From the Department of Clinical Molecular Biology,
Faculty of Medicine, Kyoto University, 54 Shogoin Kawaharacho,
Sakyo-ku, Kyoto 606-8507, Japan and the § Laboratory of
Intracellular Proteolysis, School of Biomedical Sciences, University of
Nottingham Medical School, Queens Medical Centre,
Nottingham NG7 2UH, United Kingdom
Received for publication, June 19, 2002, and in revised form, January 13, 2003
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ABSTRACT |
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Hepatocellular carcinoma ranks among the most
common malignancies in Southeast Asia and South Africa. Although there
are many modalities of treatment, the recurrence and metastasis rates
are high, and the prognosis is unsatisfactory. Gankyrin, a recently found oncoprotein, is a promising target for drug therapy because it is
overexpressed in all studied hepatocellular carcinomas. Gankyrin
contains six ankyrin repeats and interacts with Rb, Cdk4, and the S6
ATPase of the 26 S proteasome. In this study, a yeast two-hybrid screen
with gankyrin has identified MAGE-A4 as another interacting protein.
The interaction, mediated by the C-terminal half of MAGE-A4, was
reproduced in mammalian cells. The interaction was specific to MAGE-A4,
because other MAGE family proteins structurally similar to MAGE-A4,
i.e. MAGE-A1, MAGE-A2, and MAGE-A12, did not bind to
gankyrin. MAGE-A4 partially suppressed both anchorage-independent growth in vitro and tumor formation in athymic mice of
gankyrin-overexpressing cells. The ability of mutant MAGE-A4 to
interact with gankyrin correlated with the ability to suppress the
anchorage-independent growth. These results demonstrate that MAGE-A4
binds to gankyrin and suppresses its oncogenic activity. So far, the
major focus of studies on the MAGE proteins has been on their potential
for cancer immunotherapy. Our results may also shed light on novel functions for MAGE-A proteins.
Gankyrin (gann ankyrin repeat protein, also known as PSMD10 and
p28) is an oncoprotein, the expression of which is increased (1, 2)
in hepatocellular carcinomas
(HCCs).1 Gankyrin consists of
six ankyrin repeats and a 38-amino acid N-terminal extension and binds
to the retinoblastoma tumor suppressor protein (Rb), the S6 ATPase
subunit of the 26 S proteasome (PSMC4, RPT3, TBP7), and
cyclin-dependent kinase 4 (Cdk4) (1, 3, 4). Overexpression
of gankyrin increases both the phosphorylation and degradation of Rb
in vivo and oncogenically transforms NIH/3T3 cells. Gankyrin
binds to Cdk4 and counteracts the inhibitory function of the tumor
suppressors p16INK4A and p18INK4C (4). In a
rodent model of hepatocarcinogenesis, gankyrin is overexpressed from
the earliest stage of tumor development (5). These findings suggest
that gankyrin is a major player in cell cycle control and tumorigenesis
in HCCs.
The MAGE (melanoma antigen) genes were initially
identified because they encode tumor antigens that can be recognized by
cytolytic T lymphocytes derived from the blood lymphocytes of cancer
patients (6). The MAGE gene family is composed of more than
25 genes in humans and are classified as type I MAGE genes
(including MAGE-A, MAGE-B, and MAGE-C
genes) and type II MAGE genes, which include those that
reside outside of the MAGE-A, MAGE-B, and
MAGE-C genomic clusters (7, 8). The MAGE-A
subfamily comprises 12 genes (MAGE-A1 to
MAGE-A12), and is expressed in various types of
tumors but not in normal adult tissues, except for testis and placenta. The MAGE-A antigens are of particular interest for antitumor
immunotherapy because they are strictly tumor specific and are shared
by many tumors. Despite the isolation of growing numbers of
MAGE genes, their function in normal tissues remains mostly unknown.
To further characterize the molecular mechanism underlying the
oncogenic activity of gankyrin and facilitate development of a
therapeutic agent against HCCs, we have used a yeast two-hybrid screen
to identify further gankyrin interactions. We report here that MAGE-A4
binds to human gankyrin and suppresses its oncogenicity.
