[CANCER RESEARCH 59, 3157–3165, July 1, 1999]
Characterization of the GAGE Genes That Are Expressed in Various Human
Cancers and in Normal Testis1
Olivier De Backer,2 Karen C. Arden, Mauro Boretti, Valérie Vantomme, Charles De Smet, Suzanne Czekay,
Carrie S. Viars, Etienne De Plaen, Francis Brasseur, Patrick Chomez, Benoı̂t Van den Eynde, Thierry Boon, and
Pierre van der Bruggen
Ludwig Institute for Cancer Research, Brussels Branch, and Cellular Genetics Unit, Université Catholique de Louvain, B-1200 Brussels, Belgium [O. D. B., M. B., V. V., C. D. S.,
E. D. P., F. B., P. C., B. V. d. E., T. B., P. v. d. B.]; Ludwig Institute for Cancer Research, San Diego Branch, University of California, San Diego School of Medicine, La Jolla,
California 92093-0660 [K. C. A., S. C., C. S. V.]; and Department of Medicine, University of California San Diego, La Jolla, California 92093-0660 [K. C. A.]
ABSTRACT
The GAGE-1 gene was identified previously as a gene that codes for an
antigenic peptide, YRPRPRRY, which was presented on a human melanoma by HLA-Cw6 molecules and recognized by a clone of CTLs derived
from the patient bearing the tumor. By screening a cDNA library from
this melanoma, we identified five additional, closely related genes named
GAGE-2– 6. We report here that further screening of this library led to the
identification of two more genes, GAGE-7B and -8. GAGE-1, -2, and -8
code for peptide YRPRPRRY. Using another antitumor CTL clone isolated from the same melanoma patient, we identified antigenic peptide,
YYWPRPRRY, which is encoded by GAGE-3, -4, -5, -6, and -7B and
which is presented by HLA-A29 molecules. Genomic cloning of GAGE-7B
showed that it is composed of five exons. Sequence alignment showed that
an additional exon, which is present only in the mRNA of GAGE-1, has
been disrupted in gene GAGE-7B by the insertion of a long interspersed
repeated element retroposon. These GAGE genes are located in the p11.2–
p11.4 region of chromosome X. They are not expressed in normal tissues,
except in testis, but a large proportion of tumors of various histological
origins express at least one of these genes. Treatment of normal and tumor
cultured cells with a demethylating agent, azadeoxycytidine, resulted in
the transcriptional activation of GAGE genes, suggesting that their expression in tumors results from a demethylation process.
INTRODUCTION
Human cancer cells bear antigens that are recognized by CTLs
derived from the patient bearing the tumor. These antigens consist of
membrane-bound class I MHC molecules with short peptides derived
from cellular proteins. A number of these antigenic peptides are
encoded by genes that are expressed at high frequency in cancers of
various histological origins but not in normal tissues, except in male
germ cells. The expression of these genes in germ cells does not result
in the formation of antigens because these cells does not produce class
I MHC molecules (1– 4). The genes coding for these tumor-specific
shared antigens include the members of several gene families, such as
the MAGE, GAGE, BAGE, SSX, and LAGE-1/NY-ESO-1 genes (5–9).
GAGE-1 was identified as a gene coding for a tumor antigen of
melanoma cell line MZ2-MEL. Five cDNAs sharing 80 –98% nt3
identity with the GAGE-1 sequence were identified by screening a
Received 11/9/98; accepted 5/3/99.
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
This study was supported in part by grants from the Belgian Program on Interuniversity Poles of Attraction, initiated by the Belgian State, Prime Minister’s Office, Office
for Science, Technology and Culture; by the “Caisse Générale d’Epargne et de Retraite
Assurances” (Belgium); by the “Fonds Joseph Maisin” (Belgium); and by BIOMED, a
European Community Program for Research and Technological Development. V. V. was
supported by the Fonds National de la Recherche Scientifique (TELEVIE grants; Belgium).
2
To whom requests for reprints should be addressed, at Ludwig Institute for Cancer
Research, Brussels Branch, 74 avenue Hippocrate, UCL 74.59, B1200 Brussels, Belgium.
Phone: 32 2/764 74 23; Fax: 32 2/762 94 05; E-mail: [email protected].
3
The abbreviations used are: nt, nucleotide(s); PBL, peripheral blood lymphocyte;
TNF, tumor necrosis factor; FISH, fluorescence in situ hybridization; RT-PCR, reverse
transcription-PCR; LINE, long interspersed nuclear element; DAC, azadeoxycytidine.
MZ2-MEL cDNA library with a GAGE-1 probe (6), and two additional homologous cDNAs were identified by screening for genes
expressed in cell lines from the LNCaP prostate cancer model (10).
One of these cDNAs corresponds to an additional GAGE gene,
GAGE-7; the other, called PAGE-1, differs from the GAGE sequences, essentially by the presence of insertions and deletions (10).
