LRP6 expression promotes cancer cell proliferation and tumorigenesis
by altering b-catenin subcellular distribution
Yonghe Li*,1, Wenyan Lu1, Xi He2, Alan L Schwartz1,3and Guojun Bu1,4
1Department of Pediatrics, St Louis Children’s Hospital, Washington University School of Medicine, St Louis, MO 63110, USA;
2Division of Neuroscience, Children’s Hospital, Department of Neurology, Harvard Medical School, Boston, MA 02115, USA;
3Department of Molecular Biology and Pharmacology, St Louis Children’s Hospital, Washington University School of Medicine,
St Louis, MO 63110, USA;4Department of Cell Biology and Physiology, St Louis Children’s Hospital, Washington University School
of Medicine, St Louis, MO 63110, USA
The Wnt signaling pathway plays key roles in both
embryogenesis and tumorigenesis. The low-density lipo-
protein (LDL) receptor-related protein-6 (LRP6), a novel
member of the expanding LDL receptor family, functions
as an indispensable co-receptor for the Wnt signaling
pathway. Although the role of LRP6 in embryonic
development is now well established, its role in tumorigen-
esis is unclear. We report that LRP6 is readily expressed
at the transcript level in several human cancer cell lines and
human malignant tissues. Furthermore, using a retroviral
gene transfer system, we find that stable expression of
LRP6 in human fibrosarcoma HT1080 cells alters
subcellular b-catenin distribution such that the cytosolic
b-catenin level is significantly increased. This is accom-
panied by a significant increase in Wnt/b-catenin signaling
and cell proliferation. Finally, we demonstrate that LRP6
expression promotes tumorigenesis in vivo. These results
thus indicate that LRP6 may function as a potential
oncogenic protein by modulating Wnt/b-catenin signaling.
Oncogene (2004) 23, 9129–9135. doi:10.1038/sj.onc.1208123
Published online 25 October 2004
Keywords: LRP6; b-catenin; Wnt signaling; prolifera-
The Wnt canonical signaling pathway is involved in
various differentiation events during embryonic devel-
opment and can lead to tumor formation when
aberrantly activated (Orford et al., 1997; Wodarz and
Nusse, 1998; Giles et al., 2003; Lustig and Behrens,
2003). Wnts are secreted glycoproteins that bind to the
seven transmembrane receptors frizzled. A key compo-
nent of the Wnt signaling pathway is b-catenin. In the
absence of Wnts, b-catenin is phosphorylated by a
multiprotein complex, a modification that is required for
its ubiquitination and subsequent proteasomal degrada-
tion. b-catenin phosphorylation involves the sequential
actions of casein kinase I and glycogen synthase kinase
3, and is regulated by the adenomatous polyposis coli
(APC) tumor suppressor protein and the scaffold
protein axin. Upon Wnt stimulation, b-catenin phos-
phorylation and subsequent ubiquitination and degra-
dation are inhibited by a largely unknown mechanism.
Wnt signaling thus stabilizes b-catenin, which enters
the cell nucleus and associates with the T-cell factor/
lymphoid-enhancing factor (TCF/LEF) family of tran-
scription factors, leading to the transcription of Wnt
target genes, including regulators of cell proliferation,
developmental control genes, and genes implicated in
tumor progression (Orford et al., 1997; Wodarz and
Nusse, 1998; Giles et al., 2003; Lustig and Behrens,
2003; He et al., 2004).
The low-density lipoprotein receptor-related protein-5
(LRP5) and LRP6 are two members of the expanding
low-density lipoprotein receptor family (Herz and Bock,
2002; Schneider and Nimpf, 2003). Recent studies have
demonstrated that these two receptors are indispensable
elements of the canonical Wnt pathway by interacting
with several components of the Wnt signaling pathway.