Yeast Two-hybrid Assay--
A full-length human gankyrin
cDNA was cloned into pAS2-1 vector (BD Biosciences) and
co-transformed into Y190 yeast cells with a placenta or a U-2 OS
cDNA library in pACT2 vector (BD Biosciences). Yeast clones
containing interacting proteins were identified by growth on media
lacking tryptophan, leucine, and histidine, followed by assaying for
Plasmids--
FLAG-tagged MAGE-A4, HA-tagged MAGE-A4, and
HA-tagged gankyrin cDNAs were cloned into the eukaryotic expression
vectors pMKit-neo, pMKit-hygro, and pCMV4. pEGFP-C1 vector (BD
Biosciences) was used to express EGFP-S6 and EGFP-MAGE-A4 fusion
proteins. For conditional expression of FLAG-MAGE-A4, we used the
tetracycline-regulated system as described (9). The pMACS4-IRES vector
(Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) was used to express
MAGE-A4 or C-terminal 107 or 55 amino acids of MAGE-A4 with
truncated human CD4. The CD4-expressing transfectants were enriched by
using the MACSelect 4 system (Miltenyi Biotec GmbH).
Cell Culture--
Mouse NIH/3T3 embryonal fibroblasts and its
derivatives, monkey COS-7 kidney cells, human U-2 OS osteosarcoma
cells, Huh-7 hepatoma cells, PLC/PRF/5 hepatoma cells, and HEK293 human
embryonal kidney cells and its derivatives were cultured in Dulbecco's
modified Eagle medium supplemented with 10% calf or fetal calf serum
at 37 °C in a humidified atmosphere of 5% CO2 in air.
Transfection was performed by the calcium phosphate method. pl16-GK-6
cells were generated from NIH/3T3 cells so that they constitutively expressed HA-tagged human gankyrin and the tetracycline activator, rtTA
(9). In the presence of 2 µg/ml doxycycline (Dox) in culture medium,
they expressed FLAG-tagged MAGE-A4. GK-S25 cells were derived from
NIH/3T3 cells and constitutively expressed gankyrin. To assess the
anchorage dependence of growth in vitro, 5 × 102 cells were plated in 0.3% agarose on top of 0.6%
agarose in the presence or absence of Dox in a 35-mm dish. Four weeks
later, the number of colonies (>20 cells) was counted microscopically. Statistical differences between sample means were calculated by analysis of variance, followed by an unpaired Student's t test.
Western Blot Analysis and Immunoprecipitation--
Western blot
analysis and immunoprecipitation were performed as described previously
(1, 9). Antibodies used were mouse monoclonal anti-HA antibody (Roche
Diagnostics), anti-FLAG antibody (Sigma), anti-GFP antibody (BD
Biosciences), anti-actin antibody (Chemicon International, Inc.,
Temecula, CA), rabbit polyclonal anti-gankyrin antibody (Santa Cruz
Biotechnology), anti-MAGE antibody (FL-309; Santa Cruz Biotechnology),
horseradish peroxidase-conjugated goat anti-mouse antibody (DAKO,
Kyoto, Japan), and anti-rabbit antibody (DAKO). To analyze interaction
between endogenous MAGE-A4 and endogenous gankyrin, U-2 OS cell lysates
were immunoprecipitated using rabbit anti-MAGE antibody (FL-309)
immobilized to the protein G support (Seize X mammalian
immunoprecipitation kit; Pierce). The antibody is broadly reactive with
all MAGE family members according to the manufacturer. The immobilized
antibody was incubated consecutively with four aliquots of cell lysates
(total of 1 ml), the first aliquot at 4 °C overnight and the others
at 20 °C sequentially for 1 h. The bound proteins were eluted
with the elution buffer supplied in the kit. The elution was repeated
three times, and each fraction was analyzed by Western blotting using
rabbit polyclonal anti-gankyrin antibody (Santa Cruz Biotechnology.).
