[Cancer Research 61, 854-858, February 1, 2001]
© 2001 American Association for Cancer Research
A Mammalian Two-Hybrid System for Adenomatous Polyposis Coli-Mutated Colon Cancer Therapeutics1
Kenichi Wakita,
Osamu Tetsu and
Frank McCormick2
Cancer Research Institute, University of California, San Francisco, School of Medicine, San Francisco, California 94143-0128 [K. W., O. T., F. M.], and Daiichi Pharmaceutical Co. Ltd., New Product Research Laboratories III, Tokyo 134-8630, Japan [K. W.]
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ABSTRACT
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Colon cancer cells frequently lose expression of the tumor suppressor
adenomatous polyposis coli (APC). As result, ß-catenin accumulates
and activates transcription of Tcf-responsive genes. Here we describe a
novel mammalian two-hybrid system that selectively kills APC-mutated
cells. This system consists of GAL4/ß-catenin, VP16/Tcf4, and a gene
that is transcribed when GAL4 and VP16 associate. In APC-mutated human
colon cancer cells, such as SW480, GAL4/ß-catenin accumulates, and in
the presence of VP16/Tcf4, induces high levels of expression of the
reporter gene. Expression of wild-type APC reduced GAL4/ß-catenin and
intact ß-catenin levels and inhibited reporter gene expression. In
colon cancer cells such as SW48 that have wild-type APC,
GAL4/ß-catenin was degraded, and expression levels of the output gene
were low. Replacement of the reporter gene with a suicide gene resulted
in selective killing of SW480 cells. This system may be applicable for
broader use of gene therapy by targeting diseases that involve protein
degradation.
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Introduction
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Mutations in the
APC3
tumor suppressor gene occur in familial adenomatous polyposis
patients (1
, 2)
, and in sporadic colorectal cancer
(3)
. APC is necessary for complex formation between
GSK-3ß and ß-catenin (4)
and degradation of
cytoplasmic ß-catenin via the ubiquitin-proteasome pathway (5
, 6) . Functional loss of APC by genetic mutation in colorectal
cancer, therefore, causes accumulation of ß-catenin (7)
.
The fact that colorectal cancer cells that retain wild-type APC have
mutations in the GSK-3ß phosphorylation site of ß-catenin, which
also stabilizes ß-catenin protein in the cells (8)
,
implies that stabilization of ß-catenin is one of the major
consequences of APC loss. Moreover, in mouse models, both APC-mutant
mice (9)
and ß-catenin-mutated mice (10)
form intestinal polyps. In humans, mutations in APC are observed at
early stages of colon cancer development even before ras or p53
mutation (11)
.
ß-Catenin that accumulates in colorectal cancer cells forms complexes
with transcription factor Tcf4 (12
, 13)
and facilitates
expression of Tcf/lef-1-dependent gene expression such as cyclin
D1 (14)
, c-myc (15)
, and
peroxisome proliferator-activated receptor 
(16)
.
The yeast two-hybrid system has been used to study protein-protein
interactions or to identify interacting partners for many proteins
(17)
. This system consists of basically three components.
The first component is a fusion protein that contains the DNA binding
domain of a transcription factor. The second component is a fusion
protein that contains the transcription activation domain of a
transcription factor. The third component is a gene expression
construct that has the consensus sequence for the first component and
is followed by the transcription activation site, such as TATA box, for
the second component. The yeast GAL4 DNA binding domain and the herpes
simplex virus VP16 transcription activation domain are widely used in
this system. A few years after this yeast two-hybrid system was
developed, it was modified for mammalian cells (18)
. In
this report, we describe a novel application of the mammalian
two-hybrid system. We used ß-catenin and Tcf4 as binding partners to
drive expression of p53 and showed that this system kills APC-mutant
cells selectively (Fig. 1)
.
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Fig. 1. The ß-catenin/Tcf4 mammalian two-hybrid system. When
cells are transfected with the plasmids encoding GAL4/WT-ß-catenin,
VP-16/FL-Tcf4, and GAL4-responsive element-driven suicide gene, suicide
gene expression occurs only in APC-mutated cells.
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Materials and Methods
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Cells.
