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Molecular Biology and Genetics |
Johns Hopkins Oncology Center, Baltimore, Maryland 21231 (I-M. S., J. Y., T-C. H., B. V., K. W. K.); Department of Pathology, Johns Hopkins Medical Institution, Baltimore, Maryland 21287 (I-M. S.); and Howard Hughes Medical Institutes, Johns Hopkins University, Baltimore, Maryland 20815 (B. V.)
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The molecular characterization of APC function is complicated by the
fact that APC contains multiple functional domains that interact with a
variety of cytoplasmic proteins. These include ß-catenin (4
, 5)
, 
-catenin (6
, 7)
, GSK3-ß (8)
,
AXIN family proteins (9, 10, 11)
, EB-1 (12)
,
microtubules (13
, 14) , and the human homologue of the
Drosophila tumor suppressor gene discs large (hDLG; Ref.
15
). Among these factors, the study of interaction between
APC protein and ß-catenin provides the most penetrating insights into
APC function (4
, 5) . The ß-catenin regulatory domain in
the cAPC is removed or truncated by the majority of both inherited and
somatic mutations. These truncated forms of APC are ineffective in
forming APC/AXIN/GSK3/ß-catenin complexes (9, 10, 11
, 16)
,
which phosphorylate ß-catenin and lead to its degradation by the
ubiquitin-proteasome system (17, 18, 19, 20)
. The resulting
accumulation of ß-catenin allows it to complex with Tcf-4, creating a
bipartite transcription complex that activates downstream
growth-promoting genes (21
, 22) . Accordingly, colorectal
cancers exhibit high levels of constitutive ß-catenin/Tcf-4-mediated
transcription, which can be suppressed by exogenous APC
(23)
. Moreover, mutations of ß-catenin that render it
resistant to APC-mediated down-regulation have been identified in the
unusual colorectal tumors that express wild-type APC
(24)
as well as in other tumor types
(25, 26, 27, 28, 29, 30, 31)
. Finally, recent studies have identified direct
downstream targets of CRT, including the growth promoting genes c-MYC
and cyclin D1 (32, 33, 34, 35)
.
The findings reviewed above suggest that APC suppresses tumorigenesis by inhibiting CRT of growth-promoting genes and subsequently inducing apoptosis. However, direct evidence for this scenario is still lacking. In this study, we determined the ability of the ß-catenin-binding domain of APC in isolation to inhibit CRT, down-regulate expression of growth-promoting target genes, and inhibit tumor cell growth in vitro and in vivo.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Generation of Recombinant Adenovirus Expressing cAPC (Ad-CBR).
The recombinant adenovirus, Ad-CBR, which carried the cAPC, was
generated using a modified system as previously described
(36)
. The cAPC containing amino acids 958-2075
(nucleotides 2890–6240) was isolated from pCMV-APC by BglII
digestion. This fragment was subcloned into the pEGFP-C1 (Clontech,
Palo Alto, CA). The cassette containing the EGFP-tagged cAPC was
further subcloned into the shuttle vector (pShuttle) using Apal I and
Mlu I sites. Recombinant adenoviral plasmid was generated by homologous
recombination in Escherichia. coli (BJ5183). BJ5183 cells
were transformed using electroporation with pAdEasy-1 and
pShuttle/EGFP-cAPC linearized with PmeI. Successful recombinants were
identified by restriction endonuclease mapping. The recombinant
EGFP-cAPC virus (Ad-CBR) was produced in the 911 and 293 adenovirus
packaging lines, and the viral particles were purified by CsCl banding.
The control virus (Ad-EGFP) with EGFP alone was also prepared and
purified side by side. Viral titer was determined by a modified CPE end
point assay. A series of Ad-GFP infections was performed on HCT116
cells to determine the optimal MOI to avoid adenovirus-associated CPE.
Typically, viral CPE could be observed at an MOI of >100, which
resulted in >80% of cells becoming fluorescent 18 h after Ad-GFP
infection. To avoid any CPEs of viruses, we infected cells with a
minimal MOI, generating fluorescence in 20–30% of the cells
(MOI = 5–11), then flow-sorted infected cells to obtain
homogeneous populations.
Viral Infection and Cell Sorting.
Viral stocks were predialyzed using 1% agarose in microcentrifuge
tubes. Three million cells were infected with either Ad-CBR or Ad-GFP
in a 75-cm2 flask. After 18 h of incubation
at 37°C, cells were washed, trypsinized, and subjected to
fluorescence-activated cell sorting. Cells with green fluorescence were
collected for experiments or were replated in culture flasks
immediately after sorting.
Reporter Assay.
