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Immunology |
Ludwig Institute for Cancer Research, Brussels Branch, and Cellular Genetics Unit, Université Catholique de Louvain, B-1200 Brussels, Belgium
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The antigens encoded by genes that are expressed only in tumors and in germ-line cells appear to be strictly tumor specific, because the spermatogenic cells that express these genes do not express HLA molecules and are therefore incapable of presenting antigens to T cells (19, 20) . Because these genes are expressed in a large proportion of tumors of various histological types, they appear to be excellent potential sources of antigens for cancer immunotherapy. Clinical trials involving MAGE antigens are ongoing, and tumor regressions have been observed (21–24) .
For the purpose of finding new genes that present the same pattern of expression as the MAGE, BAGE, GAGE, and LAGE-1/NY-ESO-1 genes, we have recently applied subtraction of cDNA from a tumor with cDNA from a panel of normal tissues. This approach has led to the identification of a new member of the MAGE family, gene MAGE-C1 (25) , as well as LAGE-1, a cancer germ-line gene mentioned above (7) . We report here that a similar approach, this time applied to a sarcoma cell line, led to the identification of two new genes that have a pattern of expression similar to the MAGE-type genes. These genes are not homologous to the MAGE, BAGE, GAGE, and LAGE-1/NY-ESO-1 families.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Representational Difference Analysis.
Total RNA was isolated from normal tissues (uterus, breast, colon, and
heart) and from cell line LB23-SAR by the guanidine
isothiocyanate/cesium chloride procedure (26)
. Poly(A)+
RNA was prepared by binding to oligo-dT cellulose (mRNA purification
kit; Pharmacia Fine Chemicals, Piscataway, NJ), starting from 500 µg
of rhabdomyosarcoma LB23-SAR total RNA and from 455 µg of a mixture
of human uterus, breast, heart, and colon total RNA. About 4 µg of
each poly(A)+ RNA were used to synthesize double-stranded cDNA (cDNA
Synthesis Module; Amersham Life Science, Buckinghamshire, United
Kingdom). Two micrograms of each cDNA were then digested with
DpnII (New England Biolabs, Beverly, MA). The digested cDNAs
were ligated to R-Bgl adapters and PCR-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
amplification with the J-Bgl and N-Bgl adapters, as described
(27)
.
Cloning of Difference Products.
The second and third difference products were digested with
DpnII, separated from the adapters on a 1.2% agarose gel,
and purified using the QIAEX II Gel Extraction kit (Qiagen, Westburg,
Leusden, the Netherlands). They were ligated to dephosphorylated
BamHI ends of pTZ18R (Pharmacia). DH5
F'IQ bacteria (Life
Technologies, Gaithersburg, MD) were transformed by electroporation
with one-fifth of the ligation product.
Sequencing and Sequence Comparison.
Plasmid DNA was prepared by the boiling minipreparation procedure
(28)
. Sequencing was performed with the T7 Sequencing kit
(Pharmacia LKB) and [-35S]dATP (>1000
Ci/mmol; Amersham), and reaction products were separated on a 2010
Macrophor manual sequencer (Pharmacia LKB). When long read lengths were
required, we performed the sequencing reactions with the BigDye
Terminator Cycle Sequencing kit (PE Applied Biosystems, Warrington,
United Kingdom). The reaction products were separated on the ABI PRISM
310 Genetic Analyser (Perkin-Elmer).
Sequence homology searches were performed in the databases provided by the National Center for Biotechnology Information (Bethesda, MD) using the BLAST program (29) . Alignments were performed with the GeneWorks computer program (Intelligenetics, Mountain View, CA). For DNA, we used parameters 30-2-10-4000.
RT-PCR Analyses.