Interaction of transiently expressed exogenous MAGE-A4 or its mutant
with exogenous gankyrin was examined by co-transfecting COS-7 or U-2 OS
cells with plasmids expressing FLAG-tagged MAGE-A4, FLAG-tagged C-terminal 107 amino acids of MAGE-A4, HA-tagged full-length gankyrin, FLAG alone, or HA alone in various combinations. Interaction of stably
transfected gankyrin with MAGE-A4 was examined by using pl16-GK-6
cells, which were derived from mouse NIH/3T3 cells, constitutively
expressed HA-tagged human gankyrin, and inducibly expressed FLAG-tagged
MAGE-A4 in the presence of Dox. To analyze the effects of MAGE-A4 on
binding of gankyrin to Rb, S6, and Cdk4, U-2 OS cells and 293T cells
were co-transfected with plasmids expressing FLAG-gankyrin, HA-tagged
Rb, EGFP-S6 fusion protein, HA-tagged Cdk4, HA-tagged MAGE-A4,
EGFP-MAGE-A4 fusion protein, EGFP alone, or FLAG alone in various combinations.
Immunofluorescence Staining--
Immunofluorescence staining was
performed essentially as described (9, 10). COS-7 cells were replated
on chamber slides after transfection, fixed with phosphate-buffered
saline containing 4% paraformaldehyde for 30 min, and then
rendered permeable with phosphate-buffered saline containing 0.2%
Triton X-100 for 30 min at room temperature. After blocking nonspecific
antibody-binding sites with bovine serum albumin, the cells were
incubated with mouse monoclonal anti-FLAG antibody (Sigma) and rabbit
polyclonal anti-HA antibody (
Berkeley Antibody, Richmond, CA). Then the
bound antibodies were reacted with FITC-linked anti-mouse and
TRITC-linked anti-rabbit IgGs (Amersham Biosciences) and observed under
a confocal laser microscope (Olympus, Tokyo, Japan).
Tumorigenicity Assay in Mice--
Twenty-four female BALB/c
Slc-nu/nu athymic mice (4 weeks old) were injected subcutaneously with
pl16-GK-6 cells or GK-S25 cells (8 × 106 cells each)
and divided into two groups. Twelve were given Dox (2 mg/ml) in the
drinking water ad libitum. Tumor size was calculated by
measuring the length, width, and thickness with calipers.
Identification of MAGE-A4 as a Gankyrin-interacting
Protein--
We performed yeast two-hybrid assays to search for
proteins capable of physically interacting with human gankyrin. Using
full-length gankyrin as a bait, we identified 18 clones of 3.5 × 106 yeast clones transformed with human placenta or U-2 OS
cell cDNA libraries. Each clone proliferated on media containing
the histidine inhibitor 3AT and was positive for
To confirm that gankyrin interacts with the isolated MAGE-A4 fragment
in mammalian cells, COS-7 cells were co-transfected with plasmid DNAs
expressing HA-tagged human gankyrin and FLAG-tagged C-terminal 107 amino acids of MAGE-A4. When cell lysates were immunoprecipitated with
an anti-FLAG antibody, HA-gankyrin was detected in them but not in
precipitates from cells co-transfected with parental FLAG vector and
HA-gankyrin (Fig. 1B, left panels). Reciprocally, truncated MAGE-A4 was detected in the anti-HA
immunoprecipitates from cells co-transfected with plasmids expressing
HA-gankyrin and FLAG-tagged truncated MAGE-A4 (Fig. 1B,
right panels).
We next examined whether full-length MAGE-A4 interacts with gankyrin in
COS-7 cells. As shown in Fig. 1C, HA-tagged gankyrin was
immunoprecipitated with anti-FLAG antibody in lysates from cells
co-transfected and expressing FLAG-tagged full-length MAGE-A4 and
HA-tagged gankyrin. Reciprocally, FLAG-tagged MAGE-A4 was immunoprecipitated with an anti-HA antibody. Similar interactions were
observed in U-2 OS cells as well (data not shown). Furthermore, in the
COS-7 cells expressing FLAG-MAGE-A4 and HA-gankyrin, double immunofluorescence staining showed that both proteins were co-localized in the cytoplasm (Fig. 1D).
COS-7 cells are well known to vastly over-express transfected proteins,
which makes it a problem to conclude that physiologically relevant
associations occur between such expressed proteins. Therefore, we
generated an NIH/3T3-derived clone (pl16-GK-6) in which HA-gankyrin was
stably overexpressed and FLAG-MAGE-A4 was inducibly expressed in the
presence of Dox. As shown in Fig.
2A, Dox increased the level of
FLAG-MAGE-A4 but not that of HA-gankyrin in pl16-GK-6 cells. Only in
the presence of Dox was FLAG-MAGE-A4 co-immunoprecipitated with
HA-gankyrin by the anti-HA antibody and HA-gankyrin
co-immunoprecipitated with FLAG-MAGE-A4 by anti-FLAG antibody (Fig.