DOT and Dluc cells were generated from DLD1 cells by cotransfection of
pTK-hygro (Clontech) and a Tcf-4-responsive luciferase plasmid
(pGL3-OT)4
or a constitutive luciferase plasmid (pGL3-control; Promega, Madison,
WI), respectively. Clones were isolated, and the sensitivity to CRT was
determined using a dominant-negative Tcf-4
adenovirus.4
Luciferase reporter activity
in the DOT clone was constitutively high as expected for a
CRT-responsive reporter in a colorectal cancer cell line with mutated
APC. This constitutive activity was inhibited by dominant-negative
Tcf-4. In contrast, the luciferase activity in the Dluc clone was
unaffected by dominant-negative Tcf-4 as expected for expression driven
by the SV40 promoter. To assess the effects of cAPC on CRT, DOT and
Dluc cells were infected with Ad-GFP and Ad-CBR. Eighteen h after viral
infection, equal numbers of GFP-positive cells were pelleted, lysed,
and collected for luciferase assays using luciferase assay reagents
(Promega).
Western Blot Analysis.
Whole cell lysates were prepared in a solution containing 62.5
mM Tris-HCl (pH 6.8), 2% SDS, and 0.5%
ß-mecarptoethanol. Equal amounts of total protein from each lysate
were loaded and separated on 4–12% Tris-Glycine-SDS polyacrylamide
gels (Novex, San Diego, CA) and electroblotted to Millipore Immobilon-P
polyvinylidene difluoride membranes. Western blots were developed by
chemiluminescence (NEN Life Science, Boston, MA), detected by Kodak
Image Station 440CF, and analyzed by one-dimensional Image Analysis
software (NEN Life Science). Primary antibodies included anti-GFP
polyclonal antibody from Clontech (Palo Alto, CA), anti-c-MYC
monoclonal antibody (9E10) from Santa Cruz (Santa Cruz, CA), anticyclin
D1 monoclonal antibody (A-12) from Santa Cruz, and anti-
-tubulin
monoclonal antibody (TU-02) from Santa Cruz. Secondary
peroxidase-conjugated antibodies were goat antimouse IgG and goat
antirabbit IgG from Pierce (Rockford, IL).
Immunofluorescence Staining.
Cells were infected with Ad-GFP or Ad-CBR for 18 h and sorted.
Fluorescent cells were cultured on an 8-well chamber CultureSlides
(Becton Dickinson, Bedford, MA). After 8 h, cells were fixed in
3% paraformaldehyde in PBS at room temperature for 8 min, then
permeabilized with 0.3% NP40 in PBS for another 8 min. After washing
in PBS, the cells were incubated with primary mouse anti-ß-catenin
monoclonal antibody (1 µg/ml; Transduction Laboratories, Lexington,
KY) at 4°C overnight. After washing, cells were incubated with
biotinylated goat antimouse IgG (Pierce, Rockford, IL) at room
temperature for 1 h. The immunoreactivity was revealed using
Alexa568-conjugated streptavidin (Molecular Probes, Eugene, OR), and
cells were counterstained with 10 µg/ml DAPI. The cells were examined
under a Nikon fluorescence microscope (Image Systems, Columbia, MD).
Cell Growth and Colony Formation Assay.
Cells (105) were plated in one well of a
24-well plate. Cells were counted using a hemocytometer after
trypsinization on days 1, 2, 3, and 5. For colony formation assays,
each well of the 24-well plates was precoated with 100 µl of collagen
gel containing 50% type I collagen (Collaborative Biomedical Science,
Bedford, MA), 40% culture medium, and 0.75%
NaHCO3 (Halttunen). One hundred sixty µl of
collagen gel-cell suspension containing 10,000 cells, 45% type I
collagen, 40% culture medium, and 0.075% NaHCO3
were added to the wells. After solidification, each well was covered
with 1 ml of culture medium, and the plates were incubated at 37°C.
Twelve days after seeding, cells were stained with 0.05% crystal
violet (Sigma, St. Louis, MO) containing 10% buffered formalin
(Sigma).
DAPI Staining and Annexin V Staining for Apoptosis Detection.
Both attached and floating cells were harvested for staining. For DAPI
staining, 3 x 105 cells were
resuspended in 50 µl of PBS and 350 µl of staining solution
containing 0.6% NP40, 3% paraformaldehyde, and 10 µg/ml DAPI. For
annexin V staining, 105 cells were suspended in
100 µl of annexin-binding buffer containing 10 mM HEPES,
140 mM NaCl, and 2.5 mM
CaCl2. Five µl of Alexa568-conjugated annexin V
(Molecular Probes) were added and incubated at room temperature for 15
min, at which point an additional 400 µl of annexin-binding buffer
were added to each sample. Apoptotic cells were defined as those cells
containing condensed and/or fragmented nuclei after DAPI staining or
were fluorescent after annexin V staining. At least 500 cells were
counted, and the results were expressed as the percentage of apoptotic
cells in each sample.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The employment of a fusion protein ensured that
expression of GFP was coupled with APC and allowed positive
verification of cAPC expression. The growth suppressive effects of
tumor suppressor genes impose a powerful negative selection force that
can result in loss of expression of the tumor suppressor gene even in
the presence of a positive selection marker.