The expression of cDNA clones isolated from the difference products was
analyzed by
RT-PCR3
. RNA were extracted from tumor cells and normal tissues and reverse
transcribed, as described previously (30)
. RNA samples
from 5-aza-2'-deoxycytidine-treated cells were obtained as described
(31)
. Twenty-four pairs of PCR primers were derived from
the sequences of the fragments of cDNA of the enriched library. The
sequence of these primers are available from the authors. PCR
amplification of cDNA was performed for 30 or 35 cycles, whereas
amplification of ß-actin was performed for 21 cycles with primer
5'-GGCATCGTGATGGACTCCG-3' (exon 3, sense) and primer
5'-GCTGGAAGGTGGACAGCGA-3' (exon 6, antisense). PCR products were
visualized on 1.7% agarose gels stained with ethidium bromide.
For the analysis of SAGE expression, the cDNA produced from 50 ng of total RNA was PCR amplified in a TRIO-Thermoblock (Biometra, Göttingen, Germany) for 30 cycles of 1 min at 94°C, 2 min at 57°C, and 2 min at 72°C with primers sdph3.10S and sdph3.10A.
To analyze HAGE expression, PCR amplification was performed for 30 cycles of 1 min at 94°C, 2 min at 57°C, and 2 min at 72°C with primers sdp3.8.8 and sdp3.8.9.
PCR Analysis of Radiation Hybrids.
The GeneBridge 4 Radiation Hybrid Panel (Research Genetics, Inc.,
Huntsville, AL) was used to map the SAGE and HAGE
genes (32)
. Twenty-five ng of genomic DNA from each of the
93 radiation hybrid clones were PCR amplified with primers sdph3.10S
(5'-TGTACCTCTTCAAGCAAAAT-3') and sdph3.10A (5'-GTGACCCACCAGTTACAGTA-3')
that are specific for gene SAGE. PCR amplification was
performed 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 Whitehead Institute for Biomedical
Research.4
Positioning of markers from the Whitehead RH map on the chromosome
cytogenetic map was performed via the Genome Database web
site.5
The same panel was used to map gene HAGE with primers
sdp3.8.8 (5'-TATTCTTCAGATTGACGAAG-3') and sdp3.8.9
(5'-CCTTTCAATGTTATCCTGAG-3') using identical PCR conditions.
Construction and Screening of the cDNA Library.
We used a cDNA library derived from human testis sample LB451 to search
for the complete cDNA sequences of genes SAGE and
HAGE. Poly(A)+ RNA was extracted from testis cells using the
FastTrack mRNA extraction kit (InVitrogen, San Diego, CA). mRNA was
converted to cDNA with the SuperScript Choice System (Life
Technologies, Inc.) using an oligo-dT primer containing a
NotI site at its 5' end. cDNAs were then ligated to
BstXI adaptors and digested with NotI. After size
fractionation, the cDNAs were cloned into the BstXI and
NotI sites of plasmid pcDNAI/Amp. Recombinant plasmids were
electroporated into Escherichia coli DH5F'IQ and selected
with ampicillin (50 µg/ml). The library 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-amplified fragment of
genes SAGE or HAGE, using the same primers as
those used for the analysis of radiation hybrids, and labeled with
[-32P]dCTP (3000 Ci/mmol). Plasmid DNA from
one positive clone was amplified and purified for further analysis.
Northern Blot Analysis.
Four µg of poly(A)+ RNA were separated by formaldehyde agarose
gel electrophoresis, transferred to a nylon membrane by capillary
transfer, and fixed to the membrane by heating at 80°C for 2 h.
Hybridization with the 1.95-kb insert of SAGE cDNA or the
2.3-kb insert of HAGE cDNA probes was performed overnight at
60°C in 10% dextran sulfate, 1 M NaCl, 1%
SDS, and 100 µg/ml denatured herring sperm DNA. Final washing of the
membrane was done in 0.2x SSC, 0.1% SDS for 10 min at 60°C.
Autoradiography was performed overnight using BioMax MS film (Kodak).
The same membrane was stripped and hybridized with a 0.6-kb probe
for ß-actin obtained in similar conditions. Autoradiography was
performed for 2 h.
Southern Blot Analysis.
