1 (25), as well asLAGE-1, a cancer germ-line gene men-tioned above (7). We report here that a similar approach, this timeapplied to a sarcoma cell line, led to the identification of two newgenes that have a pattern of expression similar to theMAGE-typegenes. These genes are not homologous to theMAGE,BAGE,GAGE,andLAGE-1/NY-ESO-1families.

MATERIALS AND METHODS

Cell Lines. Rhabdomyosarcoma cell line LB23-SAR was derived frompatient LB23 and cultured in Iscove’s medium (Life Technologies, Inc., GrandIsland, NY) containing 10% FCS (Life Technologies) and supplemented withL-asparagine (36 mg/l),L-arginine (116 mg/l), andL-glutamine (216 mg/l).

Representational Difference Analysis.Total RNA was isolated from nor-mal tissues (uterus, breast, colon, and heart) and from cell line LB23-SAR bythe guanidine isothiocyanate/cesium chloride procedure (26). Poly(A)1RNAwas prepared by binding to oligo-dT cellulose (mRNA purification kit; Phar-macia Fine Chemicals, Piscataway, NJ), starting from 500mg of rhabdomy-osarcoma LB23-SAR total RNA and from 455mg of a mixture of humanuterus, breast, heart, and colon total RNA. About 4mg of each poly(A)1RNAwere used to synthesize double-stranded cDNA (cDNA Synthesis Module;Amersham Life Science, Buckinghamshire, United Kingdom). Two micro-grams of each cDNA were then digested withDpnII (New England Biolabs,Beverly, MA). The digested cDNAs were ligated to R-Bgl adapters andPCR-amplified to generate the rhabdomyosarcoma (tester) and uterus-breast-heart-colon (driver) representations, as described by Hubank and Schatz (27).We performed three rounds of subtractive hybridization and selective ampli-fication with the J-Bgl and N-Bgl adapters, as described (27).

Cloning of Difference Products. The second and third difference productswere digested withDpnII, separated from the adapters on a 1.2% agarose gel, andpurified using the QIAEX II Gel Extraction kit (Qiagen, Westburg, Leusden, theNetherlands). They were ligated to dephosphorylatedBamHI ends of pTZ18R(Pharmacia). DH5aF9IQ bacteria (Life Technologies, Gaithersburg, MD) weretransformed by electroporation with one-fifth of the ligation product.

Sequencing and Sequence Comparison.Plasmid DNA was prepared bythe boiling minipreparation procedure (28). Sequencing was performed with theT7 Sequencing kit (Pharmacia LKB) and [a-35S]dATP (.1000 Ci/mmol; Amer-sham), and reaction products were separated on a 2010 Macrophor manualsequencer (Pharmacia LKB). When long read lengths were required, we per-formed the sequencing reactions with the BigDye Terminator Cycle Sequencingkit (PE Applied Biosystems, Warrington, United Kingdom). The reaction productswere separated on the ABI PRISM 310 Genetic Analyser (Perkin-Elmer).

Sequence homology searches were performed in the databases provided bythe National Center for Biotechnology Information (Bethesda, MD) using theBLAST program (29). Alignments were performed with the GeneWorks

Received 12/17/99; accepted 5/16/00.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from Fonds J. Maisin (Belgium), Fe´derationBelge contre le Cancer (Belgium), Caisse Ge´nerale d’Epargne et de Retraite (CGER)-Assurances and VIVA (Belgium), the Belgian program on Interuniversity Poles ofAttraction initiated by the Belgian State, Prime Minister’s Office, Office for Science,Technology and Culture. V. M. is supported by Fonds pour la Recherche Scientifique dansl’Industrie et l’Agriculture (Belgium).

2 To whom requests for reprints should be addressed, at Ludwig Institute for CancerResearch, Universite Catholique de Louvain, 74 avenue Hippocrate, UCL 7459, B-1200Brussels, Belgium. Phone: 32-2-764-75-80; Fax: 32-2-762-94-05; E-mail: boon@licr.ucl.ac.be.

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computer program (Intelligenetics, Mountain View, CA). For DNA, we usedparameters 30-2-10-4000.