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and is sufficient for promoting ß-catenin degradation
(17)
. Infection of SW480 cells with
adenovirus-expressing GFP/cAPC (Ad-CBR) resulted in the production of a
fusion protein of the expected size (150-kDa fusion) as determined by
Western blot analysis with an anti-GFP antibody (Fig. 1B)
.
Fluorescence microscopy revealed that the GFP/cAPC fusion protein was
diffusely present in the cytoplasm in all of eight colorectal cancer
cell lines tested (Table 1
and examples in Fig. 1C
).
|
. This inhibition appeared to be specific because infection
of Dluc cells with Ad-CBR had no effect on luciferase activity (Fig. 2B)
. To determine whether the Ad-CBR suppression could be
extended to endogenous targets of CRT, we evaluated the expression of
c-MYC (32)
and cyclin D1 (33)
, two recently
described targets of the APC/ß-catenin/Tcf-4 pathway. Expression of
c-MYC and cyclin D1 was examined in three human colorectal cancer cell
lines. In DLD1 and SW480, CRT was constitutively activated because of
APC mutations (37)
, whereas in HCT116, CRT activation was
due to a ß-catenin mutation (24)
. Infection of SW480 and
DLD1 cells with Ad-CBR resulted in a marked reduction of c-MYC protein
levels as well as a reduction of cyclin D1 (Fig. 3)
. Ad-GFP infection had no inhibitory effect on either c-MYC or cyclin
D1 protein levels in these cells. As expected, HCT116 cells, which
possess a stabilizing ß-catenin mutation, were relatively resistant
to the effects of Ad-CBR (Fig. 3)
.
|
|
. A similar alteration in the subcellular distribution of
ß-catenin was observed in SW480 cells (Fig. 4B)
. The
selective depletion of nuclear ß-catenin by Ad-CBR was consistent
with the model for APC action proposed previously (17
, 26)
. The nuclear ß-catenin staining in HCT116 was
significantly less striking and appeared similar in Ad-GFP- and
Ad-CBR-infected cells (data not shown).
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. This growth inhibition was persistent, with
Ad-CBR-infected DLD1 and SW480 cells failing to reach confluence even
after 2 weeks of culture, long after mock- and Ad-GFP-infected cells
became superconfluent. Consistent with the effects of Ad-CBR on CRT in
HCT116 cells, the growth of Ad-CBR-infected HCT116 cells was only
partially inhibited (Fig. 5A)
.
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. The cells were flow-sorted to select
virally infected cells, as described in "Materials and Methods."
Infection with Ad-CBR resulted in a marked suppression of colony
formation in SW480, DLD1, LoVo, HT29, SW837, and SW1417 cells, all of
which possess mutated APC (Fig. 5B)
. Colony numbers were
reduced by at least 96% in each of these lines compared with cells
mock-infected or infected with Ad-GFP virus. Examination under
phase-contrast microscopy revealed that most of the Ad-CBR-infected
cells were pyknotic. A small number of growth-arrested single cells
remained in the gels, and these continued to express APC as judged by
fluorescence. In the few colonies that did form after Ad-CBR infection,
APC did not appear to be expressed. In contrast, cells with intact APC
but mutated ß-catenin were able to form a significant number of
colonies following Ad-CBR infection (HCT116 and SW48 in Fig. 5B
).
Ad-CBR Induces Apoptosis in Colorectal Cell Lines.
Ad-CBR-expressing cells revealed a gradual loss of the
G1 peak and an accumulation of cells in the S and
G2 phases of the cell cycle (data not shown).
Five days after infection, all six colorectal cell lines with
APC mutations demonstrated significantly increased apoptosis
after Ad-CBR infection (Fig. 6)
. In line with the effects of Ad-CBR on CRT and growth, the mutant
ß-catenin-containing cell lines, HCT116 and SW48, exhibited little
increase in apoptosis in response to Ad-CBR infection. Time course
studies revealed that the first morphological signs of apoptosis were
not evident in DLD1 and SW480 cells until 72 h after plating (data
not shown). The induction of apoptosis was confirmed by Annexin V,
which has been shown to bind to phosphatidylserine exposed on the outer
leaflet of apoptotic cell membranes (38
, 39)
. The
proportion of DLD1 and SW480 cells staining with Alexa568-labeled
annexin V was in good agreement with the fraction of cells displaying
morphological signs of apoptosis (>90% of annexin V-labeled cells
displayed apoptotic nuclei). The induction of apoptosis by Ad-CBR was
equally evident in DLD1 cells grown as xenografts (data not
shown).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The biological effects of the cAPC are likely related to abrogation of
the APC/ß-catenin/Tcf-4 signaling pathway. This conclusion is based
on the fact that Ad-CBR inhibits ß-catenin nuclear translocation
(Fig. 4)
, suppresses ß-catenin/Tcf-4-mediated transcription in
reporter assays (Fig. 2)
, and down-regulates the expression of targets
of the APC/ß-catenin/Tcf-4 pathway (Fig. 3)
. Cellular proliferation
and colony formation are dramatically suppressed by Ad-CBR in cell
lines that contain mutations in the APC gene, but are only
partially inhibited in lines containing mutations of ß-catenin that
render it resistant to APC degradation.