Ten µg of genomic DNA were digested with restriction enzymes
BamHI, EcoRI, HindIII, and
PstI. They were separated by agarose gel electrophoresis,
transferred to nylon membranes by the capillary transfer method, and
fixed by heating at 80°C for 2 h. Hybridization with the
-32P-radiolabeled PCR probes was performed in
5x SSC, 5x Denhardt’s solution, 0.1% SDS, and 100 µg/ml denatured
herring sperm DNA for 16 h at 65°C. Final washing of the
membranes was done in 2x SSC, 0.1% SDS for 20 min at 65°C.
Autoradiography was performed overnight using BioMax MS film (Kodak).
Rapid Amplification of the 5' cDNA End of Gene
SAGE.
The amplification of the 5' cDNA end of SAGE was performed
with the 5' rapid amplification of cDNA ends system, version 2.0 (Life
Technologies, Inc.). We used antisense primers Gsp1
(5'-CTGGTACTCATGGGTGGAAT-3'), Gsp2 (5'-GTTAATAAGCTCTGGTGTAA-3'), and
GspN (5'-CATATCAGCTGTTGCCAAAT-3'). The amplified products were cloned
using the Original TA Cloning kit (InVitrogen). The resulting clones
were screened with the labeled sdph3.10.1 oligonucleotide
(5'-ACTCACAATGTCTGTGAAGA-3'). We sequenced 24 positive clones with
primer GspN.
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RESULTS |
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Forty-two different sequences of the second and third difference products were analyzed. Twenty-two sequences showed ubiquitous expression, 12 of these coding for mitochondrial or ribosomal components or for known abundant proteins involved in cell proliferation. Eighteen clones were transcribed in some normal tissues but had a level of expression in LB23-SAR cells that was significantly higher. Among those, six were not recorded in databases. Finally, two sequences that we named SAGE and HAGE appeared to be highly expressed in testis and in LB23-SAR but were completely or nearly completely silent in normal tissues. Because these genes had a pattern of expression similar to that of the MAGE, BAGE, GAGE, and LAGE-1/NY-ESO-1 genes, we felt that they had the potential to code for tumor-specific antigens, and we characterized them fully.
Gene SAGE.
We analyzed the expression pattern of gene SAGE in
tumor samples and normal tissues. The PCR product was 171 bp when
derived from cDNA templates. It was easily distinguished from the
750-bp product derived from genomic DNA, eliminating the risk of false
positives attributable to DNA contamination of the RNA. SAGE
was expressed in many samples of bladder, lung, and head and neck
carcinomas (Table 1
). In contrast, normal tissues were found to express <0.1% of the
amount expressed by LB23-SAR. The only positive tissue was testis, as
has been observed for the other MAGE-type genes (Fig. 1
). The expression of several MAGE genes can be induced with
5-aza-2'-deoxycytidine in normal and tumoral cell types that do not
normally express these genes (25, 31)
. We examined whether
gene SAGE could also be activated in nonexpressing cells
after treatment with this agent. We found that gene SAGE was
induced in fibroblasts and in tumoral cell lines of various types (data
not shown).
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) indicating that we had not obtained a full-length cDNA clone. To
obtain the full-length cDNA, we used the PCR amplification of the 5'
cDNA end. This led to a 3069 bp sequence. The largest open reading
frame comprised 2712 bp and encoded a putative protein of 904 amino
acids; this protein is composed of 15 repeated motifs of 47 amino acids
(Fig. 3
).
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) suggesting that SAGE is a member of a family of genes.
Using the GeneBridge 4 Radiation Hybrid Panel, we established that gene
SAGE was located between markers WI-6213 and
WI-5285 on the chromosome X, in the q28 region, like the
MAGE-A and LAGE-1/NY-ESO-1 genes (Fig. 5
).
