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Metabolism and Cancer Susceptibility Section, Basic Research Laboratory, Division of Basic Sciences, National Cancer Institute [J. M. M., N. G. H., S. P. D., J. M. P], and Intramural Research Support Program, Science Applications International Corporation-Frederick [D. F. W.], National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21702
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ABSTRACT |
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Introduction |
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We have recently reported that ß-catenin/LEF-1 DNA binding complex may play a role mediating the overexpression of prostaglandin endoperoxide H synthase-2 induced by NO (9) . To further explore the connection between Apc, ß-catenin, and NO, we examined the formation of nuclear ß-catenin/LEF-1 DNA binding complex as well as the increase of cytoplasmic ß-catenin in response to NO-releasing drugs in two nontransformed and nontumorigenic murine colonic epithelial cells with distinct Apc genotypes. In this pair of cell lines with similar genetic backgrounds, one carries the ApcMin/+ mutation and is associated with defective degradation and the greater accumulation of ß-catenin. These conditionally immortal cells are designated YAMC (Apc+/+) derived from a SV40LT antigen parental mouse and IMCE (ApcMin/+) derived from the F1 hybrids resulting from the mating of ApcMin/+ and SV40LT antigen transgenic mice. Because both YAMC and IMCE express the heat-labile SV40LT antigen that allows them to proliferate at 33OC, they revert to a nontransformed phenotype at the restrictive temperature of 39OC, at which the proliferation of these cells ceases (10, 11, 12) . The temperature-sensitive SV40LT mutant antigen becomes inactivated and nonfunctional when cells are transferred to 39°C for 72 h before each experiment. The genotype and expression of APC protein have been confirmed in these cells by allele-specific PCR and by Western immunoblotting, respectively (10) . The epithelial nature of these cells was demonstrated by staining with antikeratin antisera (11) . Furthermore, these cells have been used to demonstrate that the ApcMin/+ mutation in IMCE cells can cooperate with stably transduced oncogenic ras to produce the transformed, tumorigenic phenotype (e.g., growth in soft agar; tumor formation in athymic mice; 13 ). All of the experiments in this study were carried out under nonpermissive and nonproliferative conditions.
Data from the present study suggest that NO may increase cytoplasmic ß-catenin levels and, therefore, enhance the differential formation of ß-catenin/LEF-1 DNA binding complexes in YAMC and IMCE cells under these conditions. Significantly, the increased formation of nuclear ß-catenin/LEF-1 DNA binding complex via accumulation of free ß-catenin in the cytoplasm is differentially expressed based on their respective Apc genotypes.
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Materials and Methods |
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from Life Technologies, Inc.
(Grand Island, NY); neonatal calf serum from Gemini Bio-Products
(Calabasas, CA), ITS+ from Collaborative Biomedical (Bedford, MA). The
NO donors NOR-1 and SNAP were purchased from Calbiochem (San
Diego, CA). All other chemicals and reagents were purchased from Sigma
unless indicated otherwise.
Cell Culture.
Experiments were carried out using the conditionally immortalized
murine colonic epithelial cell (9, 10, 11, 12)
. Both YAMC and IMCE
cells express the heat-labile SV40 large T antigen with an
IFN--inducible promoter. The temperature-sensitive SV40 large T
antigen is inactivated and becomes nonfunctional if the temperature is
changed to 39°C. All of the cells were grown in
75-cm2 culture flasks coated with type I collagen
(5 µg/cm2) in RPMI 1640 supplemented with 5%
neonatal calf serum, ITS+ (6.25 µg/ml insulin, 6.25 µg/ml
transferrin, 6.25 ng/ml selenious acid, 5.35 mg/ml linoleic acid, and
1.25 mg/ml BSA), 5 IU/ml murine IFN-, 100,000 IU/liter penicillin,
and 100 mg/liter streptomycin. They were cultured under transforming
(permissive) conditions in a 33°C incubator with 5%
CO2. On attaining confluency, all of the cells
were then transferred to nontransforming (nonpermissive) conditions at
39°C in serum-free and IFN--free media for 72 h before each
experiment.
Preparation of Nuclear Extracts and EMSAs.