Human cancer cells lines were obtained from The American Type Culture
Collection (Rockville, MD). Human colon cancer cell lines SW480 and
SW48 were maintained in L-15 medium (Life Technologies, Inc.,
Gaithersburg, MD) supplemented with 10% heat-inactivated FBS (Life
Technologies, Inc.) in a 0.8% CO2 incubator at 37°C. The
human osteosarcoma cell line U2-OS and embryonic kidney cell line 293
were maintained in RPMI 1640 (Life Technologies, Inc.) supplemented
with 10% FBS in a 5% CO2 incubator at 37°C.
Plasmids.
The pBIND, pBIND/Id, pACT, and pG5Luc plasmids were purchased from
Promega Corp. (Madison, WI). pp53-EGFP was purchased from Clontech
(Palo Alto, CA). The GAL4 fusion ß-catenin expression plasmid,
pBIND/ß-catenin, and VP16 fusion Tcf4 expression plasmids, pACT/Tcf4,
were prepared as follows. The human ß-catenin cDNA with
BamHI linker was prepared with PCR and cloned into
BamHI site of pBIND. The human Tcf4 cDNA with
BamHI linker was prepared with PCR and cloned into
BamHI site of pACT. The pcDNA3/p53-EGFP was prepared by
generating KpnI and XbaI fragments of the
pp53-EGFP, into the KpnI/XbaI site of pcDNA3.1
(Invitrogen, Carlsbad, CA). The pG5/p53-EGFP plasmid was constructed by
inserting the p53-EGFP cDNA fragment of pp53-EGFP to the
BglII and HindIII sites of pG5Luc. The pG5/EGFP
plasmid was constructed by deleting p53 cDNA from the pG5/p53-EGFP
plasmid. The p53-Luc, pAP-1-Luc, and pCRE-Luc plasmids were purchased
from Stratagene (La Jolla, CA).
Immunostaining.
Cells grown to 50–80% confluency were trypsinized and washed twice
with PBS, and lysed in TE buffer (10 mM Tris, 1
mM EDTA) containing protease inhibitors (Complete Mini;
Boehringer Mannheim). Cell lysate samples were separated with 4–20%
acrylamide linear gradient SDS-PAGE gel (Bio-Rad, Cambridge, MA),
transferred onto nitrocellulose membrane, incubated with primary
antibodies and horseradish peroxidase-conjugated secondary antibodies,
and detected with an enhanced chemiluminescence kit (ECL; Amersham
Pharmacia Biotech, Uppsala, Sweden). Anti-ß-catenin (sc-7199), GAL4
(sc-577), VP16 (sc-7546), and p53 (sc-126) antibodies were purchased
from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-APC (OP44)
antibody was from Calbiochem Novabiochem (San Diego, CA). Anti-Tcf4
antibody was obtained from Upstate Biotechnology, Inc. (Lake Placid,
NY). Horseradish-conjugated antimouse or antirabbit IgG antibodies were
from Amersham Pharmacia Biotech.
Two-Hybrid Assay.
The pBIND/ß-catenin, pACT/Tcf4 and pG5/luc plasmids were
cotransfected to cells using TransFast (Promega) according to the
manufacturer’s recommended protocol. Briefly, cells were seeded in
24-well plates at a density of 5 x 104
cells/well and cultured for 24 h. Total plasmids (1 µg/well)
were transfected and cultured for another 24 h. Finally,
luciferase activity was measured with a commercially available kit
(Promega). The transfection efficiency was normalized by
Renilla luciferase activity, which was simultaneously
expressed from the pBIND plasmids.
Fluorescence-activated Cell Sorter Analysis.
The pG5/p53-EGFP or pG5/EGFP plasmid was cotransfected with
pBIND/WT-ß-catenin and pACT/FL-Tcf4 plasmids to SW480 cells. After
48–72 h incubation, cells were trypsinized and washed twice with PBS.
Cells were suspended in PBS containing 10 µM propidium
iodide and analyzed on a FACScan flow cytometer (Becton Dickinson, San
Jose, CA). Cell viability profile of EGFP-positive cells was analyzed
by the uptake of propidium iodide.