2A, bottom two panels). About 40% of total MAGE-A4 and 5% of total gankyrin present in pl16-GK-6 cells after transfection were estimated to be in a complex (data not shown). Western blot analysis demonstrated that the level of
stably overexpressed gankyrin in pl16-GK-6 cells was less than those
observed in human U-2 OS osteosarcoma and Huh-7 hepatoma cells (Fig.
2B, top panels). The level of MAGE-A4
in the presence of Dox was probably less than that in U-2 OS
osteosarcoma and PLC/PRF/5 hepatoma cells (Fig. 2B,
bottom panels). Further confirmation is necessary
for this observation, because the anti-MAGE antibody used was reactive
with MAGE proteins other than MAGE-A4. We next examined the interaction
between endogenous gankyrin and MAGE-A4 in U-2 OS cells from which we
isolated the original truncated MAGE-A4 cDNA clones. As shown in
Fig. 2C, the endogenous gankyrin was co-immunoprecipitated
with MAGE proteins by anti-MAGE antibody. Taken together, these results
strongly suggest that gankyrin and MAGE-A4 interacts in human cancer
cells.
Because gankyrin binds to Rb, the S6 subunit of the 26 S proteasome,
and Cdk4 (1, 3, 4), the effects of overexpression of MAGE-A4 on the
binding of gankyrin to these proteins were examined. When U-2 OS cells
were co-transfected with plasmids expressing FLAG-tagged gankyrin and
HA-tagged Rb, HA-Rb was immunoprecipitated with anti-FLAG antibody as
expected (Fig. 3A).
Overexpression of EGFP-MAGE-A4 fusion protein by co-transfection did
not affect the amount of immunoprecipitated HA-Rb. When U-2 OS cells
were co-transfected with plasmids expressing FLAG-tagged gankyrin and EGFP-S6 fusion protein, EGFP-S6 was immunoprecipitated with anti-FLAG antibody as expected (Fig. 3B). Overexpression of HA-tagged
MAGE-A4 by co-transfection did not affect the amount of
immunoprecipitated EGFP-S6. Similarly, no effect of EGFP-MAGE-A4 was
observed on the binding of gankyrin to Cdk4 (data not shown). Similar
results were obtained with 293T cells (data not shown).
Specific Binding of C-terminal Portion of MAGE-A4 to Full-length
Gankyrin--
To characterize the interacting domains of gankyrin and
MAGE-A4, we made various deletion mutants and analyzed their
interactions in the yeast two-hybrid system. We demonstrated previously
that full-length is necessary for the interaction with Rb (1).
Similarly, no deletion mutant of gankyrin interacted with MAGE-A4,
indicating that all ankyrin repeats and the N-terminal extension are
necessary for the binding (Fig.
4A, and data not shown).
The MAGE-A4 clone originally isolated by the two-hybrid screen
contained only the C-terminal 107 amino acids (position 211 to 317, Fig. 1, A and B, and Fig. 4B). Further
N-terminal truncation up to residue 226 did not prevent the interaction
with gankyrin. By contrast, a truncation of 10 amino acids from the C
terminus of MAGE-A4 abolished its binding to gankyrin (Fig.
4B). The interaction was specific to MAGE-A4, because the
corresponding regions of other MAGE proteins, although structurally
quite similar to MAGE-A4, did not interact with gankyrin (Fig. 4,
C and D). These results indicate that the
C-terminal region of MAGE-A4 containing the HLA-A2-presented peptides
(11, 12) (GVYDGREHTV and YLEYRQVPV) specifically interacts with gankyrin.
Effects of MAGE-A4 on Tumorigenic Activity of Gankyrin--
To
investigate the biological effects of the interaction of gankyrin and
MAGE-A4, we used the NIH/3T3-derived pl16-GK-6 cells in which gankyrin
was stably overexpressed and MAGE-A4 expression could be induced with
Dox (Fig. 2A). These gankyrin-transformed cells formed
colonies in soft agar (Fig.