First, LRP5 and LRP6 act as co-receptors for Wnts,
which interact with both frizzled and LRP5/LRP6 in
order to initiate canonical Wnt signaling (Pinson et al.,
2000; Tamai et al., 2000; Wehrli et al., 2000). Second,
LRP5, when activated by Wnt proteins, recruits the
scaffold protein axin to the membrane, and therefore
prevents it from participation in the degradation of
b-catenin (Mao et al., 2001). Third, individual members
of the Dickkopf (Dkk) family of secreted proteins can
either antagonize or stimulate Wnt signaling through
interaction with LRP6 (Wu et al., 2000; Bafico et al.,
2001; Bao et al., 2001, 2002; Semenov et al., 2001; Brott
and Sokol, 2002). Fourth, a context-dependent activator
and inhibitor of Wnt signaling, WISE, is able to compete
with Wnt for binding to LRP6 (Itasaki et al., 2003).
LRP6 is widely expressed in many tissues including
heart, brain, placenta, lung, kidney, pancreas, spleen,
testis, ovary, and the mucosal lining of the colon (Brown
et al., 1998). Despite extensive studies on the expression
Received 13 May 2004; revised 22 July 2004; accepted 22 July 2004;
published online 25 October 2004
*Correspondence: Y Li, Department of Pediatrics, Washington
University School of Medicine, CB 8208, 660 South Euclid Avenue,
St Louis, MO 63110, USA; E-mail: email@example.com.
Oncogene (2004) 23, 9129–9135
& 2004 Nature Publishing Group All rights reserved 0950-9232/04 $30.00
and alterations of other components of the Wnt/b-
catenin signaling pathway in human cancer, little is
known regarding the expression and function of LRP6
in human cancer. By using a retroviral gene transfer
system, we show herein that overexpression of LRP6
alters b-catenin subcellular distribution, enhances cancer
cell Wnt/b-catenin signaling, and promotes cell prolif-
eration in vitro and tumorigenesis in nude mice.
LRP6 expression in human cancer cell lines and tissues
To define a potential role for LRP6 in human cancer,
LRP6 expression in a variety of human cancer cell lines
and human normal and malignant tissues was examined.
We used semiquantitative RT–PCR techniques to
determine LRP6 transcript level in human colon cancer
cell lines SW620 and DLD-1, breast cancer cell lines
MDA-MB-231 and MDA-MB-468, lung cancer cell lines
H441 and H520, and fibrosarcoma cell line HT1080.
In order to confirm the absence of a genomic DNA
contamination from the RNA preparations, a negative
control with PCR amplification but without reverse
transcription was included in each assay (data not
shown). Human normal tissues including lung, colon,
kidney, and small intestine, and human malignant tissues
including lung tumor, colon tumor, kidney tumor, and
breast tumor were also analysed for LRP6 expression. As
seen in Figure 1, all cancer cell lines, as well as normal
and malignant tissues, express LRP6. These results
indicate that LRP6 is readily expressed in human cancers.
Stable expression of LRP6 in HT1080 cells
Compared to the other cell lines examined, fibrosarcoma
HT1080 cells express LRP6 at the lowest level (Figure 1).
Thus, we transduced LRP6 complementary DNA
(cDNA) into HT1080 cells in order to study the roles
of LRP6 in Wnt/b-catenin signaling. Transduced
HT1080 cells were propagated in medium containing
G418. After selection for 10 days, resistant colonies
(over 200) were pooled. Expression of LRP6 in
transformants was examined by immunoblotting with
the hemagglutinin (HA) antibody or a monoclonal
antibody that recognizes both LRP5 and LRP6. As seen
in Figure 2a, immunoblot analysis of LRP6-transduced
cells demonstrated that the transduced LRP6 was
expressed and recognized by both monoclonal LRP5/6
antibody and anti-HA antibody. Flow cytometric
analysis of nonpermeabilized cells demonstrated that
all the G418-resistant colonies were transduced with
LRP6, and that abundant LRP6 expression was
observed at the cell surface (Figure 2b).