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Results
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Functional Expression of Two-Hybrid Proteins in a Colon Cancer
Cell Line
We constructed four fusion protein expression-plasmids,
pBIND/WT-ß-catenin, pBIND/MT-ß-catenin, pACT/FL-Tcf4, and
pACT/DN-Tcf4, which express GAL4/WT-ß-catenin, GAL4/MT-ß-catenin,
VP16/FL-Tcf4, and VP16/DN-Tcf4, respectively (Fig. 2A)
. GAL4/WT-ß-catenin is a fusion protein that consists of the DNA
binding domain of yeast GAL4 protein and full-length human ß-catenin.
GAL4/MT-ß-catenin has a truncated form of ß-catenin, which lacks
the GSK-3ß recognition site. VP16/FL-Tcf4 is a fusion protein that
consists of the transcription activation domain of herpes simplex virus
VP16 and full-length human Tcf4. VP16/DN-Tcf4 has a truncated form of
Tcf4, which lacks the ß-catenin binding site. We transfected
pBIND/WT-ß-catenin, pBIND/MT-ß-catenin, pACT/FL-Tcf4, or
pACT/DN-Tcf4 to SW480 cells and detected those fusion protein
expression levels using immunoblotting. Expressed GAL4/ß-catenin
fusion proteins were recognized by both anti-GAL4 and anti-ß-catenin
antibodies, as well as VP16/Tcf4 proteins, which were recognized by
both anti-VP16 and anti-Tcf4 antibodies (Fig. 2B)
. The
cotransfection of pBIND/WT-ß-catenin, pACT/FL-Tcf4, and pG5/Luc led
to an expression of the output gene, firefly luciferase (Fig. 2C)
. This output gene expression level was strictly
controlled by the expression of the GAL4/ß-catenin and VP16/FL-Tcf4
combination, because the replacement of pBIND/WT-ß-catenin plasmid to
pBIND/Id or pACT/FL-Tcf4 plasmid to pACT/DN-Tcf4 failed to increase
firefly luciferase activity.
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Fig. 2. A, structure of the two-hybrid components. The
pBIND/WT-ß-catenin plasmid is designed to produce chimera protein
that consists of yeast GAL4 DNA binding domain and wild-type
ß-catenin. The pBIND/MT-ß-catenin plasmid produces a chimeric
protein consisting of a GAL4 DNA binding domain and a GSK-3ß
phosphorylation site (amino acids 29-48)-deleted ß-catenin. The
pACT/FL-Tcf4 plasmid produces a chimeric protein that consists of
herpes simplex virus VP16 transcription activation domain and
full-length Tcf4. The pACT/DN-Tcf4 plasmid produces chimeric protein
that consists of VP16 transcription activation domain and ß-catenin
binding site (amino acids 2-53)-deleted Tcf4. B, expression
of GAL4/ß-catenin and VP16/Tcf4 in SW480 Cells. Cells were
transfected with pBIND/ß-catenin or pACT/Tcf4 plasmid and cultured
for 24 h. Then expression levels of fusion proteins were detected
by Western blot. C, specificity of the two-hybrid system.
SW480 cells were transfected with various combinations of GAL4 fusion
protein, VP16 fusion protein, and pG5/luc plasmids and cultured for
24 h. The luciferase activity of each sample was measured by a
luminometer. WT, wild type; MT, mutated;
FL, full length; DN, dominant
negative.
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Regulation of ß-Catenin/Tcf4 Two-Hybrid System by APC.
We next examined whether the GAL4/WT-ß-catenin protein was degradable
in an APC-dependent manner. Wild-type APC expression plasmid was
cotransfected with pBIND/WT or MT-ß-catenin, and pACT/FL-Tcf4 and
pG5/Luc activity was measured. The addition of APC in the
pBIND/WT-ß-catenin system resulted in a drastic decrease of firefly
luciferase activity, whereas pBIND/MT-ß-catenin system was not
affected by APC expression (Fig. 3A)
. Next, the protein levels of GAL4/ß-catenin and VP16/Tcf4 were
detected in the same condition as reporter gene assay. As a positive
control, pcDNA3/myc-ß-catenin was cotransfected with the pcDNA/APC
plasmid. In the presence of APC protein, both GAL4/WT-ß-catenin and
myc-tagged WT-ß-catenin protein levels were reduced, whereas neither
GAL4/MT-ß-catenin nor myc-tagged MT-ß-catenin protein levels were
changed (Fig. 3B)
. Neither VP16/Tcf4 nor intact Tcf4 protein
levels were affected by APC expression.