In addition, more distant genes were recently identified by searching
expressed sequence tag sequences (11). Contrary to the GAGE genes,
these genes, also named PAGE, are expressed in normal male and
female reproductive tissues, prostate, testis, fallopian tube, uterus, and
placenta as well as in prostate cancer, testicular cancer, and uterine
cancer (11). The GAGE and PAGE putative proteins do not show
significant homology with any polypeptide recorded in data banks,
and nothing is known about their function. Here, we report the
identification of two additional GAGE genes, GAGE-7B and -8, the
definition of the exon-intron structure of the GAGE genes, and their
chromosomal localization. We also report that GAGE-3, -4, -5, -6 and
-7B code for a new antigenic peptide presented by melanoma cells.
MATERIALS AND METHODS
Cell Lines. Melanoma cell line MZ2-MEL was derived from an abdominal
metastasis of patient MZ2, and a number of subclones were obtained. Subclone
MZ2-MEL.3.0 was obtained by limiting dilution. Subline MZ2-MEL.43 was
derived by limiting dilution from MZ2-MEL.3.0 cells that had survived mutagen treatment (12, 13). Subline MZ2-MEL.3.1 was obtained by extending the
culture of subclone MZ2-MEL.3.0 for .150 generations. Subline MZ2MEL.F2, which does not express antigen F, was selected from subclone
MZ2-MEL.3.1 with autologous CTL clone 76/6 (13). Subline MZ2MEL3.11HLA-A29 was obtained by transfecting the HLA-A29 gene into
subline MZ2-MEL.3.1. Melanoma cell lines were grown as described previously (13, 14). Autologous CTL clones 76/6 and 22/23 were derived from
blood mononuclear cells of patient MZ2 and grown in conditions similar to
those described previously (15). PBLs were isolated using Lymphoprep (Nycomed Pharma, Oslo, Norway) and grown in Iscove’s medium (Life Technologies, Inc., Grand Island, NY) supplemented with 10% FCS (Life Technologies, Inc.), 100 units/ml interleukin 2 (Biogen, Geneva, Switzerland), and 0.3%
(v/v) phytohemagglutinin-P (Difco Laboratories, Detroit, MI). The fibroblast
culture was derived from a human lung sample and maintained in Iscove’s
medium supplemented with 10% FCS, L-arginine (116 mg/ml), L-asparagine
(36 mg/ml), and L-glutamine (216 mg/ml).
Transfection of COS-7 Cells. Transfection experiments were performed
by the DEAE-dextran-chloroquine method (16). Briefly, 1.5 3 104 COS-7
cells were transfected with 50 ng of plasmid pcDNA1/Amp (Invitrogen,
Carlsbad, CA) containing cDNA of HLA-A29 or Cw6 and 50 ng of each of the
GAGE cDNA cloned in pcDNA1/Amp. The HLA cDNAs were isolated from
a cDNA library prepared with RNA extracted from subline MZ2-MEL.43.
Transfected COS-7 cells were tested in a CTL stimulation assay after 24 h.
CTL Stimulation Assay. Transfectants were tested for their ability to
stimulate the production of TNF by CTL (15). Briefly, in microwells containing target cells, 1500 22/23 CTLs or 1000 76/6 CTLs were added to 100 ml of
Iscove’s medium (Life Technologies, Inc.) containing 10% human serum and
20 units/ml recombinant human interleukin 2. After 24 h, the supernatant was
collected, and its TNF content was determined by testing its cytotoxic effect on
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cells of WEHI-164 clone 13 (17) in a 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide colorimetric assay (15, 18)
Antigenic Peptides and CTL Assay. Peptides were synthesized on solid
phase using N-(9-fluorenyl)methoxycarbonyl for transient NH2-terminal protection, as described previously (19), and characterized by mass spectrometry.
All peptides were .90% pure, as indicated by analytical high-performance
liquid chromatography. Lyophilized peptides were dissolved at 20 mg/ml in
DMSO, diluted to 2 mg/ml in 10 mM acetic acid, and stored at 220°C. Peptides
were tested in a chromium release assay, as described previously (20). In this
peptide sensitization assay, target cells were 51Cr-labeled for 1 h at 37°C and
washed extensively. One thousand target cells were then incubated in 96-well
microplates in the presence of various concentrations of peptide for 15 min at
37°C before CTLs were added at an effector:target ratio of 5:1. Chromium
release was measured after 4 h at 37°C.
Construction and Screening of cDNA Libraries. The cDNA library was
derived from MZ2-MEL.43, as described previously (6).
Construction and Screening of Human Genomic Libraries. Genomic
DNA isolated from PBLs of patient MZ2 was partially digested with Sau3AI,
size-fractionated by NaCl density gradient centrifugation, and ligated with
BamHI-/EcoRI-digested LambdaGEM-11 cloning vector (Promega, Madison,
WI). The cosmid library was constructed with DNA from the renal cell
carcinoma cell line LE9211-RCC, as described previously (21). Screening of
the libraries was performed using standard techniques (22) and the PCRgenerated GAGE-1 probe.