LRP6 expression increases cytosolic b-catenin level and
TCF/LEF transcriptional activity
As b-catenin is a key molecule in the Wnt/b-catenin
signalingpathway, we hypothesizedthatLRP6
malignant tissues. (a) Expression of LRP6 in human cancer cell
lines. Total RNA was extracted from the indicated cell lines, and
RT–PCR was performed with primers for LRP6 and GAPDH. (b)
Expression of LRP6 in human normal tissues and malignant
tissues. RT–PCR was performed with primers for LRP6 and
GAPDH with the total RNA of the indicated tissues. This
experiment is a representative of two such experiments performed
with similar data
Expression of LRP6 in human cancer cell lines and
transduced HT1080 cells. (a) Western blot analysis of LRP6-
transduced HT1080 cells and control cells. Cell lysates were
analysed via 6% SDS–PAGE under reducing conditions and
Western blotted with anti-LRP5/6 or anti-HA antibodies as
indicated. (b) Flow cytometry analysis of LRP6-transduced
HT1080 cells and control cells. The negative controls without the
primary antibody are indicated with light lines, whereas the signals
from receptor staining are drawn with dark lines
Western blot and flow cytometric analyses of LRP6-
LRP6 is a potential oncogenic protein
Y Li et al
expression would increase the cytosolic b-catenin level
and enhance Wnt/b-catenin signaling activity in human
cancer cells. To test this, LRP6-transduced HT1080 cells
and control cells were fractionated into membrane and
cytosolic fractions, and the b-catenin levels in these
fractions, as well as in the whole-cell extract, were
examined by Western blotting. It was interesting to note
that LRP6-transduced cells expressed a much higher
level of cytosolic b-catenin, but a lower level of
(Figure 3a). The level of b-catenin in whole-cell extract
was unchanged. Fluorescence microscopy revealed that
overexpression of LRP6 reduced b-catenin localization
at the plasma membrane, especially at the junctions
between cells (Figure 3b). Taken together, these results
indicate that LRP6 expression in HT1080 cells induces
b-catenin disassociation from the plasma membrane and
accumulation in the cytoplasm.
The altered intracellular distribution of b-catenin in
LRP6-tranduced HT1080 cells was also found to be
associated with a corresponding increase in endogenous
TCF/LEF transcriptional activity. The TOP-FLASH
luciferase reporter contains TCF-binding sites and can
be directly activated by the b-catenin/TCF complex
(Korinek et al., 1997). We found that LRP6-transduced
cells exhibited 3.9-fold greater activity than control cells
To further examine the roles of LRP6 in b-catenin
localization and activity, we transiently transfected
Wnt1 cDNA or b-catenin cDNA into HT1080 cells
stably transduced with LRP6 cDNA or control vector.
After transfection with Wnt1 cDNA, the level of
cytosolic b-catenin in LRP6-transduced cells was much
higher than that in control cells, while the level of
membrane-associated b-catenin in LRP6-transduced
cells was lower than that of control cells. Similarly,
after transfection with b-catenin cDNA, the level
was significantly higher than that in control cells,
although the level of membrane-associated b-catenin
in LRP6-transduced cells was not changed (Figure 4a).