5A). When the cells were cultured in the presence of Dox, the numbers of colonies was decreased to 65% of those in the absence of Dox (Fig. 5B). When the
mouse NIH/3T3-derived GK-S25 cells stably overexpressing gankyrin were transfected with plasmids expressing the C-terminal 55 amino acids of
MAGE-A4 that did not bind to gankyrin (Fig. 4B), no decrease in the number of colonies were observed (Fig. 5C). By
contrast, the C-terminal 107 amino acids of the MAGE-A4 that binds to
gankyrin inhibited the colony formation, suggesting that the ability to bind to gankyrin correlates with the ability to suppress
anchorage-independent growth in vitro.
The anchorage-independent phenotype in cell culture has been closely
correlated with the ability of cells to form tumors in animals (13). We
therefore evaluated the effect of MAGE-A4 on tumor formation of
gankyrin-transformed cells in athymic nude mice. After being
subcutaneously inoculated with pl16-GK-6 cells, the animals were
divided into two groups, one of which was administered Dox in drinking
water. In the absence of Dox, all mice developed tumors 5 weeks after
the inoculation of pl16-GK-6 cells (Fig. 6). By contrast, the tumors appeared
later and grew slower in mice given Dox. Dox by itself showed no
suppressive effects on tumor formation by GK-S25 cells (data not
shown). Taken together, these results demonstrate that MAGE-A4 directly
binds to gankyrin and suppresses its tumorigenic activity.
Rb and p53 play critical roles in transducing a variety of growth
inhibitory signals to the cell cycle control machinery via distinct
mechanisms (14-16). The concurrent inactivation of these two pathways
occurs frequently in human cancers and argues that unscheduled entry
into the cell cycle and escape from cell cycle arrest/apoptosis are two
critical events that a cell cycle requires to become cancerous.
Gankyrin destabilizes Rb and is commonly overexpressed in HCCs (1, 2).
Gankyrin therefore plays an important role in hepatocarcinogenesis and
is a promising target for therapeutic drug development. Here, we
demonstrated that MAGE-A4 binds to gankyrin and suppresses its
oncogenic activity. The interaction was identified by the yeast
two-hybrid assay and reproduced in mammalian cells. Because endogenous
gankyrin is associated with endogenous MAGE protein(s) in human cancer
cells in which MAGE-A4 transcripts were detected, it is highly likely
that the interaction is physiologically relevant. MAGE-A4 is a member
of the MAGE family, a large group of proteins that contain a well
conserved ~200 amino acid region known as the MAGE homology domain
(7, 8). Although its general function is unknown, the MAGE homology
domain of necdin binds to E2F-1 (17). In the present study, the C
terminus of MAGE-A4, in addition to the C-terminal half of MAGE
homology domain, was found to be necessary for MAGE-A4 to bind to
gankyrin. This binding was specific for MAGE-A4, because similar amino
acid sequences from other MAGE-A family members did not bind to gankyrin.
Despite their normally restricted physiological expression,
the MAGE genes are expressed in a wide variety of cancer
cells. In the case of HCC, more than 60% of the carcinomas express
MAGE-A1 and/or MAGE-A3 transcripts, which have
been regarded as tumor-specific markers (18, 19). Expression of other
MAGE family members, including MAGE-A4, has also
been detected in HCCs (20). When overexpressed in the cytosol of cancer
cells, MAGE proteins are proteolytically processed, transported to the
endoplasmic reticulum, and then presented on the cell surface as
antigenic major histocompatibility complex-associated peptides (8).
Thus, attention has been focused on the potential of MAGE as a target
for cancer immunotherapy. The C-terminal region of MAGE-A4 that binds
to gankyrin contains decapeptides presented by HLA-A2 (11, 12). Whether
binding of gankyrin affects degradation of MAGE-A4 and/or the
presentation of antigenic peptide remain to be investigated.
The physiological roles played by the MAGE gene family are
unknown with a few exceptions (8). Necdin has an important role in
development and/or maintenance of discrete cells in the nervous system.
Overexpression of necdin causes cell cycle arrest through mechanisms
that may involve physical interactions with E2F-1 or p53 (17). NRAGE
(MAGE-D1), which binds to several proteins including the p75
neurotrophin receptor and inhibitor of apoptosis proteins (IAP), blocks
cell cycle progression and enhances apoptosis (21, 22). Magphinin
(MAGE-D4) is suggested as regulating cell proliferation during
gametogenesis, and its ectopic expression suppresses cell proliferation
(23). In contrast to NRAGE, necdin, and magphinin, which belong to the
type II MAGE genes, MAGE-A4 did not affect the
cell cycle progression, proliferation rate, nor apoptosis by itself.