DNA Sequencing. Fragments of l clones containing GAGE genes were
subcloned in plasmids and sequenced using a D Taq Cycle-sequencing kit
(United States Biochemical, Cleveland, OH), and primers were 59-labeled with
[g-33P]ATP. Sequences were also obtained directly from l clones and cosmids.4
Genomic Southern Blot Analysis. Transfer and hybridization of genomic
DNA were performed using standard techniques (22). The GAGE-specific
probe was a PCR-amplified fragment corresponding to nt 18 –309 of the
GAGE-1 cDNA. This fragment was purified from low melting point agarose
and labeled by incorporation of [a-32P]dCTP (3000 Ci/mmol) upon primer
extension (23). This probe hybridizes to all known GAGE genes because of
their high homology. Washing conditions were: 0.23 SSC-0.1% SDS at 65°C
for the human blot and 23 SSC-0.1% SDS at 60°C for the zoo blot, which was
obtained from Clontech Laboratories (Palo Alto, CA).
Analysis of Somatic Cell Hybrids. DNA isolated from the human 3 rodent hybrids was digested with EcoRI and used to prepare Southern blots, as
described previously (24). The hybrids designated with the GM prefix were
derived from human cell lines containing translocations between chromosome
X and an autosome, resulting in the presence of only a portion of the X
chromosome. They were obtained from the Human Genetic Mutant Cell
Repository (Camden, NJ). The PCR analysis was performed with primers
VDE23 (sense, 59-ACGGAAACCTTGAGTGAC-39, nt 453– 470 of the
GAGE-1 cDNA sequence) and VDE25 (antisense, 59-GCAGAAGGCTGTAAAGCT-39, nt 613– 630 of the GAGE-1 cDNA sequence), which amplify a
GAGE genomic fragment of 0.7 kb. Thirty-five cycles of PCR were performed,
each cycle consisting of a denaturation at 94°C for 60 s, an annealing at 50°C
for 60 s and an extension at 72°C for 60 s. PCR was preceded by a 3-min
incubation at 94°C and followed by a 72°C final soak for 10 min. Amplified
products were electrophoresed through 2% agarose gels and visualized by
ethidium bromide staining.
FISH. Chromosome preparations were obtained from phytohemagglutininstimulated normal PBLs cultured for 72 h. To obtain banded chromosomes, we
inoculated some harvests with 5-bromodeoxyuridine, as described previously
(25). Cytogenetic harvests and slide preparations were performed using standard methods. The slides were stored at 280°C before use. FISH with metaphase chromosomes was performed as described previously (26). Briefly, the
slides were denatured for 2 min in 70% formamide-23 SSC (pH 7.0) at 70°C
and dehydrated in ice-cold ethanol. A cosmid that contained gene GAGE-7B
was used as a probe. The digoxigenin-labeled cosmid DNA (100 ng) was
preannealed with 10 mg of COT-1 DNA (Life Technologies, Inc.) and dissolved in Hybrisol VII (Oncor; 50% formamide-23 SSC), denatured at 75°C
for 5 min, and applied to the slides. Hybridization was allowed to proceed
overnight in a humid chamber at 37°C. The slides were then washed using the
formamide wash procedure and revealed with the FITC-digoxigenin detection
kit and amplification protocol for dual-color FISH (Oncor). Slides were counterstained by mounting in an antifade solution containing 1 mg/ml
phenylenediamine and 0.3 mg/ml propidium iodide. Metaphase spreads were
examined and photographed using a Zeiss Axiophot microscope and the
appropriate UV-filter combinations. We considered signals to be specific only
if they were detected on each chromatid of a single chromosome. Chromosome
identification was performed by simultaneous hybridization with the a-satellite
repeat probe or by R-banding using the 5-bromodeoxyuridine incorporation, as
described previously (25).
RT-PCR Assay for the Expression of GAGE Genes. Total RNA was
extracted by the guanidine-isothiocyanate procedure, as described (27). Reverse transcription was performed on 2 mg of total RNA in the presence of 2
mM oligo(dT)15 primer in a reaction volume of 20 ml. One-twentieth of the
cDNA product was used for PCR amplification. For specific amplification of
the GAGE-1, -2, and -8 cDNAs, primers were VDE44 (sense, 59-GCACAAGACGCTACGTAG-39) and VDE24 (antisense, 59-CCATCAGGACCATCTTCA-39). For specific amplification of the GAGE-3, -4, -5, -6, and -7B cDNAs,
primers were VV1 (sense, 59-GACCAAGGCGCTATGTAC-39) and VDE24
(antisense, 59-CCATCAGGACCATCTTCA-39). Both amplifications were
performed as follows: after a first denaturation step at 94°C for 5 min, 30
cycles of amplification were performed (1 min at 94°C, 2 min at 58°C, and 2
min at 72°C), and then a final extension step terminated the reaction. PCR
products were analyzed by agarose gel electrophoresis. RNA integrity was
checked by reverse transcription and amplification of the b-actin mRNA.