RT-PCR Analyses. The expression of cDNA clones isolated from thedifference products was analyzed by RT-PCR3. RNA were extracted fromtumor cells and normal tissues and reverse transcribed, as described previously(30). RNA samples from 5-aza-29-deoxycytidine-treated cells were obtained asdescribed (31). Twenty-four pairs of PCR primers were derived from thesequences of the fragments of cDNA of the enriched library. The sequence ofthese primers are available from the authors. PCR amplification of cDNA was

performed for 30 or 35 cycles, whereas amplification ofb-actin was performedfor 21 cycles with primer 59-GGCATCGTGATGGACTCCG-39(exon 3,sense) and primer 59-GCTGGAAGGTGGACAGCGA-39(exon 6, antisense).PCR products were visualized on 1.7% agarose gels stained with ethidiumbromide.

For the analysis ofSAGEexpression, the cDNA produced from 50 ng oftotal RNA was PCR amplified in a TRIO-Thermoblock (Biometra, Gottingen,Germany) for 30 cycles of 1 min at 94°C, 2 min at 57°C, and 2 min at 72°Cwith primers sdph3.10S and sdph3.10A.

To analyzeHAGE expression, PCR amplification was performed for 30cycles of 1 min at 94°C, 2 min at 57°C, and 2 min at 72°C with primerssdp3.8.8 and sdp3.8.9.

PCR Analysis of Radiation Hybrids. The GeneBridge 4 Radiation HybridPanel (Research Genetics, Inc., Huntsville, AL) was used to map theSAGEandHAGE genes (32). Twenty-five ng of genomic DNA from each of the 93radiation hybrid clones were PCR amplified with primers sdph3.10S (59-TGTACCTCTTCAAGCAAAAT-39) and sdph3.10A (59-GTGACCCAC-CAGTTACAGTA-39) that are specific for geneSAGE. PCR amplification wasperformed for 30 cycles of 1 min at 94°C, 2 min at 57°C, and 2 min at 72°C.PCR results were submitted for analysis to the web site of the WhiteheadInstitute for Biomedical Research.4 Positioning of markers from the WhiteheadRH map on the chromosome cytogenetic map was performed via the GenomeDatabase web site.5 The same panel was used to map geneHAGEwith primerssdp3.8.8 (59-TATTCTTCAGATTGACGAAG-39) and sdp3.8.9 (59-CCTT-TCAATGTTATCCTGAG-39) using identical PCR conditions.

Construction and Screening of the cDNA Library. We used a cDNAlibrary derived from human testis sample LB451 to search for the completecDNA sequences of genesSAGEandHAGE. Poly(A)1RNA was extractedfrom testis cells using the FastTrack mRNA extraction kit (InVitrogen, SanDiego, CA). mRNA was converted to cDNA with the SuperScript ChoiceSystem (Life Technologies, Inc.) using an oligo-dT primer containing aNotIsite at its 59end. cDNAs were then ligated toBstXI adaptors and digested withNotI. After size fractionation, the cDNAs were cloned into theBstXI andNotIsites of plasmid pcDNAI/Amp. Recombinant plasmids were electroporatedinto Escherichia coliDH5aF9IQ and selected with ampicillin (50mg/ml). Thelibrary was divided into three fractions: A (54,000 independent clones); B(150,000 clones); and C (300,000 clones). Recombinant bacteria (250,000)from fractions B and C were screened by colony hybridization with a PCR-

3 The abbreviations used are: RT-PCR, reverse transcription-PCR;SAGE,sarcomaantigen gene;HAGE, helicose antigen gene.

4 Internet address: http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl.5 Internet address: http://www.ncbi.nlm.nih.gov/SCIENCE96.

Fig. 1. RT-PCR analysis of geneSAGEexpression. Total RNA from the indicatednormal tissues or tumor samples was submitted to 30 cycles of RT-PCR amplificationwith SAGE-specific primers. The 171-bp PCR products were visualized on a 1.7% agarosegel stained with ethidium bromide.

Fig. 2. Northern blot hybridized with the 1.95-kb insert ofSAGEcDNA. Each lane wasloaded with 4mg of poly(A)1 RNA from tumor cell lines.