2 However, MAGE-A4
showed anti-tumorigenic effects on gankyrin-overexpressing cells.
Because this effect was observed in vitro as well as
in vivo in athymic nude mice, it is cell autonomous and not
mediated by T cells.
In mammals, the execution of G1 Cdks, namely Cdk4, Cdk6,
and Cdk2, is essential for onset of the S phase (24). A major target for the G1 Cdks is Rb. Phosphorylation of Rb leads to
activation of the E2F-DP1 transcriptional factor complex that controls
the expression of genes essential for onset of the S phase (14). Although there appears to be cell type- and genotype-specific differences in the control of anchorage-dependent growth, a
recent study has demonstrated that the G1 Cdks and Cdc6
constitute major cell cycle targets for the regulation of the
G1-S transition by anchorage and oncogenic stimulation
(25). Gankyrin binds to Cdk4, evades inhibition by p16INK4A
(3, 4), increases phosphorylation and degradation of Rb, and transforms
cells to grow in an anchorage-independent manner (1). Because the
suppressive effect of MAGE-A4 on anchorage-independent growth
correlated with its binding to gankyrin, it is possibly mediated by
gankyrin and/or dependent on association with gankyrin. However, we
were unable to detect effects of MAGE-A4 on the degradation of
Rb.3 MAGE-A4 did not reduce
the binding of gankyrin to Rb, S6, or Cdk4. Molecules involved in the
anti-tumorigenic activity of MAGE-A4 are yet to be elucidated.
Recently, continued activity of a specific oncogene has been found to
be necessary to maintain the cancer phenotype in some cancer cells,
which suggests a possibility that suppression of gankyrin alone has a
therapeutic effect in HCCs (26). Further clarification of the
mechanisms underlying the effects of MAGE-A4 on gankyrin and
anchorage-independent growth will facilitate development of novel
therapeutics against HCCs and also shed light on normal physiological
functions of MAGE-A proteins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-galactosidase activity. The interaction was confirmed by
co-transformation into Y190 cells with pAS2-1-gankyrin and their
growth on the selection medium with 25 mM 3-aminotriazole (3AT). For analysis of interactions, full-length and various mutant cDNAs of gankyrin (GenBankTM accession number D83197)
and MAGE-A4 (GenBankTM accession number U10687) were
generated by the polymerase chain reaction and cloned into a pAS2-1 or
pACT2 vector, respectively. cDNAs corresponding to the C-terminal
regions of MAGE-A1 (amino acids 203-309, GenBankTM
accession number NM_004988), MAGE-A2 (amino acids 210-314,
GenBankTM accession number NM_005361), and MAGE-A12 (amino
acids 210-314, GenBankTM accession number XM_010079) were
also cloned into pACT2.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-galactosidase
staining (Fig. 1A, and data
not shown). DNA sequencing analysis of the rescued plasmids revealed
that two of them encoded the C-terminal 107 amino acids of MAGE-A4.
Consistent with our previous findings (3), the remaining 16 clones
encoded different C-terminal sequences of the S6 ATPase subunit of the
26 S proteasome. MAGE-A4 did not interact with the GAL4 DNA binding
domain alone (Fig. 1A), indicating that it interacted with
gankyrin.
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Fig. 1.