Nuclear extracts were prepared from YAMC and IMCE cells according
to the method described previously (9)
. Briefly, cells
were rinsed once with cold PBS followed by trypsinization. After
centrifugation at 1000 x g for 5 min, they
were resuspended in 5-pellet volumes of hypotonic buffer
containing 0.2 mM phenylmethylsulfonyl fluoride
and 0.5 mM DTT. They were then chilled on ice for
10 min followed by lysis with a PT 3000 Polytron (Brinkmann,
Littau, Switzerland) for 30 s at 14,000 rpm and centrifuged at
4000 x g for 15 min. The pellet was
resuspended in 0.5-pellet volumes of low-salt buffer. An equal
volume of high-salt buffer was added drop-wise to the gently stirred
suspension. The nuclear extracts were subjected to centrifugation at
16,000 x g for 30 min followed by overnight
dialysis. Nuclear protein (5 µg) was added to a 20-µl reaction mix
containing 300 ng of poly(dI·dC); binding buffer [10
mM HEPES (pH 7.6), 60 µM
KCl, 1 mM EDTA, 1 mM DTT,
and 12% glycerol]; with or without double-stranded mouse LEF-1
oligonucleotide (Life Technologies, Inc.), CACCCTTTGAAGCTC with 5'
overhang, as a specific competitor. Samples were incubated on ice for
10 min. Then LEF-1 oligonucleotide, radio-labeled using T4 kinase (Life
Technologies, Inc.) and [-32P]ATP (NEN,
Boston, MA), was added at 1.5–2 x 104 cpm per reaction and incubated at room
temperature for 30 min. DNA loading dye (Quality Biological,
Gaithersburg, MD) was added to stop the reaction. Samples were run on a
4% polyacrylamide (37.5:1; Protogel; National Diagnostics, Atlanta,
GA) gel at 189V for 2.5 h in 0.5 x Tris-borate
EDTA running buffer. Gels were dried and exposed to XAR-5 film
(Kodak). For super-shift studies, 3–5 µg of nuclear lysate were
mixed in a 20-µl reaction mixture as described for EMSA, and that
mixture was incubated on ice for 10 min. Antibodies, 12 µg of
polyclonal anti-ß-catenin and 500 ng of monoclonal anti-E-cadherin,
or rabbit IgG, were then added to the respective reaction tubes.
Reactions were incubated on ice for 15 min.
-32P-labeled murine LEF-1 oligonucleotide
probe was added at 1.5–2 x 104
cpm per reaction and incubated at room temperature for 30 min.
Western Blot Analysis.
Briefly, cells were washed twice with cold PBS and were harvested
either under denaturing conditions by scraping in boiling 2x Laemmli
sample buffer (Bio-Rad, Hercules, CA) or under nondenaturing conditions
by using a radioimmunoprecipitation assay buffer set (RIPA; Boehringer
Mannheim). For total cell lysates under denaturing conditions, samples
were heated at boiling temperature for an additional 5 min in Laemmli
sample buffer. Homogenates were then prepared by sonication (1 min
each; Sonifier; Branson, Danbury, CT). After centrifugation at
2000 x g for 5 min, the supernatants were
used as the protein source. To make protein preparations that would
contain only soluble cytoplasmic fractions, cells were lysed in
radioimmunoprecipitation assay buffer under nondenaturing conditions at
4°C. They were then incubated on ice for 15 min on a shaker. The
homogenized soluble supernatants were prepared by centrifugation at
100,000 x g for 30 min at 4°C. The protein
concentration was determined by the bicinchoninic acid method (Pierce,
Rockford, IL). Electrophoresis samples were prepared by mixing the
respective protein preparations with 2x Laemmli sample buffer. To each
well of SDS-polyacrylamide gel, 15–30 µl of cell lysate (about
15–30 µg of total protein) were applied and electrophoresed in
0.75-mm thick 7.5% Tris-Glycine gel (Bio-Rad, Hercules, CA). Proteins
were transferred to nitrocellulose membrane using a semidry blotter
(Bio-Rad) at 15V for 30 min. Blots were then probed with the respective
primary antibodies at the manufacturer’s suggested dilution followed
by a secondary antimouse IgG antibody conjugated to horseradish
peroxidase (1:2000). Detection was by an ECL kit (Amersham,
Arlington Heights, IL). Blots were routinely stripped by Encore Blot
Stripping Kit (Novus Molecular, Inc., San Diego, CA) and reprobed with
anti-actin monoclonal antibody to serve as loading controls.
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Results |
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, IMCE cells had a slightly higher basal expression of this complex than
did YAMC under these conditions. This is consistent with the defective
ß-catenin degradation machinery attributable to the
ApcMin/+ mutation in IMCE cells. It
suggests that some ß-catenin accumulates in resting IMCE cells
instead of being degraded rapidly as occurs in YAMC, and, consequently,
more ß-catenin/LEF-1 DNA binding complex formed in the nucleus. As we
reported recently (9)
, the formation of the
ß-catenin/LEF-1 DNA binding complex was greatly enhanced by the two
NO donors, NOR-1 and SNAP, which both release NO spontaneously when
dissolved in aqueous solution and do not share any structural
similarity. The amount of ß-catenin/LEF-1 DNA binding complex induced
by either NOR-1 or SNAP appeared to be concentration- (Fig. 1A
) and time-dependent (Fig. 1, B and C
), which indicated the specificity of the NO effects. In
all of the EMSA experiments, an excess amount of unlabeled LEF-1 probe
effectively competed away the ß-catenin/LEF-1 DNA binding complex,
which demonstrated the participation of LEF-1 in the complex and
indicated its specificity. More importantly, the difference in the
ß-catenin/LEF-1 DNA binding complex formation between YAMC and IMCE
cells widened markedly in response to NO, which indicated that APC may
be involved.