At the cellular level, expression of the cAPC eventually results in apoptosis of colorectal cancer cells containing APC mutations. This observation is consistent with those in a previous report, demonstrating apoptosis 60 h after induction of full-length APC expression (2) . In both cases, the delay in appearance of apoptotic cells suggests that APC-induced apoptosis may not be a direct result of suppression of CRT. It should also be noted that the above cited observations and those reported here were made with tumor human cell lines maintained in culture and that additional experiments will be necessary to confirm that they accurately reflect the growth effects of APC in primary human tumors.
Although the ß-catenin-binding domain in the cAPC is sufficient for growth suppression by APC, it may not recapitulate all of the functions of this gene. For example, the carboxyl-terminal third of APC can associate with the human homologue of the Drosophila tumor suppressor gene discs large (hDLG; Ref. 15 ) and EB-1 (12) . The latter has recently been implicated in the spindle checkpoint (40 , 41) . In addition, a carboxyl-terminal fragment of APC has been shown to induce assembly and bundling of microtubules in vitro and has a role in directed cell migration (13 , 14 , 42) . Like other canonical tumor suppressor genes, it is likely that APC functions at several levels to regulate cell growth and suppress neoplastic transformation. However, the finding that the middle third of APC is sufficient to inhibit tumor cell growth focuses further attention on the APC/ß-catenin interaction. Future experiments to understand the upstream regulators and downstream transducers of this interaction should shed further light on tumorigenesis associated with defects in the APC pathway.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH Grant CA57345 and Genzyme
Molecular Oncology (Genzyme; to K. W. K.). B. V. and
K. W. K. are consultants to Genzyme. B. V. is an
investigator of the Howard Hughes Medical Institutes. 
2 To whom requests for reprints should be
addressed, at Johns Hopkins Oncology Center, Room 588, Cancer Research
Building, 1650 Orleans Street, Baltimore, MD 21231.
3 The abbreviations used are: APC, adenomatous
polyposis coli; Tcf, T-cell factor; DAPI,
4',6-diamidino-2-phenylindole; cAPC, central third of APC; CBR,
catenin-binding region; CRT, catenin-regulated transcription; GFP,
green fluorescent protein; EGFP, enhanced GFP; CPE, cytopathic effect;
MOI, multiplicity of infection.
Received 8/ 2/99. Accepted 1/19/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A. M. Carothers, K. A. Melstrom Jr., J. D. Mueller, M. J. Weyant, and M. M. Bertagnolli Progressive Changes in Adherens Junction Structure during Intestinal Adenoma Formation in Apc Mutant Mice J. Biol. Chem., October 12, 2001; 276(42): 39094 - 39102. [Abstract] [Full Text] [PDF]
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H. Roh, D. W. Green, C. B. Boswell, J. A. Pippin, and J. A. Drebin Suppression of {beta}-Catenin Inhibits the Neoplastic Growth of APC-Mutant Colon Cancer Cells Cancer Res., September 1, 2001; 61(17): 6563 - 6568. [Abstract] [Full Text] [PDF]
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J. M. Mariadason, M. Bordonaro, F. Aslam, L. Shi, M. Kuraguchi, A. Velcich, and L. H. Augenlicht Down-Regulation of {beta}-Catenin TCF Signaling Is Linked to Colonic Epithelial Cell Differentiation Cancer Res., April 1, 2001; 61(8): 3465 - 3471. [Abstract] [Full Text]
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Y. Zhang, W.-J. Qiu, D.-X. Liu, S. Y. Neo, X. He, and S.-C. Lin Differential Molecular Assemblies Underlie the Dual Function of Axin in Modulating the Wnt and JNK Pathways J. Biol. Chem., August 17, 2001; 276(34): 32152 - 32159. [Abstract] [Full Text] [PDF]
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Z. You, D. Saims, S. Chen, Z. Zhang, D. C. Guttridge, K.-l. Guan, O. A. MacDougald, A. M.C. Brown, G. Evan, J. Kitajewski, et al. Wnt signaling promotes oncogenic transformation by inhibiting c-Myc-induced apoptosis J. Cell Biol., April 29, 2002; 157(3): 429 - 440. [Abstract] [Full Text] [PDF]
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