Identification of MAGE-A4 as a
gankyrin-binding protein. A, interaction in
yeasts. Yeast cells were co-transformed with the human full-length
gankyrin cDNA, p53 cDNA, or none (Gal4-BD) in pAS2-1 vector in
combination with the isolated partial MAGE-A4 cDNA, S6 cDNA,
SV40 T-antigen cDNA, or none (Gal4-AD) in pACT2 vector as
indicated. They were then plated onto media lacking tryptophan and
leucine (SD/ W
L) and media lacking
tryptophan, leucine, and histidine and supplemented with 3AT
(SD/
W
L
H+3AT). B, interaction of
isolated clones in COS-7 cells. Lysates were prepared after
co-transfection with vectors expressing HA-tagged gankyrin, FLAG-tagged
C-terminal 107 amino acids of MAGE-A4 (FLAG-MAGE
N), FLAG alone, and
HA alone as indicated. The lysates and those immunoprecipitated with
antibodies to FLAG or HA were analyzed by Western blotting using the
indicated antibodies. Arrowheads indicate mobilities of
specific bands. C, interaction of full-length MAGE-A4 in
COS-7 cells. Lysates were prepared after co-transfection with vectors
expressing HA-tagged gankyrin, FLAG-tagged full-length MAGE-A4, FLAG
alone, and HA alone as indicated, and analyzed as described for
panel B. D, co-localization of
gankyrin and MAGE-A4. COS-7 cells co-transfected with HA-gankyrin and
FLAG-MAGE-A4 were incubated with rabbit anti-HA and mouse anti-FLAG
antibodies, visualized with TRITC-linked anti-rabbit and FITC-linked
anti-mouse IgGs, and observed under confocal microscope. Note
cytoplasmic co-localization (yellow) of gankyrin
(red) and MAGE-A4 (green).
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Fig. 2.
Interaction of MAGE-A4 with gankyrin in
mammalian cells. A, interaction of inducibly
overexpressed MAGE-A4 with stably overexpressed human gankyrin in mouse
cells. pl16-GK-6 cells were cultured in the presence (+) or absence
( ) of Dox, and lysates were prepared from them. 5% of the input
lysates (top two panels) and one-quarter of the immune
complexes precipitated by anti-HA antibody or anti-FLAG antibody
(bottom two panels) were analyzed by
Western blotting using anti-HA antibody or anti-FLAG antibody as
indicated. Arrowheads indicate mobilities of the specific
bands. B, levels of overexpressed HA-gankyrin and
FLAG-MAGE-A4 in pl16-GK-6 cells in the presence of Dox compared with
those in human cancer cells. Cell lysates were prepared from pl16-GK-6
cells, U-2 OS cells, Huh-7 cells, and PLC cells as indicated and
analyzed by Western blotting using anti-gankyrin antibody (top
left panel), anti-HA antibody (top right
panel), anti-MAGE antibody (bottom
left panel), and anti-FLAG antibody
(bottom right panel). Mobilities of
HA-gankyrin, endogenous gankyrin, FLAG-MAGE-A4, and endogenous MAGE
proteins are indicated on the left. C,
interaction of endogenous MAGE proteins with endogenous gankyrin.
Lysates from U-2 OS cells were immunoprecipitated with anti-MAGE
antibody immobilized to the protein G support. 2% of input, eluate
fractions 1, 2, and 3, and flow-through were analyzed by Western
blotting using anti-gankyrin antibody. The mobility of endogenous
gankyrin is indicated on the right.
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Fig. 3.
Effects of MAGE-A4 on binding of Rb and S6 to
gankyrin. A, no effect on binding of Rb to gankyrin.
U-2 OS cells were co-transfected with plasmids expressing FLAG-gankyrin
and HA-Rb together with increasing amount of plasmids expressing the
EGFP-MAGE-A4 fusion protein as indicated. Total amount of plasmids was
adjusted to 5 µg/60-mm dish with empty vectors expressing FLAG alone
or EGFP alone. 48 h after transfection, lysates were prepared from
them. One-quarter of immune complexes precipitated by anti-FLAG
antibody (top panel) and 10% of the input
lysates (bottom four panels) were
analyzed by Western blotting using antibodies as indicated.
B, no effect on binding of S6 to gankyrin. U-2 OS cells were
co-transfected with plasmids expressing FLAG-gankyrin and EGFP-S6
fusion protein together with increasing amount of plasmids expressing
the HA-MAGE-A4 fusion protein as indicated. Total amount of plasmids
was adjusted to 5 µg/60-mm dish with empty vectors expressing FLAG
alone or HA alone. 48 h later, lysates were prepared from them.
One-quarter of immune complexes precipitated by anti-FLAG antibody
(top panel) and 10% of the input lysates
(bottom four panels) were analyzed by
Western blotting using antibodies as indicated.
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Fig. 4.
Specific interaction of C-terminal part of
MAGE-A4 with gankyrin. A, interaction between
full-length MAGE-A4 and various gankyrin deletion mutants analyzed by
the yeast two-hybrid assay. Top row, full-length gankyrin.