Table 1 Expression of gene SAGE tested by RT-PCR in samples of normal tissuesand tumors

Total RNA was converted to cDNA; the cDNA corresponding to 50 ng of total RNAwas then amplified by PCR with primers sdph3.10S and sdph3.10A (Fig. 3) and 0.625 unitof TaKaRa Taq for 30 cycles (94°C for 1 min, 57°C for 2 min, 72°C for 2 min).

Normal tissues Tumoral samples

Ovary 2a Cutaneous melanoma 2/47Kidney 2 Primary 1/25Adrenal glands 2 Metastatic 1/22Uterus 2 Uveal melanoma 0/5Breast 2 Neuroblastoma 0/2Sperm 2 Bladder carcinoma 15/128 (12%)Skin 2 Breast carcinoma 1/19Brain 2 Lung carcinoma NSCLCb 29/130 (22%)Testis 111 Epidermoid carcinoma 27/115 (23%)Heart 2 Bronchiolo-alveolar carcinoma 0/2Prostate 2 Adenocarcinoma 2/13Stomach 2 Sarcoma 1/20Lung 2 Brain tumors 0/9Colon 2 Prostate adenocarcinoma 0/10Bladder 2 Head and neck carcinoma 17/98 (17%)Liver 2 Colorectal carcinoma 0/19Bone marrow 2 Leukemia 1/25PBL (peripheral blood

lymphocytes)2 Renal tumors 1/20

Retina 2 Uterine tumors 0/5Skeletal muscle 2 Esophagial carcinoma 3/15

Myeloma 1/5Mesothelioma 0/4Thyroid tumors 0/5

a Normal tissues represent,1/1000 of the level of expression of LB23-SAR.b NSCLC, non-small-cell lung carcinoma.

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amplified fragment of genesSAGEor HAGE, using the same primers as thoseused for the analysis of radiation hybrids, and labeled with [a-32P]dCTP (3000Ci/mmol). Plasmid DNA from one positive clone was amplified and purifiedfor further analysis.

Northern Blot Analysis. Four mg of poly(A)1 RNA were separated byformaldehyde agarose gel electrophoresis, transferred to a nylon membrane bycapillary transfer, and fixed to the membrane by heating at 80°C for 2 h. Hybrid-ization with the 1.95-kb insert ofSAGEcDNA or the 2.3-kb insert ofHAGE

Fig. 3. Nucleotide and amino acid sequences of geneSAGE. Primers areunderlined. The repetitive motif of 47 amino acids is inbold.

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cDNA probes was performed overnight at 60°C in 10% dextran sulfate, 1M NaCl,1% SDS, and 100mg/ml denatured herring sperm DNA. Final washing of themembrane was done in 0.23 SSC, 0.1% SDS for 10 min at 60°C. Autoradiog-raphy was performed overnight using BioMax MS film (Kodak). The samemembrane was stripped and hybridized with a 0.6-kb probe forb-actin obtainedin similar conditions. Autoradiography was performed for 2 h.

Southern Blot Analysis. Ten mg of genomic DNA were digested withrestriction enzymesBamHI, EcoRI, HindIII, and PstI. They were separated byagarose gel electrophoresis, transferred to nylon membranes by the capillarytransfer method, and fixed by heating at 80°C for 2 h. Hybridization with thea-32P-radiolabeled PCR probes was performed in 53 SSC, 53Denhardt’s solu-tion, 0.1% SDS, and 100mg/ml denatured herring sperm DNA for 16 h at 65°C.Final washing of the membranes was done in 23 SSC, 0.1% SDS for 20 min at65°C. Autoradiography was performed overnight using BioMax MS film (Kodak).