Aza-2*-deoxycytidine Treatment and mRNA Expression Analysis.
Cells were incubated for 72 h in culture medium containing 1 mM 5-aza-29deoxycytidine (Sigma, Deisenhofen, Germany). Fibroblasts were treated at
passage 4. Total RNA purification and cDNA synthesis were performed as
described (28). The primers used for PCR amplification of the GAGE cDNAs
were VDE24 and VDE44 (6).
RESULTS
The GAGE Gene Family Contains at Least Eight Members. We
previously identified six closely related cDNAs, named GAGE-1– 6,
by probing a cDNA library made with RNA from melanoma cell line
MZ2-MEL.43 (6). A second screening of the MZ2-MEL.43 cDNA
library yielded two additional cDNAs. One of these differed only by
a single base from the sequence of GAGE-7 described by Chen et al.
(10) and encoded the same putative protein. Because we did not know
whether this new cDNA corresponded to a new gene or to another
allele of GAGE-7, we named it GAGE-7B. The additional cDNA was
named GAGE-8. Fig. 1 shows that the sequences of GAGE-2, -3, -4,
-5, -6, -7B, and -8 differ from each other mainly by single nt substitutions. The GAGE-1 cDNA differs from the seven others by the
presence of a 143-bp insertion (Fig. 1).
Because most GAGE cDNAs present only a few single-nt differences, there was a possibility that some of them were allelic products.
However, the MZ2-MEL.43 cells must contain a single allele of each
GAGE gene because they contain a single X chromosome, where the
GAGE genes are located (see below). Indeed, both karyotyping and
absence of Xist expression indicated that, despite their female origin,
MZ2-MEL.43 cells contain a single X chromosome.5 We conclude
that a minimum of eight GAGE genes are expressed in MZ2-MEL.43
cells.
Genomic Structure of the GAGE Genes. A GAGE genomic clone
was isolated from a phage library constructed with DNA from patient
MZ2. The sequence of the insert revealed that it contained gene
GAGE-7B and that this gene comprises five exons (Fig. 2; see also
Fig. 1). An open reading frame coding for a product of 117 amino
acids spans exons 2–5 (Fig. 1).
The fourth intron contained two regions (Fig. 2, 49A and 49B) that
4
The sequence data reported in this paper have been deposited with the GenBank
database (accession nos. AF055473, AF055474, and AF055475).
5
C. De Smet, unpublished results.
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Fig. 1. A, alignment of the nt sequences of GAGE-1– 8 cDNAs. The sequences are ordered according to their degree of homology to the GAGE-1 sequence. z, nt that are identical
to the GAGE-1 sequence. Double-headed arrows, limits of the exons. Initiation and termination codons are in boldface type. Horizontal arrows, locations of the primers VDE44,
VDE24, and VV1 used for the analysis of GAGE expression by RT-PCR. B, comparison between GAGE putative proteins. z, amino acid residues that are identical to the GAGE-1
sequence; u, antigenic peptides presented by HLA-Cw6 and -A29 molecules.
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Fig. 2. Structure of gene GAGE-7B. A schematic representation of gene GAGE-7B is shown. Boxes, exons. Horizontal boldface arrow, LINE L1 element inserted between the two
), coding region. The draft also represents a proposed structure for gene GAGE-1 with its undisrupted exon 49.
parts (49A and 49B) of the disrupted exon 49. (
showed strong sequence homology with region 49, which is present
only in the GAGE-1 cDNA (Figs. 1 and 2). A genome database search
revealed that the region of 561 bp located between 49A an 49B
corresponds to a truncated L1 retroposon belonging to the LINE
family (29). This LINE element is flanked by a perfect 13-bp target
site duplication and contains part of the reverse transcriptase coding
region, the 39 untranslated region, and the poly(A) tail of the original
retroposon.
We concluded that GAGE-1 should contain an additional exon
corresponding to the region disrupted by the LINE insertion in the
GAGE-7B intron. This hypothesis was supported by the presence of an
acceptor splicing site at the 59 end of 49A and a donor splicing site at
the 39 end of 49B. To confirm the existence of an undisrupted exon 49
in gene GAGE-1, we performed a PCR with MZ2 genomic DNA
using a sense primer in 49A and an antisense primer in 49B. As
expected, in addition to the fragment of 0.7 kb corresponding to the
genes with a LINE insertion, the PCR produced a 140-bp fragment,
the sequence of which corresponded exactly to the insertion of
GAGE-1 (data not shown). This confirmed that GAGE-1 contains an
additional exon that is skipped in the other GAGE genes because it has
been disrupted by a LINE insertion.