As expected, LRP6-transduced HT1080 cells display
greater Wnt1 or b-catenin-induced TCF/LEF transcrip-
tional activity than control cells. After transfection with
Wnt1 or b-catenin cDNA, LRP6-transduced cells
exhibit 2.6- and 7.9-fold increases in TCF/LEF tran-
scriptional activity, respectively, whereas the corre-
sponding control HT1080 cells exhibit only 2.0- and
3.2-fold increases in TCF/LEF transcriptional activity
LRP6 expression enhances HT1080 cell proliferation
Having established that LRP6 expression results in
increases in the cytosolic b-catenin level, and TCF/LEF
transcriptional activity in HT1080 cells, we then
investigated the role of LRP6 in cell proliferation. The
effect of LRP6 expression on HT1080 cell proliferation
was examined in a 7-day proliferation assay. Figure 5a
shows that there was a significant difference between
LRP6-transduced cells and control cells in their
proliferation rates (Po0.01). After 7 days of culture,
an approximately 1.6-fold increase in the number of cells
cytosolic b-catenin, and TCF/LEF transcriptional activity. (a)
LRP6-transduced HT1080 cells and control cells were fractionated
into membrane and cytosolic fractions, and the b-catenin levels in
these fractions and the whole-cell extract were examined by
Western blotting using b-catenin antibody. Loading in each lane
was normalized to protein content. (b) LRP6-transduced HT1080
cells and control cells were labeled with anti-b-catenin antibody,
followed by detection with Alexafluor488 goat anti-mouse IgG and
confocal microscopy. (c) LRP6-transduced HT1080 cells and
control cells were co-transfected with a TCF/LEF transcriptional
activity reporter plasmid (TOP-FLASH), and the luciferase activity
was measured 48h after transfection. This experiment is a
representative of two such experiments performed with similar
data. *Po0.01 compared to pLNCX2 control cells
LRP6-transduced cells display increased levels of
b-catenin-induced TCF/LEF transcriptional activity than control
cells. (a) LRP6-transduced HT1080 cells and control cells were
transiently transfected with Wnt1, b-catenin, or empty pcDNA3
vector. The cells were fractionated into membrane and cytosolic
fractions 48h after transfection. b-Catenin levels in these fractions
and the whole-cell extract, as well as Wnt1 levels in whole-cell
extract, were examined by Western blotting using b-catenin
antibody or Wnt1 antibody. (b) LRP6-transduced HT1080 cells
and control cells were co-transfected with Wnt1, b-catenin, or
empty pcDNA3 vector, and a TCF/LEF transcriptional activity
reporter plasmid (TOP-FLASH). The luciferase activity was
measured 48h after transfection. This experiment is a representa-
tive of three such experiments performed with similar data.
*Po0.01 compared to pcDNA3 control
LRP6-transduced HT1080 cells display greater Wnt1 or
LRP6 is a potential oncogenic protein
Y Li et al
was found in LRP6-transduced cells compared to
control cells. The population doubling time of LRP6-
transduced cells was 17.870.7h (i.e. the standard
deviation; n¼3), while that of control cells was
21.370.8h (Po0.05 when compared to LRP6-trans-
duced cells). These results indicate that LRP6 expression
significantly increases anchorage-dependent growth of
As anchorage-independent growth correlates well
with tumorigenicity in vivo (Freedman and Shin, 1974),
we examined this parameter using a soft agar colony
assay. As seen in Figure 5b, the soft agar assay
demonstrated that LRP6-transduced cells form greater
numbers of colonies compared to control cells.
LRP6 expression promotes HT1080 cell growth in vivo
Results from the in vitro assays described above suggest
that tumorigenicity in vivo may also be enhanced by
LRP6 expression. To examine this possibility, we
performed tumorigenesis assays by comparing tumor
volume in nude mice after 7, 14, 21, or 28 days following
tumor cell injection. We found that LRP6-transduced
cells are more tumorigenic than control cells (Figure 6).
The difference in tumor size between LRP6-transduced
cells and control cells increased as tumor size increased.
By the termination of the experiment (28 days following
cell injection), LRP6-transduced cells had formed
tumors approximately twofold larger than those derived
from control cells (Figure 6).
Genetic and biochemical studies have demonstrated that
LRP6, by interacting with several components of the
pathway, is an indispensable element of the Wnt
signaling pathway (Herz and Bock, 2002; Schneider
and Nimpf, 2003; He et al., 2004). Our present study
provides the first direct evidence that genetic manipula-
tion of LRP6 expression enhances Wnt/b-catenin
signaling and the malignant phenotype of tumor cells.
We found that increased LRP6 expression elevated the
cytosolic b-catenin level and TCF/LEF transcriptional
activity, and promoted cell proliferation in culture,
colony formation in soft agar, and tumor formation in
soft agar by LRP6-transduced HT1080 cells. (a) Effect of LRP6
expression on HT1080 cell proliferation in culture. LRP6-
transduced HT1080 cells and control cells were plated in six-well
plates, and counted with trypan blue at days 3, 5, and 7. The mean
cell numbers were plotted with s.d. given as error bars. (b) Effect of
LRP6 expression on HT1080 cell colony formation on soft agar.