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Fig. 3. A, APC-dependent expression of the two-hybrid
output gene in SW480 cells. SW480 cells were transfected with various
concentrations of the pcDNA/APC plasmid in the presence of pBIND/WT or
MT-ß-catenin plasmid, pACT/FL-Tcf4 plasmid, and pG5/luc plasmid.
After 24 h in culture, cells were lysed, and luciferase activity
was measured. B, detection of two-hybrid component proteins
in SW48 cells. SW480 cells were transfected with various concentrations
of the pcDNA/APC plasmid in the presence of pBIND/WT or MT-ß-catenin
plasmid, pACT/FL-Tcf4 plasmid, and pG5/luc plasmid. After 24 h in
culture, cells were lysed, and two-hybrid component proteins were
detected by Western blotting. WT, wild type; MT,
mutated.
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Regulation of the ß-Catenin/Tcf4 Two-Hybrid System in Various
Cell Types.
To confirm these results, we performed the two-hybrid assay using human
osteosarcoma U-2OS and human embryonic kidney 293 cells, which express
wild-type APC. We examined the expression levels of cytoplasmic
ß-catenin and APC first (Fig. 4A)
. Both U-2OS and 293 showed low levels of cytoplasmic ß-catenin. These
two cell lines also showed an APC band at the expected molecular size.
These results indicated that the degradation mechanism of ß-catenin
was intact in these cell lines. SW480, in which APC is mutated, did not
show an APC band at the expected size. SW48, in which ß-catenin is
mutated, exhibited high levels of ß-catenin despite the presence of
wild-type APC. Fig. 4B
shows the expression levels of
GAL4/WT or MT-ß-catenin proteins in SW480 cells and SW48 cells. There
was a specific lower band observed only in SW48 cells that expressed
GAL4/WT-ß-catenin, which implied that GAL4/WT-ß-catenin but not
GAL4/MT-ß-catenin was degraded in the APC wild-type cell line. All of
the cell lines examined that have wild-type APC showed lower output
gene expression levels than SW480 cells (Fig. 4C)
. Moreover,
SW48 cells showed a similar amount of the output gene expression levels
to SW480 cells, when transfected with the two-hybrid system using
GAL4/MT-ß-catenin (data not shown). These results imply that APC
controls the output gene expression levels.
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Fig. 4. A, expression levels of cytoplasmic ß-catenin
and APC in human cell lines. SW480 cells, SW48 cells, U-2OS cells, and
293 cells (1.0 x 106 cells) were lysed with
TE buffer containing protease inhibitors (Complete Mini; Boehringer
Mannheim), and a soluble fraction was collected by centrifugation. Each
sample was loaded to 4–20% gradient acrylamide gels and transferred
to nitrocellulose membrane. Separated protein was detected with
anti-ß-catenin antibody or anti-APC antibody. B, detection
of GAL4 fusion protein degradation products. SW480 cells and SW48 cells
were transfected with pBIND/WT or MT-ß-catenin plasmid and cultured
for 24 h. Cells were lysed with SDS-PAGE sample buffer and loaded
to 4–20% gradient poly acrylamide gels. Separated protein was
transferred to nitrocellulose membrane, and GAL4 protein was detected
with anti-GAL4 antibody. C, selective output gene expression
of two-hybrid system dependent on the APC profile of the cell lines.
Human cell lines were transfected with various concentrations of
pBIND/WT-ß-catenin plasmid in the presence of pACT/FL-Tcf4 and
pG5/luc plasmids. The luciferase activity of each point was measured
after 24 h in culture.
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Application of the Mammalian Two-Hybrid System to in
Vitro Gene Therapy.