By screening a cosmid library with a GAGE probe (corresponding
to nt 18 –309 of the GAGE-1 cDNA), we obtained a cosmid clone
containing gene GAGE-8. This gene had the same exon-intron structure as the GAGE-7B gene, including the LINE insertion.
The same probe was used to hybridize a Southern blot, made with
PstI-digested human genomic DNA from 11 men. The probe revealed
four bands (of 0.9, 1.4, 2.8, and 3.7 kb) in all of the DNAs, whereas
two other bands (of 4.3 and 5.2 kb) were present in only some
subjects, indicating the existence of polymorphism (Fig. 3). The bands
of 0.9 and of 5.2 kb were expected from the sequence of gene
GAGE-7B. The other bands should correspond to other members of
the family. In addition, the strong intensity of the 0.9- and 2.8-kb
bands suggests the stacking of several DNA fragments corresponding
to different genes. Thus, this hybridization pattern confirmed the
existence of several GAGE genes.
Southern hybridization with the GAGE probe revealed the presence
of homologous genes in the monkey but not in the other mammals
analyzed (mouse, rat, dog, cow, and rabbit). This indicated either that
these species do not contain sequences that are homologous to GAGE
genes or that these sequences are not sufficiently homologous to be
detected by hybridization, even in low-stringency conditions.
PAGE-1 Belongs to the GAGE Gene Family. When compared to
the GAGE-2 sequence, PAGE-1 shows two insertions (35 and 126 bp)
and two short deletions (24 and 12 bp; Fig. 4). The insertion of 35 bp
is located precisely between the first and the second exon and its
sequence is homologous to the 59 end of the first intron in the GAGE-8
gene. Thus, it seems that, in PAGE-1, the use of a splicing donor site
located downstream from the site used in the GAGE genes lengthens
the first exon of 35 bp. The last 59 bp of the second insertion are a
repetition of a sequence located just upstream from this insertion (Fig.
4). GAGE-2 shares 78% of identical residues with PAGE-1, whereas
an identity of .56% was reported by Chen et al. (10). This difference
is probably due to the addition of the gaps in the calculations of Chen
et al. (10). The resemblance between PAGE-1 and the GAGE genes
indicates clearly that it belongs the GAGE family. Therefore, we
suggest renaming PAGE-1 as GAGE-9.
Chromosomal Localization of the GAGE Genes. The chromosomal localization of the GAGE genes was initially determined by
Southern blot analysis of a panel of hamster or mouse 3 human
somatic cell hybrids. Hybridization of human DNA with the GAGE-1
probe (nt 18 –309) detected a single EcoRI band of 4.3 kb, indicating
that the EcoRI sites defining this fragment are conserved in all GAGE
genes (Fig. 5). No hybridization signal was observed with hamster or
mouse DNA or with the 23 hybrids that covered the 22 human
autosomes. A hybridization signal was detected only with the DNA
from hybrid GM06318B that contained the human X chromosome
(Fig. 5). The absence of a signal with the DNA from GM06317 that
contained the human Y chromosome suggested that the GAGE genes
do not reside in a pseudoautosomal region.
Fig. 3. Autoradiograph of a Southern blot of PstI-digested DNA from 11 male people
hybridized with a probe corresponding to nt 18 –309 of the GAGE-1 cDNA.
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Fig. 4. Comparison between the PAGE-1 and GAGE-2 cDNA sequences. The correspondence between the 35-bp insertion of PAGE-1 and the first 35 bp of the intron-1 of GAGE-8
is also shown. u, conserved residues. The two insertions present in the PAGE-1 sequence are boxed. Horizontal arrows, repeated sequence present in the PAGE-1 cDNA.
Fig. 5. Autoradiograph of a Southern blot of EcoRI-digested DNA from human 3 hamster hybrid cells hybridized with a probe corresponding to nt 18 –309 of the
GAGE-1 cDNA. DNAs were as follows: Lane 1 (from left to right), GM10498 containing
human chromosome 17; Lane 2, GM11010 containing human chromosome 18; Lane 3,
GM10449 containing human chromosome 19; Lane 4, GM10478 containing human
chromosomes 4 and 20; Lane 5, GM11441 containing human chromosomes 5 and 20;
Lane 6, GM08854 containing human chromosome 21; Lane 7, GM10888 containing
human chromosome 22; Lane 8, GM06318 containing human chromosome X; Lane 9,
GM06317 containing human chromosome Y; Lane 10, human; Lane 11, hamster; Lane
12, mouse.
To refine the localization of the GAGE locus, hybrid cell lines
containing only fragments of the human X chromosome were analyzed by hybridization and by PCR with GAGE primers. No signal
was observed in hybrid GM09412 containing the Xpter–p21 region
and in hybrid GM10095 containing the Xq13– qter region, indicating
that the GAGE genes are located in Xp21–Xq13.