Rapid Amplification of the 5* cDNA End of GeneSAGE. The amplifi-cation of the 59cDNA end of SAGE was performed with the 59rapidamplification of cDNA ends system, version 2.0 (Life Technologies, Inc.). We

used antisense primers Gsp1 (59-CTGGTACTCATGGGTGGAAT-39), Gsp2(59-GTTAATAAGCTCTGGTGTAA-39), and GspN (59-CATATCAGCTGT-TGCCAAAT-39). The amplified products were cloned using the Original TACloning kit (InVitrogen). The resulting clones were screened with the labeledsdph3.10.1 oligonucleotide (59-ACTCACAATGTCTGTGAAGA-39). We se-quenced 24 positive clones with primer GspN.

RESULTS

Genes Overexpressed in a Rhabdomyosarcoma.To obtain acDNA library enriched for tumor-specific sequences, we used the repre-sentational difference analysis method (27). Rhabdomyosarcoma LB23-SAR was used as the source of tester cDNA to be subtracted with driver

Fig. 4. Southern blot hybridized with the 1.95-kb insert ofSAGEcDNA. Each lane wasloaded with 10mg of genomic DNA from LB23-SAR cell line digested with the indicatedrestriction enzymes.

Fig. 5. Localization of geneSAGEand other tumor-specific genes on the human Xchromosome. The diagram is a schematic representation of the X chromosome, with itsG-banding pattern.Left, name of the cytogenetic bands.

Fig. 6. RT-PCR analysis of geneHAGE expression. Total RNA from the indicatednormal tissues or tumor samples were submitted to 30 cycles of RT-PCR amplificationwith HAGE-specific primers. The 431-bp PCR products were visualized on a 1.7%agarose gel stained with ethidium bromide.

Table 2 Expression of gene HAGE tested by RT-PCR in normal tissues andtumoral samples

Total RNA was converted to cDNA; the cDNA corresponding to 50 ng of total RNAwas then amplified by PCR with primers sdp3.8.8 and sdp3.8.9 (Fig. 8) and 0.625 unit ofTaKaRa Taq for 30 cycles (94°C for 1 min, 57°C for 2 min, 72°C for 2 min).

Normal tissues Tumoral samples

Ovary 2a Cutaneous melanoma 5/29 (17%)Kidney 2 Primary 3/14Adrenal glands 2 Metastatic 2/15Uterus 2 Uveal melanoma 2/3Breast 2 Neuroblastoma 0/2Sperm 2 Bladder carcinoma 7/29 (24%)Skin 2 Breast carcinoma 1/19Brain 2 Lung carcinoma NSCLCb 11/34 (32%)Testis 111 Epidermoid carcinoma 6/19Heart 2 Bronchiolo-alveolar

carcinoma0/2

Prostate 2 Adenocarcinoma 5/13Stomach 2 Sarcoma 3/15Lung 2 Brain tumors 3/8Colon 2 Prostate adenocarcinoma 2/9Bladder 2 Head and neck carcinoma 4/20 (20%)Liver 2 Colorectal carcinoma 6/19Bone marrow 2 Leukemia 2/22 (9 %)PBL (peripheral blood

lymphocytes)2 Renal tumors 1/16

Retina 2 Uterine tumors 0/5Skeletal muscle 2 Esophagial carcinoma 4/15

Myeloma 1/5Mesothelioma 2/4Thyroid tumors 1/5

a Normal tissues represent,1/500 of the level of expression of LB23-SAR.b NSCLC, non-small-cell lung carcinoma.

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cDNA that was a mixture of human uterus, breast, heart, and coloncDNA. Tester and driver were subjected to PCR amplification. Theamplified tester was then ligated to a pair of adapters and hybridized toa large excess of driver. The hybridization product was amplified by PCRusing the tester-specific adapters as primers. Under these conditions, onlytester-tester homoduplexes, corresponding to LB23-SAR specific se-quences, were amplified exponentially. This first difference product wassubmitted to two additional rounds of subtraction and amplification,generating the second and third difference products, which were cloned.

Forty-two different sequences of the second and third differenceproducts were analyzed. Twenty-two sequences showed ubiquitousexpression, 12 of these coding for mitochondrial or ribosomal com-ponents or for known abundant proteins involved in cell proliferation.Eighteen clones were transcribed in some normal tissues but had alevel of expression in LB23-SAR cells that was significantly higher.Among those, six were not recorded in databases. Finally, two se-quences that we namedSAGE and HAGE appeared to be highlyexpressed in testis and in LB23-SAR but were completely or nearlycompletely silent in normal tissues. Because these genes had a patternof expression similar to that of theMAGE, BAGE, GAGE, andLAGE-1/NY-ESO-1genes, we felt that they had the potential to codefor tumor-specific antigens, and we characterized them fully.