We next switched the output gene from luciferase to p53-EGFP. We
expressed p53-EGFP in SW480 cells in the presence of a p53-responsive
element-driven luciferase plasmid to examine whether the p53-EGFP
fusion protein was functionally equivalent to wild-type p53.
Transfected p53-EGFP selectively activated the p53-responsive
element-driven luciferase, whereas negative control lacZ did not
activate p53-responsive element-driven luciferase (Fig. 5A)
. p53-EGFP expression was observed as a green light under the
fluorescence microscope (data not shown) and was also recognized as a
Mr 80,000 protein by anti-p53 antibody
(Fig. 5B)
. The viability of those p53-EGFP-expressing cells
was analyzed by flow cytometry. The two-hybrid plasmid-transfected
cells were harvested after 48 or 72 h incubation. About 20% of
the pG5/p53-EGFP plasmid-transfected cells survived, whereas 
80% of
the pG5/EGFP-transfected cells survived (Fig. 5C)
. In this
experiment, pG5/EGFP-transfected cells showed a higher intensity of GFP
light than pG5/p53-EGFP-transfected cells (data not shown).
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Fig. 5. A, activation of p53-responsive-luciferase
expression by p53-EGFP. pcDNA/p53-EGFP was transfected with p53-Luc,
pAP-1-Luc, or pCRE-Luc (Stratagene) in the presence of pRL/TK (Promega)
to SW480 cells and cultured for 24 h. Cells were harvested, and
luciferase activity was measured. Bars, SD.
B, detection of p53-EGFP in SW480 cells. SW480 cells were
cotransfected with pBIND/WT-ß-catenin, pACT/FL-Tcf4, and pG5/p53-EGFP
and cultured for 24 h. Produced p53-EGFP was detected by Western
blot using anti-p53 antibody. C, cell killing experiment
using two-hybrid system. SW480 cells were transfected with
pBIND/WT-ß-catenin, pACT/FL-Tcf4, and pG5/p53-EGFP and cultured for
48–72 h. Cells were trypsinized and washed with PBS. Then, cells were
suspended in PBS containing 10 µM propidium iodide. The
cell death profiles of EGFP-positive cells were analyzed by measuring
the uptake of propidium iodide using a flow cytometer.
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Discussion
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Gene therapy offers the promise of selective tumor cell killing
based on molecular defects that cause cancer. Many technical issues
must be resolved before the full potential of gene therapy is realized,
but there is reason to believe that this will be a successful
therapeutic approach in the future (19)
.
In this report, we describe a novel approach that could be applied to
kill tumor cells selectively. This approach targets cancer cells that
are caused by a specific protein-degradation disorder and uses the
mammalian two-hybrid system to control suicide gene expression.
Accumulation of ß-catenin in the cytoplasm occurs in most colon
cancer cells, and 80% of those are caused by dysfunctional APC. Our
system takes advantage of the fact that ß-catenin is not degraded
efficiently in APC-mutated cells, but the principle could be used in
other disorders that involve abnormal protein degradation. This system
can also be used to identify signaling pathways that regulate
association of ß-catenin and Tcf as well as forming the basis of a
screen for inhibitors of this interaction. Indeed, the mammalian
two-hybrid system has general utility for monitoring protein-protein
interactions in cells and manipulating these interactions for
therapeutic purposes.
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ACKNOWLEDGMENTS
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We thank P. Polakis (Genentech, South San Francisco, CA) for
wild-type ß-catenin cDNA, H. Clevers (University Medical Center
Utrecht, Utrecht, Netherlands) for full-length Tcf4 cDNA, and R. L. White (University of Utah, Salt Lake City, UT) for
pcDNA3/APC. We thank W. Hyun for technical assistance with FACScan
analysis. We thank L. Barna for technical support.
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FOOTNOTES
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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 work was supported in part by Daiichi
Pharmaceutical Co. Ltd. Japan. 
2 To whom requests for reprints should be
addressed, at 2340 Sutter Street, San Francisco, CA 94115. E-mail: mccormick{at}cc.ucsf.edu
3 The abbreviations used are: APC, adenomatous
polyposis coli; EGFP, enhanced green fluorescent protein.
Received 8/ 8/00.
Accepted 12/13/00.
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ABSTRACT
Introduction