The position of the GAGE locus was also determined by FISH.
Several cosmids containing GAGE genes were used as probes. All of
them hybridized in the p11.2–p11.4 region of chromosome X (Fig. 6).
Antigenic Peptides Encoded by the GAGE Genes. A panel of
stable autologous CTL clones was obtained previously against human
melanoma cell line MZ2-MEL by stimulating blood mononuclear
cells from patient MZ2 with irradiated autologous tumor cells (12).
Tumor cells, selected in vitro for resistance to some of these CTL
clones, remained sensitive to others, indicating that several different
antigens were present on the MZ2-MEL cells (13). One of these
antigens, named MZ2-F, was the antigenic peptide YRPRPRRY,
presented by HLA-Cw6 (Fig. 1). This peptide is encoded by GAGE-1,
GAGE-2, and also GAGE-8. We showed previously that cells expressing GAGE-1 or GAGE-2 are recognized by the CTLs directed against
this peptide (6). We found that the antigenic peptide can also be
processed from the GAGE-8 protein because COS-7 cells transfected
with HLA-Cw6 and GAGE-8 stimulated the release of TNF by 76/6
CTLs (Fig. 7).
A number of other CTL clones, including CTL 22/23, were shown
to lyse several MZ2-MEL subclones but not MZ2-MEL.3.1, a line that
had been passaged in vitro for .150 generations and had lost one
HLA haplotype coding for HLA-Cw*1601, -A29, and -B44 (Fig. 8).
The sequences encoding these HLA molecules were isolated from a
cDNA library of patient MZ2 and stably transfected into MZ2MEL.3.1. The cells transfected with HLA-A29 were lysed by the
22/23 CTLs, demonstrating that the presenting HLA molecule is A29
(Fig. 8). We then transfected COS-7 cells with HLA-A29 cloned in
pcDNA1/Amp and a panel of expression plasmids containing cDNA
coding for various tumor specific shared antigens, including members
of the MAGE, BAGE, and GAGE families (6, 7, 30). Cells cotransfected with the cDNA of GAGE-3, -4, -5, -6, and -7B stimulated the
release of TNF by 22/23 CTLs (Fig. 7). We searched the sequences of
Fig. 6. a, partial metaphase spread showing hybridization of the GAGE-containing
cosmid c7A to the proximal Xp11.2–p11.4 region (arrowhead). The X chromosome is
labeled at the centromere with a X-specific a-satellite probe. b, additional chromosome X
homologues showing signals in Xp11.2–p11.4.
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highest fraction of positive tumors were found in melanoma as well as
in esophageal and lung carcinomas (Table 1). In addition, GAGE-1, 2,
and 8 mRNAs were also detected in 15% of prostate adenocarcinomas
(n 5 20), 10% of breast carcinomas (n 5 153), and 17% of sarcomas
(n 5 12). The expression of GAGE-3, -4, -5, -6, and -7B was not
determined in these tumors. No expression of GAGE was found in
colorectal carcinomas and renal carcinomas (data not shown).
Expression of GAGE Genes Can Be Induced by DAC Treatment. To assess whether expression of GAGE genes can be induced
by demethylation, we treated several cultured tumor and normal cells
with DAC. RT-PCR analysis revealed the induction of GAGE-1, -2,
and -8 in treated cells from the sarcoma cell line LB23 and from the
melanoma cell lines Mi665 and SK23-MEL. GAGE-1, -2, and -8 and
GAGE-3, -4, -5, -6, and -7B mRNAs were detected after treatment of
phytohemagglutinin-stimulated PBLs (data not shown).
DISCUSSION
Fig. 7. Stimulation of MZ2-CTL clones by COS-7 cells transiently transfected with
GAGE cDNA and the DNA coding for the HLA-presenting molecule. COS-7 cells were
distributed in microwells (1.5 3 104) and transfected the day after by the DAE-dextran
method with 50 ng of HLA encoding plasmid and 50 ng of the plasmids containing the
GAGE cDNA. One day after the transfection, 1000 76/6 CTLs or 1500 22/23 CTLs were
added into the microwells containing the transfected cells. TNF production was estimated
after overnight coculture by testing the toxicity of the supernatants for the TNF-sensitive
WEHI-164 clone 13 cells. MZ2-MEL.43 cells were used as positive control.
the corresponding proteins for a peptide that was not encoded by
GAGE-1, 2, or 8 and that contained consensus anchor residues for
HLA-A29, namely E in position 2 and Y in position 9 (31). Only the
nonapeptide YYWPRPRRY (Fig. 1) fulfilled these conditions. It was
tested in a cytotoxicity assay with CTL 22/23 and produced halfmaximal lysis of autologous EBV-B target cells at 610 nM (Fig. 9).