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2 kb). With the exception of testis,
which showed a high level of expression, a very low level of expression
was observed with normal tissues (Fig. 6
). The level of expression of gene HAGE in normal tissues,
including skeletal muscle, was 0.2% of the level of expression
found in the LB23-SAR sarcoma cell line. When we analyzed tumor
samples, we found that gene HAGE was expressed in a large
fraction of tumor samples of various histological types, well above the
level observed in normal tissues (Table 2
). About 5% of the positive tumor samples tested showed a level of
expression >10% of the level of LB23-SAR, and 7% showed a level of
expression between 1 and 10%. Like gene SAGE, the
expression of gene HAGE could also be induced in cells
treated with 5-aza-2'-deoxycytidine (data not shown).
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2.2 kb on a
Northern blot (Fig. 7
) suggesting that the isolated cDNA was full length. The largest open
reading frame comprised 1944 bp and encoded a putative protein of 648
amino acids (Fig. 8
).
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. All of the motifs that are present in all of the members
of the DEAD box family are conserved in the HAGE protein.
We used the 2.3-kb insert for Southern analysis of human genomic DNA,
and it hybridized with several fragments of DNA obtained with four
different restriction enzymes (Fig. 9
). It is probable that the probe hybridizes with other members of the
DEAD-box family because their sequences are well conserved.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As already observed for other MAGE-type genes, SAGE and HAGE expression was induced by 5-aza-2'-deoxycytidine, suggesting that demethylation plays a role in the activation of these genes in tumors. There is evidence that, in cells containing the relevant transcription factors, DNA methylation can on its own be effective for gene silencing (34, 35) . One mechanism by which methylation can affect gene expression is that binding of transcription factors to their target sequences can be inhibited by methylation of CpGs sites located in these sequences (36) . Another mechanism for transcriptional silencing induced by methylation is through the modification of the structure of the chromatin. Methylation of DNA helps to stabilize the chromatin in an inactive configuration and therefore inhibits gene transcription (37) . This inactive state of the chromatin is associated with underacetylated histones, suggesting that DNA methylation and histone deacetylation are linked. In line with this hypothesis, it has been shown recently that histone deacetylases form complexes with methyl-binding proteins such as MeCP2, MBD2, and MBD3 (38–40) .
We suggested previously that the activation of MAGE genes in tumor cells results from the genome-wide demethylation that occurs in these cells (31) . However, tumor cells with demethylated genomes do not activate all of the MAGE-related genes (41) . This is probably attributable to random demethylation and chromatin modification, which vary from cell to cell and lead to different patterns of expression of MAGE-related genes, even in tumors of the same histological type. We report here that MAGE and SAGE genes are both expressed in head and neck carcinomas, bladder carcinomas, and epidermoid carcinomas of the lung, but that in melanomas MAGE genes are expressed whereas SAGE is rarely expressed. MAGE-1 and MAGE-4 were found to be regulated by ubiquitous transcription factors (42, 43) , but for other genes, such as SAGE, we cannot exclude that some of the factors regulating their expression are tissue specific and hence are present only in certain types of tumors.
Gene SAGE is unrelated to any known sequence and appears to be a member of a new family of several genes. It is not expressed at all in normal tissues, with the exception of testis. The expression of MAGE and LAGE genes in testis appears to be restricted to germ-line cells (19) ,6 and we consider it likely that this will apply to SAGE also. Like MAGE-A genes (44) , gene SAGE maps to the q28 region of chromosome X. It is noteworthy that several other genes that are expressed in a wide array of tumors and in male germ-line cells map to chromosome X. In addition to MAGE-A and LAGE genes, this is the case for P1A in the mouse and for MAGE-B, MAGE-C1, GAGE, and SSX2 genes in humans (25, 45–47) . The significance of this localization is not clear.
The second gene that we identified was named HAGE because it encodes a protein that shows 55% similarity with the human p68 protein, a DEAD-box protein whose ATP-dependent RNA helicase activity has been demonstrated (48, 49) . The protein encoded by gene HAGE seems to be a new member of the family of DEAD-box proteins (33) , which contain the highly conserved Asp-Glu-Ala-Asp (D-E-A-D) motif. These proteins are involved in many aspects of RNA metabolism, spermatogenesis, embryogenesis, and cell growth. The amino acids that are highly conserved in all of the DEAD-box proteins are also present in protein HAGE, suggesting that HAGE may also be an ATP-dependent RNA helicase. Gene HAGE is not located on chromosome X but on chromosome 6.