LRP6-transduced HT1080 cells and control cells were suspended in
0.3% agar with complete DMEM and then layered on top of 0.5%
bottom agar in six-well plates. Colonies were counted after 3 weeks.
This experiment is a representative of three such experiments
performed with similar data. *Po0.01 compared to pLNCX2
Cell proliferation in culture and colony formation on
LRP6-transduced HT1080 cells and control cells into mice. For
each implantation, cells were injected subcutaneously into the back
of each 4-week-old, female athymic mouse. Nine mice were used in
each group. Tumor size was measured at the indicated times. Each
point represents the average volume of the nine tumors with s.d.
given as error bars
Subcutaneous tumor growth after s.c. implantation of
LRP6 is a potential oncogenic protein
Y Li et al
b-Catenin is an intracellular multifunctional protein.
In complex with the transmembrane adhesive receptor
E-cadherin, it becomes plasma membrane-associated
and mediates intercellular adhesion. A cytosolic pool of
b-catenin interacts with DNA-binding proteins and
participates in Wnt signal transduction (Hinck et al.,
1994; Gottardi et al., 2001; Klingelhofer et al., 2003). At
the heart of the canonical Wnt pathway is the
stabilization of cytosolic b-catenin, which activates
target genes by binding to the TCF/LEF family of
transcription factors. In the present study, we found that
overexpression of LRP6 in HT1080 cells resulted in an
increase of cytosolic b-catenin and a decrease of
membrane-associated b-catenin. These results suggest
that LRP6 modulate Wnt/b-catenin signaling primarily
by altering intracellular distribution of b-catenin with an
unidentified mechanism. Currently, we do not rule out
the possibility that part of the cytosolic increase of b-
catenin level in LRP6-transduced cells might result from
inhibition of b-catenin degradation by the ubiquitin/
proteasome pathway. This possibility will be examined
in future studies.
Previous studies have provided strong evidence that
the single transmembrane LRP5/6 receptors and the
seven transmembrane frizzled receptors cooperate in
Wnt/b-catenin signaling (Pinson et al., 2000; Tamai
et al., 2000; Wehrli et al., 2000; Mao et al., 2001). Recent
studies also showed that LRP5 or LRP6 truncated from
the amino-terminal ends (i.e. lacking the extracellular
domain) are capable of mediating Wnt/b-catenin signal-
ing independent of either a Wnt ligand or frizzled
receptor (Mao et al., 2001; Liu et al., 2003; Brennan
et al., 2004). Furthermore, it was demonstrated that a
PPPSP motif, which is reiterated five times in the LRP6
intracellular domain, is necessary and sufficient to
trigger Wnt/b-catenin signaling (Brennan et al., 2004;
Tamai et al., 2004). In the present study, we demon-
strated that LRP6 modulates Wnt signaling by altering
intracellular distribution of b-catenin. Thus, the me-
chanisms by which LRP5/6 modulates the Wnt signal
are complex. It was proposed that, under normal
conditions when Wnt ligand and receptors are at the
physiological levels, both frizzled and LRP5/6 receptors
are required to achieve efficient intracellular coupling to
Wnt/b-catenin signaling (Liu et al., 2003). However,
under certain pathological conditions such as in
malignancy, high levels of LRP5/6 alone might be able
to induce Wnt/b-catenin signaling by modulating sub-
cellular b-catenin distribution.