Expression of the GAGE Genes. RT-PCR with specific primers
allowed us to analyze the expression of the genes coding for one or the
other antigenic peptide. GAGE transcripts were detected in a significant proportion of malignant tumors of various histological types. The
The GAGE genes belong to the category of genes exemplified by
the MAGE genes. These genes are silent in most normal adult tissues
but are expressed in cancers of various histological origin (5). Because
the expression of these genes is shared by many different tumors, the
antigens they code are promising targets for cancer immunotherapy.
The only normal tissues that have been found to express these MAGEtype genes are testis and, in some instances, placenta, two tissues
regarded as immunologically privileged sites (32). In testis, expression of the MAGE-type genes in the male germ cells should not result
in antigen expression because these cells do not express class I MHC
molecules (33). This is supported by the observation that in mice, a
CTL response against a tumor antigen encoded by P1A, which is also
expressed in testis, did not generate antitestis autoimmune effects (4).
What could explain the expression pattern of the MAGE-type
genes? The activation of MAGE-A1 in cancer cells is due to the
demethylation of its promoter, a process that appears to be the
consequence of a genome-wide demethylation occurring in certain
tumors (34). It is tempting to speculate that demethylation could also
explain the activation of the other MAGE-type genes in tumors. The
activation of genes MAGE-A1, -A2, -A3, -A4, -B1, and -B2, BAGE,
GAGE, and P1A in cells treated with the demethylating agent DAC
Fig. 8. Lysis of melanoma sublines of patient
MZ2 by autologous CTL clones. Tumor cells were
Cr-labeled and incubated with 76/6 or 22/23
CTLs at various effector-to-target ratios. Chromium release was measured after 4 h. MZ2MEL.F2 is an antigen-loss variant obtained by
immunoselection with 76/6 CTLs, which had lost
expression of HLA-Cw6. MZ2-MEL.3.11HLAA29 was obtained by stable transfection of subline
MZ2-MEL.3.1 with cDNA of HLA-A29 cloned in
pcDNAI/Amp.
51
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THE GAGE GENE FAMILY
Table 1 Expression of the GAGE genes in tumors
No. of samples
Tumor
Total no.
tested
Cutaneous melanoma (primaries)
Cutaneous melanoma (metastases)
Esophageal squamous cell carcinoma
Esophageal adenocarcinoma
Lung squamous cell carcinoma
Lung adenocarcinoma
Head and neck carcinoma
Bladder carcinoma (superficial)
Bladder carcinoma (infiltrating)
Leukemia
79
211
18
5
83
42
92
35
40
76
Expression of GAGE-1, -2, and -8a
Expression of GAGE-3, -4, -5, -6, and -7Ba
1
1
1
2
2
1
2
2
% of samples
expressing GAGE-1, -2, and -8 and/or
GAGE-3, -4, -5, -6, and -7B
22
79
7
1
28
13
21
1
8
0
1
8
1
0
4
2
3
0
2
0
10
26
1
0
7
6
5
0
6
3
46
98
9
4
44
21
63
34
24
73
42
54
50
20
47
50
31
3
40
4
a
See Fig. 1 for the sequence and the location of the primers used for RT-PCR analysis. A more sensitive RT-PCR protocol explains the higher percentage of tumors expressing
GAGE genes with respect to the data reported previously (6).
Fig. 9. Lysis by CTL 22/23 of autologous EBV-B cells incubated with peptide
YYWPRPRRY. MZ2-EBV cells were 51Cr-labeled and incubated with 22/23 CTLs at an
E:T ratio of 5:1 in the presence of the peptide at the indicated concentrations. Chromium
release was measured after 4 h.
supports this hypothesis (34 –36).6 Genes such as MAGE-B3 and
MAGE-B4, which are poorly induced or not induced at all by DAC,
are not expressed in tumor cells (37). The demethylation hypothesis
predicts that demethylated tumors ought to express frequently several
MAGE-type genes. This was observed within the MAGE and GAGE
families, whose members are frequently expressed together by the
same tumors.7 We also frequently observed that the same tumors
expresses genes belonging to different families of MAGE-type genes.7
However, the coexpression is not absolute and certain tumors express
only one or a few MAGE-type genes. Demethylation is also likely to
account for expression in testis, as a decrease in the level of DNA
methylation was observed during spermatogenesis (38, 39). Undermethylation was also observed in placenta (40). In testis, it is not
known whether the different MAGE-type genes are activated at the
same stage of spermatogenesis. This question could be addressed by
in situ hybridization or by replacing these genes by reporters in
transgenic mice.
Chen et al. (10) reported that GAGE-7 was expressed in all of
the analyzed the tumorigenic and nontumorigenic LNCaP sublines,
whereas expression of PAGE-1 was increased 5-fold in the metastatic cell sublines with respect to the nontumorigenic LNCaP. In
addition, these authors reported that neither GAGE-7 nor PAGE-1
6
7
show regulation by androgens in the prostate cancer cell lines.