GeneSAGE. We analyzed the expression pattern of geneSAGEintumor samples and normal tissues. The PCR product was 171 bp whenderived from cDNA templates. It was easily distinguished from the750-bp product derived from genomic DNA, eliminating the risk of falsepositives attributable to DNA contamination of the RNA.SAGEwasexpressed in many samples of bladder, lung, and head and neck carci-nomas (Table 1). In contrast, normal tissues were found to express,0.1% of the amount expressed by LB23-SAR. The only positive tissuewas testis, as has been observed for the otherMAGE-type genes (Fig. 1).The expression of severalMAGE genes can be induced with 5-aza-29-deoxycytidine in normal and tumoral cell types that do not normallyexpress these genes (25, 31). We examined whether geneSAGEcouldalso be activated in nonexpressing cells after treatment with this agent.We found that geneSAGEwas induced in fibroblasts and in tumoral celllines of various types (data not shown).

To obtain a full-length cDNA for geneSAGE, we screened a testislibrary. A 1950-bp cDNA clone hybridized with the 171 bp PCRproduct that we used as a probe. This insert was sequenced andcomparison with sequences available in databases revealed that it wasunrelated to any known sequence. This 1.95-kb insert was used as aprobe and was found to hybridize with a single messenger of about 3.5kb on a Northern blot prepared with poly(A)1 RNA from tumor celllines and normal tissues (Fig. 2), indicating that we had not obtaineda full-length cDNA clone. To obtain the full-length cDNA, we usedthe PCR amplification of the 59cDNA end. This led to a 3069 bpsequence. The largest open reading frame comprised 2712 bp andencoded a putative protein of 904 amino acids; this protein is com-posed of 15 repeated motifs of 47 amino acids (Fig. 3).

In a Southern blot analysis, the labeled 1.95-kb insert hybridizedwith several bands (Fig. 4), suggesting thatSAGEis a member of afamily of genes. Using the GeneBridge 4 Radiation Hybrid Panel, weestablished that geneSAGEwas located between markersWI-6213and WI-5285 on the chromosome X, in the q28 region, like theMAGE-AandLAGE-1/NY-ESO-1genes (Fig. 5).

GeneHAGE. The expression of geneHAGEwas tested in RT-PCRassays with a large panel of tumor samples and normal tissues. We usedPCR primers that enabled us to distinguish the amplification productderived from cDNA templates (431 bp) from that of genomic DNA (;2kb). With the exception of testis, which showed a high level of expres-sion, a very low level of expression was observed with normal tissues(Fig. 6). The level of expression of geneHAGE in normal tissues,including skeletal muscle, was;0.2% of the level of expression found inthe LB23-SAR sarcoma cell line. When we analyzed tumor samples, wefound that geneHAGEwas expressed in a large fraction of tumor samplesof various histological types, well above the level observed in normaltissues (Table 2). About 5% of the positive tumor samples tested showeda level of expression.10% of the level of LB23-SAR, and 7% showeda level of expression between 1 and 10%. Like geneSAGE, the expres-sion of geneHAGEcould also be induced in cells treated with 5-aza-29-deoxycytidine (data not shown).

To obtain a full-lengthHAGEcDNA, we used colony hybridizationwith a HAGE probe to screen the same testis cDNA library that weused for geneSAGE. The only cDNA clone hybridizing with the probecontained a 2.3-kb insert. This insert used as a probe hybridized witha single messenger of;2.2 kb on a Northern blot (Fig. 7), suggestingthat the isolated cDNA was full length. The largest open readingframe comprised 1944 bp and encoded a putative protein of 648amino acids (Fig. 8).