Locations of ankyrin repeats are shown as ANK1 to ANK6. Plus
(+) and minus ( ) signs indicate the presence
and absence of the interaction with MAGE-A4, respectively.
B, interaction between full-length gankyrin and various
MAGE-A4 deletion mutants analyzed by the yeast two-hybrid assay.
Top row, full-length MAGE-A4. Striped
box, MAGE homology domain. The numbers on
top of the boxes indicate
the amino acid positions, and the numbers inside
the boxes indicate the length of each mutant.
Plus (+) and minus (
) signs indicate
the presence and absence of the interaction with gankyrin,
respectively. C, comparison of the C-terminal amino acid
sequence of MAGE-A4 to those of MAGE-A1, MAGE-A2, and MAGE-A12. Amino
acid residues identical to MAGE-A4 are highlighted, and the regions
that give rise to the MAGE-A4 peptides that are presented on the cell
surface (13, 14) are indicated by lines. D, interaction
between full-length gankyrin and C-terminal regions of various MAGE-A
proteins shown in panel C analyzed by the yeast
two-hybrid assay. Yeast cells were co-transformed with the indicated
plasmids and plated onto media lacking tryptophan and leucine
(SD/
W
L) and media lacking tryptophan,
leucine, and histidine and supplemented with 3AT
(SD/
W
L
H+3AT). Note that only the C-terminal
potion of MAGE-A4 interacted with gankyrin.
View larger version (29K):
[in a new window]
Fig. 5.
Effects of MAGE-A4 and its mutants on
anchorage-independent growth of gankyrin-transformed cells in
vitro. A and B, effects of
MAGE-A4 on colony formation in soft agar of pl16-GK-6 cells stably
overexpressing gankyrin. Dox induces the expression of FLAG-MAGE-A4 in
them. pl16-GK-6 cells were cultured in soft agar in the absence ( ) or
presence (+) of Dox, photographed (A), and the numbers of
colonies were counted under a microscope (B). Shown are
mean ± S.E. of triplicates. *, p < 0.01 versus Dox (
) group. C, effects of mutant
MAGE-A4 on colony formation in soft agar of GK-S25 cells overexpressing
gankyrin. GK-S25 cells were transfected with bicistronic vectors
expressing CD4 in combination with full-length MAGE-A4 (FL),
C-terminal 107 amino acids of MAGE-A4 (107aa), C-terminal 55 amino acids of MAGE-A4 (55aa) or no protein
(mock). CD4-expressing cells were enriched and analyzed for
colony formation. Results are mean ± S.E. of triplicates. *,
p < 0.05; **, p < 0.01 versus mock group.
View larger version (65K):
[in a new window]
Fig. 6.
Effects of MAGE-A4 on anchorage-independent
growth of gankyrin-transformed cells in vivo.
nu/nu mice were subcutaneously injected with pl16-GK-6 cells stably
overexpressing gankyrin. Then, they were divided into two groups, one
of which was given Dox in drinking water. A, photos of mice
given Dox (+) or vehicle alone ( ) for 6 weeks. Arrowhead
indicates the tumor. B, tumor volumes (mean ± S.E.,
n = 6 each) in mice given Dox (
) or vehicle alone
(
).*, p < 0.05 versus Dox (
)
group.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
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We thank Dr. Manabu Sugai for helpful suggestions.
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FOOTNOTES |
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* This work was partially supported by Grants-in-aid from the Ministry of Science, Culture, Sports, and Education of Japan, the Yasuda Anti-Cancer Foundation, and the Smoking Research Foundation of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 81-75-751-3751; Fax: 81-75-751-3750; E-mail: jfujita@virus.kyoto-u.ac.jp.
Published, JBC Papers in Press, January 13, 2003, DOI 10.1074/jbc.M206104200
2 T. Nagao and J. Fujita, unpublished observation.
3 T. Nagao, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: HCC, hepatocellular carcinoma; Rb, retinoblastoma tumor suppressor protein; Cdk, cyclin-dependent kinase; MAGE, melanoma antigen; 3AT, 3-aminotriazole; HA, hemagglutinin; Dox, doxycycline; EGFP, enhanced green fluorescent protein; FITC, fluorescein isothiocyanate; TRITC, tetramethylrhodamine isothiocyanate.
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