These analyses of expression were done essentially by Northern
blot, using the PAGE-1 and GAGE-7 cDNAs as probes. However,
given its high homology with the other GAGE genes, the GAGE-7
probe was probably not specific for GAGE-7 but hybridized with
mRNA from all of the GAGE genes. Thus, we believe that the
“GAGE-7 expression” reported by these authors probably corresponds to the expression of one or several undetermined members
of the GAGE family. The same may also be true for the PAGE-1
probe, which contained blocks of sequence with .85% identity
with GAGE sequences.
Besides their expression patterns, most of the MAGE-type genes
share a common location on the X chromosome. The MAGE genes are
clustered in three loci: Xq28 for the MAGE-A family (30), Xp21.3 for
the MAGE-B family (41), and Xq27 for MAGE-C1 (42); the GAGE
genes are located in Xp11.2-p11.4, the SSX genes map to Xp11.2 (43);
NY-ESO-1 maps to Xq28 (8); and P1A has also been localized on the
X chromosome (P. Chomez, personal communication). It is unclear at
this stage whether there is a functional reason for the location of all
these genes on the X chromosome.
Hybridization of Southern blots made with DNA from different
eukaryotic species (including six mammalian species) did not reveal
sequences homologous with GAGE genes, except in the monkey. This
indicates that the GAGE genes are poorly conserved, even within
mammals. This feature is shared with gene P1A, which appears to be
devoid of a human homologue,8 and with the MAGE genes, which
share only ;60% identity with their mouse homologues with respect
to the coding region (28, 37, 44). This rapid evolution suggests that
these genes have undergone species-specific adaptive selection. Such
a selection has been demonstrated for genes involved in intergenomic
conflicts as occur between host and pathogen. Another group of genes
exhibiting a high level of sequence divergence includes those transcribed in reproductive tissues. For example, the Y chromosomeencoded SRY sex determination gene encodes a protein that contains
domains that evolve rapidly (45, 46). The X chromosome-encoded
Pem homeobox gene, which is selectively expressed in placenta,
testis, epididymis, and ovary, has also been shown to undergo high
rates of sequence divergence (47).
GAGE-1 has an additional exon (exon 49) with respect to the other
GAGE genes. In the other genes, exon 49 is skipped because it has
been disrupted by the insertion of a LINE retroposon. This suggests a
scenario in which a GAGE ancestor gene has been duplicated before
the insertion of a LINE element in one of the two resulting copies.
GAGE-2– 8 would then be generated through duplication of the disrupted copy. Because exon 49 encodes the 29 COOH-terminal resi-
V. Martelange, unpublished results.
F. Brasseur, personal communication.
8
O. De Backer and B. Lethé, unpublished results.
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THE GAGE GENE FAMILY
dues of the putative protein, its skipping leads to their substitution by
7 unrelated amino acids encoded by exon 5. Because nothing is
presently known about the function of the GAGE proteins, the effects
of this substitution are obscure. Exon skipping associated with LINE
insertions has been observed in the distrophin gene (48, 49) and in the
sodium channel gene Scn8a (50). Two possible causes have been
proposed to explain this skipping. The two reported insertions in the
dystrophin gene were 0.6 and 2 kb long, increasing exon length
beyond the usual limit of 400 bp, which is thought to interfere with the
exon definition step of splicing (51). Such a size effect could explain
exon skipping in the GAGE genes, in which the LINE insertion
resulted in an exon length of 707 bp. The insertion in Scn8a resulted
in an exon of 300 bp, which is within the normal range. Exon skipping
was in this case ascribed to the poly(U) sequence of the LINE element
because insertion of polypyrimidine tracts is known to inhibit splicing
(52).
Two antigenic peptides derived from GAGE proteins have been
identified. One derives from proteins GAGE-1, -2, and -8 and is
presented by HLA-Cw6. The other derives from GAGE-3, -4, -5, -6,
and -7B and is presented by HLA-A29, an allele that is expressed by
only 6% of Caucasians. Patients eligible for anti-GAGE immunotherapy can be identified by HLA typing and by testing by RT-PCR the
expression of the two groups of GAGE genes in their tumor samples.
In addition to the two identified antigenic peptides, the GAGE proteins are likely to comprise other peptides combined with various
class I molecules to form antigens. Their identification ought to
increase the fraction of patients who are eligible for anti-GAGE
immunotherapy.
ACKNOWLEDGMENTS
The technical support of Madeleine Swinarska is gratefully acknowledged.
We thank Simon Mapp for help with manuscript preparation.
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Characterization of the GAGE Genes That Are Expressed in
Various Human Cancers and in Normal Testis
Olivier De Backer, Karen C. Arden, Mauro Boretti, et al.
Cancer Res 1999;59:3157-3165.
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