For MAGE-1, it has been shown that tumor cell lines
could be lysed by a relevant CTL clone if the level of expression of
this gene exceeded the threshold of 10% of that found in the MZ2-MEL
reference tumor cell line, i.e., more than three RNA
molecules/cell (50)
. We have indications (data not shown)
that the level of expression of gene HAGE in cell line
LB23-SAR is 10 times lower than the level of expression of
MAGE-1 in 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 recognizes a peptide encoded by gene HAGE has
been obtained. This CTL clone could then also be used to test the
presence of antigens on normal tissues, where the level of expression
of gene HAGE is at least 100 times lower than 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 described in
retinoblastoma and neuroblastoma cell lines (51–53)
.
Moreover, 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 recognized
by a CTL clone on a UV-induced sarcoma (54)
. The human
melanoma antigen LB33-A is produced by a point mutation in a new gene,
MUM-3, which is expressed ubiquitously and shows homology
with members of the RNA helicase gene
family.7
These observations suggest that mutated or overexpressed helicases may
contribute to tumoral transformation or progression.
Genes such as SAGE and HAGE, with expression that appears to be restricted to tumors and germ-line cells, are potentially coding for tumor-specific antigens recognized by T lymphocytes. But these antigens remain to be identified. Approaches to establish which antigenic peptides recognized by T cells are encoded by such genes have recently made considerable progress. In vitro stimulation of CD8+ T lymphocytes with dendritic cells infected with canarypoxes or adenoviruses carrying sequences of MAGE genes has led to the definition of a large number of new epitopes encoded by these genes (55, 56) . Moreover, stimulation of CD4+ T cells with dendritic cells pulsed with MAGE proteins has led to the identification of several new antigenic peptides presented by class II molecules (57) . These results significantly enlarge our possibilities for therapeutic vaccination with tumor-specific antigenic peptides, because it is now certain that almost every cancer patient whose tumor expresses a MAGE gene will have at least one HLA molecule presenting a MAGE antigenic peptide. The same approach could be applied to genes SAGE and HAGE to identify tumor-specific antigens derived from them. Consecutive immunization with several antigens might ensure a more effective rejection of tumor cells because this should reduce the emergence of antigen-loss variants arising by mutations in the genes producing the antigenic peptides or by down-regulation of their expression. SAGE and HAGE antigens could also be used to immunize patients against tumors that do not express MAGE genes.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by grants from Fonds J.
Maisin (Belgium), Fédération Belge contre le Cancer
(Belgium), Caisse Générale d’Epargne et de Retraite
(CGER)-Assurances and VIVA (Belgium), the Belgian program on
Interuniversity Poles of Attraction 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 dans
l’Industrie et l’Agriculture (Belgium). 
2 To whom requests for reprints should be
addressed, at Ludwig Institute for Cancer Research, Université
Catholique de Louvain, 74 avenue Hippocrate, UCL 7459, B-1200 Brussels,
Belgium. Phone: 32-2-764-75-80; Fax: 32-2-762-94-05; E-mail: boon{at}licr.ucl.ac.be
3 The abbreviations used are: RT-PCR,
reverse transcription-PCR; SAGE, sarcoma antigen 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.
6 B. Lethé, personal communication.
7 J. F. Baurain, D. Colau, N. van Baren, C.
Landry, V. Martelange, M. Vikkula, T. Boon, and P. G. Coulie. High
Frequency of Autologous Anti-Melanoma CTL Directed Against an Antigen
Generated by a Point Mutation in a New Helicase Gene, submitted for
publication.<./>
Received 12/17/99. Accepted 5/16/00.
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REFERENCES |
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