Comparison with sequences available in databases revealed a ho-mology with p68, an ATP-dependent RNA helicase that is a memberof the DEAD-box proteins (33). They share several conserved aminoacids, including the D-E-A-D motif, which provides their name (Fig.8). All of the motifs that are present in all of the members of theDEAD box family are conserved in theHAGE protein.

We used the 2.3-kb insert for Southern analysis of human genomicDNA, and it hybridized with several fragments of DNA obtained withfour different restriction enzymes (Fig. 9). It is probable that the probehybridizes with other members of the DEAD-box family because theirsequences are well conserved.

The chromosomal location of geneHAGEwas established by PCRanalysis of DNA samples of the radiation hybrid panel. GeneHAGEwas found to be located on the long arm of chromosome 6, betweenmarkersGATA11B08andD6S284in the region q12-q13.

DISCUSSION

We have identified two new genes,SAGEandHAGE, with patternsof expression similar to that of genesMAGE, BAGE, GAGE, and

Fig. 7. Northern blot hybridized with the 2.3-kb insert ofHAGEcDNA. Each lane wasloaded with 4mg of poly(A)1 RNA from tumor cell lines.

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LAGE-1/NY-ESO-1,i.e., a strong expression in many tumor samplesand in testis and either no expression or very little expression (at least100 times lower) in other normal tissues (3–8). This confirms theusefulness of representational difference analysis for the identificationof genes with tumor-specific expression.

As already observed for otherMAGE-type genes,SAGEandHAGEexpression was induced by 5-aza-29-deoxycytidine, suggesting that dem-ethylation plays a role in the activation of these genes in tumors. There isevidence that, in cells containing the relevant transcription factors, DNAmethylation can on its own be effective for gene silencing (34, 35). Onemechanism by which methylation can affect gene expression is thatbinding of transcription factors to their target sequences can be inhibitedby methylation of CpGs sites located in these sequences (36). Anothermechanism for transcriptional silencing induced by methylation isthrough the modification of the structure of the chromatin. Methylation of

DNA helps to stabilize the chromatin in an inactive configuration andtherefore inhibits gene transcription (37). This inactive state of the chro-matin is associated with underacetylated histones, suggesting that DNAmethylation and histone deacetylation are linked. In line with this hy-pothesis, it has been shown recently that histone deacetylases formcomplexes with methyl-binding proteins such as MeCP2, MBD2, andMBD3 (38–40).

We suggested previously that the activation ofMAGE genes intumor cells results from the genome-wide demethylation that occursin these cells (31). However, tumor cells with demethylated genomesdo not activate all of theMAGE-related genes (41). This is probablyattributable to random demethylation and chromatin modification,which vary from cell to cell and lead to different patterns of expres-sion ofMAGE-related genes, even in tumors of the same histologicaltype. We report here thatMAGEandSAGEgenes are both expressed

Fig. 8. Nucleotide and amino acid sequences of geneHAGE. Theboxescorrespond to motifs typical of the DEAD-box family of helicases: the D-X-X-X-X-A-X-X-X-X-G-K-Tsequence at position 281–293 is the typical A-motif of ATP-binding proteins; the D-E-A-D box at position 394–406 represents a special version of the B-motif of ATP-binding proteins;the S-A-T motif at position 427–429 is also conserved in all DEAD-box proteins, but no function can yet be attributed to this motif; and, finally, the H-R-I-G-R motif at position573–577 seems to be involved in the polynucleotide binding and/or unwinding activity.

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in head and neck carcinomas, bladder carcinomas, and epidermoidcarcinomas of the lung, but that in melanomasMAGE genes areexpressed whereasSAGEis rarely expressed.MAGE-1andMAGE-4were found to be regulated by ubiquitous transcription factors (42,43), but for other genes, such asSAGE, we cannot exclude that someof the factors regulating their expression are tissue specific and henceare present only in certain types of tumors.

GeneSAGEis unrelated to any known sequence and appears to be amember of a new family of several genes. It is not expressed at all innormal tissues, with the exception of testis. The expression ofMAGEandLAGEgenes in testis appears to be restricted to germ-line cells (19),6 andwe consider it likely that this will apply toSAGEalso. LikeMAGE-Agenes (44), geneSAGEmaps to the q28 region of chromosome X. It isnoteworthy that several other genes that are expressed in a wide array oftumors and in male germ-line cells map to chromosome X. In addition toMAGE-AandLAGEgenes, this is the case forP1A in the mouse and forMAGE-B,MAGE-C1,GAGE,andSSX2genes in humans (25, 45–47).The significance of this localization is not clear.