Activation of Wnt/b-catenin signaling is now recog-
nized as an important event that leads to tumorigenesis
in several malignancies, especially colorectal cancer
(Giles et al., 2003; Lustig and Behrens, 2003). Activation
has been shown to occur through mutations of either the
APC tumor-suppressor gene or b-catenin itself. The
consequence of either APC inactivation or b-catenin
mutation is similar: failure of proper b-catenin degrada-
tion leads to its cytosolic accumulation, nuclear
translocation, and constitutive activation of b-catenin-
responsive genes (Giles et al., 2003; Lustig and Behrens,
2003). In the current study, we show that human cancer
cell lines and malignant tissues readily express LRP6,
and that LRP6 overexpression in human fibrosarcoma
cells promotes Wnt/b-catenin signaling and tumorigen-
esis. These results suggest that LRP6 is a potential
oncogenic protein by modulating Wnt/b-catenin signal-
ing. LRP6 expression in all the examined normal tissues,
malignant tissues, and cultured cell lines may suggest
that LRP6 is either ubiquitously expressed or essential
for cell survival. Although our data do not prove that
LRP6 is associated with tumorigenesis, they are
consistent with the notion that LRP6 is an obligate
receptor for the canonical Wnt signaling pathway.
Recently, it has been demonstrated that expression of
LRP5, a sister receptor of LRP6, is a common event in
osteosarcoma, and may serve as a potential novel
marker for disease progression in high-grade osteosar-
coma (Hoang et al., 2004a). Furthermore, overexpres-
osteosarcoma Saos-2 cells significantly reduces cell
invasion capacity and cell motility (Hoang et al.,
2004b). Future studies on LRP6 expression in malignant
tissues should allow us to determine whether alteration
of LRP6 expression occurs in primary tumors, and, if
so, whether LRP6 expression correlates with the
Materials and methods
Human cancer cell lines HT1080, HCT116, DLD-1, MDA-
MB-231, MDA-MB-468, H441, and H520 were obtained from
the American Type Culture Collection (Manassas, VA, USA),
and cultured in DMEM with 10% fetal calf serum. LRP6
transcript level in human cancer cell lines and malignant
tissues was analysed by RT–PCR as described by Qiang et al.
(2003) with minor modifications. Briefly, total RNA was
isolated from cell cultures using RNA-Bee (Tel-Test, Friend-
wood, TX 77546). Total RNA of human normal tissues (lung,
colon, kidney, and small intestine) and malignant tissues (lung,
colon, kidney, and breast) was from Clontech (Palo Alto, CA,
USA). First-strand cDNA synthesis was performed using
ProSTARTMUltro HF RT–PCR Kit (Strategene) primed with
oligo(dT) primer in a 10ml reaction mixture containing 0.3mg
total RNA. For semiquantitative analysis of LRP6 expression,
the PCR was carried out using 1ml of cDNA in a total volume
of 50ml over 32 cycles according to the manufacturer’s
instructions, and GAPDH was used as a control. The forward
and reverse primers for LRP6 are 50-GATTATCCAGAAGG-
CATGGCAG-30(þ2113 to þ 2134) and 50-TCCCATCAC-
CATCTTCCA-30(þ2827 to þ2848), respectively. The PCR
product was loaded onto a 1.2% Agarose gel and stained with
Stable expression of LRP6
Human LRP6 cDNA was subcloned into a retroviral vector
pLNCX2 (Clontech Laboratories, Inc., Palo Alto, CA, USA)
using standard procedures. A HA epitope was inserted into the
construct, located at the amino terminus after the signal
peptide, to facilitate immunodetection of the LRP6 protein in
infected cells. The HA tag does not interfere with LRP6
processing and its function, as we found that after transfection
HA-LRP6 is properly processed to the cell surface, and can
LRP6 is a potential oncogenic protein
Y Li et al
bind and internalize several LRP6 ligands (data not shown).
The integrity of the subcloned DNA sequence was confirmed
by DNA sequencing. RetroPack PT67 packaging cells
pLNCX2-LRP6 or pLNCX2 alone using FUGENE 6 (Roche
Molecular Biochemicals). Supernatants from transfected PT67
cells were incubated with 50% confluent HT1080 cells in the
presence of Polybrene (4mg/ml, final concentration, Sigma
Chemical Co., St Louis, MO, USA). LRP6-transduced
HT1080 cells and control cells were propagated in medium
containing G418 (Life Technologies, Inc.) at 700mg/ml. After
G418 selection for 10 days, resistant colonies were pooled. The
expression of total cellular LRP6 was determined by Western
blotting using both anti-HA antibody and monoclonal anti-
LRP5/6 antibody (BioVision, Mountain View, CA, USA).
Proper folding and trafficking of the receptor to the cell
surface were confirmed by examining cell surface LRP6 levels
via flow cytometry as described before (Li et al., 2000, 2002).
were transfected with
Subcellular fractionation and b-catenin Western blotting
To examine the role of LRP6 in b-catenin subcellular
b-catenin were compared in cells transduced with either
LRP6 cDNAor vector alone.
six-well plates, and used at B80–90% confluence. After
washing in ice-cold PBS, cells were collected and homogenized
in a glass Dounce homogenizer in buffer consisting of 100mM
Tris–HCl, pH 7.4, 140mM NaCl, 2mM DTT, 2mM PMSF,
and 1? Completet protease inhibitors (500ml/well). The
homogenate was centrifuged for 10min at 500g, and the
resulting supernatant was designated as the whole-cell extract.
This whole-cell extract was then centrifuged at 100000g at 41C
for 90min, after which the supernatant was designated as the
cytosolic fraction, while the pellet was dissolved in 1?
Laemmli sample buffer (62.5mM Tris–HCl, pH 6.8, 2%
(w/v) SDS, 10% (v/v) glycerol, and 5% b-mercaptoethanol),
and designated as the membrane fraction. The levels of
b-catenin in the whole-cell extract, cytosolic fraction, and
membrane fraction were then examined by quantitative
Western blotting using b-catenin-specific antibody from Cell
LRP6 transduced HT1080 cells and control cells were fixed in
4% paraformaldehyde, labeled with mouse monoclonal
b-catenin antibody, and detected with Alexafluor488 goat
anti-mouse IgG. The immunofluorescence was detected by a
laser-scan confocal microscope (Olympus Fluoview 500).
Luciferase reporter assay
HT1080 cells were plated into six-well plates. For each well,
0.5mg of the TOP-FLASH TCF luciferase construct (Upstate
Biotechnology) was cotransfected with 0.5mg of b-catenin-
expressing vector, 0.5mg of Wnt1-expressing vector, or empty
pcDNA3 vector. A b-galactosidase-expressing vector (Prome-
ga, Madison, WI, USA) was included as an internal control for
transfection efficiency. After 48h, cells were lysed and both
luciferase and b-galactosidase activities were determined with
enzyme assay kits (Promega). The luciferase activity was
determined with a luminometer using the Dual Luciferase
Assay system (Promega). Luciferase activity was normalized to
the activity of the b-galactosidase.
Cell proliferation assays
Cells were seeded into six-well plates (5?104cells per well).
Media were changed every other day, and cells were harvested
and counted every day from day 1 to 7 using the trypan blue
exclusion assay. Doubling times (DT) for LRP6-transduced
cells and control cells during periods of logarithmic growth
were determined using the formula DT¼ln2/(growth rate).
Soft agar colony assays
Cells were cultured in six-well plates covered with an agar layer
(DMEM medium with 0.5% agar and 5% FBS). The middle
layer contained 2?103cells in DMEM with 0.33% agar and
5% FBS, and this cell layer was overlaid with medium only to
prevent drying of the agar gels. Triplicate plates were prepared
for each cell line. After 3 weeks of incubation, colonies larger
than 0.1mm in diameter were scored.
Female athymic nude mice (4–5 weeks old) were obtained from
HT1080 cells (6?106cells) were suspended in 0.2ml of
serum-free DMEM with 50% matrigel matrix, and injected
s.c. into one flank of the mice. Tumors were measured every 7
days, and tumor volumes were calculated using width (a) and
length (b) measurements (a2b/2, where aob).
This work was supported in part by grant from the American
Heart Association (0330118N) to YL, and grants from the
National Institutes of Health to GB. GB is an Established
Investigator of the American Heart Association. We are
grateful to Dr Christof Niehrs for providing the LRP6 cDNA,
and Dr Theodore C Simon for providing the cDNAs for
human Wnt1 and b-catenin, and for the helpful discussion
during the course of this study.
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