The second gene that we identified was namedHAGE because itencodes a protein that shows 55% similarity with the human p68protein, a DEAD-box protein whose ATP-dependent RNA helicaseactivity has been demonstrated (48, 49). The protein encoded by geneHAGE seems to be a new member of the family of DEAD-boxproteins (33), which contain the highly conserved Asp-Glu-Ala-Asp(D-E-A-D) motif. These proteins are involved in many aspects ofRNA metabolism, spermatogenesis, embryogenesis, and cell growth.The amino acids that are highly conserved in all of the DEAD-boxproteins are also present in protein HAGE, suggesting that HAGEmay also be an ATP-dependent RNA helicase. GeneHAGE is notlocated on chromosome X but on chromosome 6.

ForMAGE-1, it has been shown that tumor cell lines could be lysed bya relevant CTL clone if the level of expression of this gene exceeded thethreshold of 10% of that found in the MZ2-MEL reference tumor cellline, i.e.,more than three RNA molecules/cell (50). We have indications(data not shown) that the level of expression of geneHAGE in cell line

LB23-SAR is;10 times lower than the level of expression ofMAGE-1in MZ2-MEL. This level may be sufficient to produce antigenic peptides,but it will only be possible to test this once a CTL clone that recognizesa peptide encoded by geneHAGE has been obtained. This CTL clonecould then also be used to test the presence of antigens on normal tissues,where the level of expression of geneHAGE is at least 100 times lowerthan in LB23-SAR. The HAGE protein is not the first case of a DEAD-box protein that is overexpressed in tumors; this has already been de-scribed in retinoblastoma and neuroblastoma cell lines (51–53). More-over, it is worth noting that of 42 tumor antigens discovered until now,two are derived from mutated helicases. A mutated murine helicase,named p68, was found to lead to the expression of an antigen recognizedby a CTL clone on a UV-induced sarcoma (54). The human melanomaantigen LB33-A is produced by a point mutation in a new gene,MUM-3,which is expressed ubiquitously and shows homology with members ofthe RNA helicase gene family.7 These observations suggest that mutatedor overexpressed helicases may contribute to tumoral transformation orprogression.

Genes such asSAGEandHAGE,with expression that appears to berestricted to tumors and germ-line cells, are potentially coding for tumor-specific antigens recognized by T lymphocytes. But these antigens re-main to be identified. Approaches to establish which antigenic peptidesrecognized by T cells are encoded by such genes have recently madeconsiderable progress.In vitro stimulation of CD81T lymphocytes withdendritic cells infected with canarypoxes or adenoviruses carrying se-quences ofMAGE genes has led to the definition of a large number ofnew epitopes encoded by these genes (55, 56). Moreover, stimulation ofCD41T cells with dendritic cells pulsed with MAGE proteins has led tothe identification of several new antigenic peptides presented by class IImolecules (57). These results significantly enlarge our possibilities fortherapeutic vaccination with tumor-specific antigenic peptides, because itis now certain that almost every cancer patient whose tumor expresses aMAGE gene will have at least one HLA molecule presenting a MAGEantigenic peptide. The same approach could be applied to genesSAGEandHAGE to identify tumor-specific antigens derived from them. Con-secutive immunization with several antigens might ensure a more effec-tive rejection of tumor cells because this should reduce the emergence ofantigen-loss variants arising by mutations in the genes producing theantigenic peptides or by down-regulation of their expression.SAGEandHAGEantigens could also be used to immunize patients against tumorsthat do not expressMAGEgenes.

ACKNOWLEDGMENTS

We thank Marie-Claire Letellier and Claudine Blondiaux for technicalassistance, Pierre van der Bruggen for critical reading of the manuscript, andSaıda Khaoulali and Simon Mapp for editorial assistance.

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