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). Nonspecific antibodies, namely anti-E-cadherin antibody and rabbit
preimmune IgG, were used as controls in the super-shift assays and did
not cause any change in the migration pattern. As shown earlier,
unlabeled LEF-1 probe effectively competed away the ß-catenin/LEF-1
DNA binding complex (Fig. 2
). This study directly demonstrates the
presence of ß-catenin in the DNA binding complex in conjunction with
LEF-1. More interestingly, the difference in ß-catenin/LEF DNA
binding complex formation between YAMC and IMCE was evident and was
markedly amplified by NO treatment. Both NOR-1 and SNAP showed similar
effects on the induction of this complex formation, although NOR-1
appeared to be more potent than SNAP (Fig. 2B
). Because
these two agents have different kinetics in terms of NO release, they
may, therefore, have different patterns of concentration- and
time-dependence on the complex formation as suggested in Fig. 2B
. Nevertheless, ß-catenin was detected by super-shift in
the complex formed in response to either drug.
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). No E-cadherin was detected in these soluble cytoplasmic
fractions (data not shown), which suggests that the assayed ß-catenin
was no longer bound to E-cadherin. In contrast, the amounts of total
ß-catenin in whole-cell lysates were examined and did not change
after various treatments with the NO donors, NOR- 1 (5
µM) and SNAP (10 µM;
see Fig. 3B
). Although this finding does not conclusively
reveal the mechanism responsible for the increase in the free
cytoplasmic pool of ß-catenin, it implies that NO may facilitate the
nuclear ß-catenin/LEF-1 complex formation by increasing the amount of
free cytoplasmic ß-catenin available for its translocation into the
nucleus. Because these nontransformed and nontumorigenic cells have
little free ß-catenin in the cytoplasm, as demonstrated in Fig. 3A
, it is likely that NO caused the disruption of the
association between ß-catenin and membrane-bound E-cadherin, thereby
releasing it into the cytoplasm. As we know, YAMC and IMCE cells differ
in ß-catenin degradation because of their genetic difference in
Apc. We found that IMCE cells had much higher amounts of
free ß-catenin accumulated in the cytoplasm in response to NO (Fig. 3A
). Importantly, the total amount of ß-catenin was not
different between the two cell lines nor did either amount
change after NO exposure (Fig. 3B
). These data suggest that
the accumulation of ß-catenin in cytoplasm was probably the net
result of dissociation from the membrane and the degradation by APC,
GSK-3ß, and Axin. The NO-stimulated turnover of ß-catenin was
further examined in a time course study (Fig. 4
). NOR-1 treatment, as early as 10 min, markedly increased free
ß-catenin accumulation in the cytoplasm of both YAMC and IMCE cells,
a finding that supports the dissociation of ß-catenin from the
membrane and militates against alteration of de novo
synthetic mechanisms. Corroborating the results shown in Fig. 3
, we
found that cytoplasmic levels of ß-catenin reached much higher levels
in IMCE cells than in YAMC cells. It seems likely that the
defective degradation apparatus for ß-catenin in IMCE cells results
in a higher level of ß-catenin accumulation once ß-catenin is freed
from membrane. The mechanism by which NO causes the dissociation
of ß-catenin from membrane-bound E-cadherin remains unknown but
is currently under vigorous investigation in this laboratory.
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Although free ß-catenin is kept at a very low level in the
cytoplasm and nucleus by APC mediated degradation, an abundant amount
of ß-catenin is normally associated with the transmembrane
glycoprotein E-cadherin. The E-cadherin-bound ß-catenin is also
called the "insoluble" ß-catenin in that it is not free to
interact with other proteins in the cytoplasm. It has been reported,
however, that the dissociation of ß-catenin from E-cadherin can
increase free soluble ß-catenin in the cytoplasm, and, importantly,
the association of ß-catenin and E-cadherin can be affected by a
variety of signals directly or indirectly (17, 18, 19)
. It is
now known that activation of the membrane-bound metalloprotease can
cause the loss of ß-catenin from E-cadherin and cell-cell contacts
and can, thereby, activate the ß-catenin-mediated intracellular
signaling pathway (20)
. The metalloprotease-induced
dissociation and intracellular translocation of ß-catenin can be
facilitated by intracellular calcium influx (20)
.
Interestingly, it is also documented that NO can activate this
membrane-bound metalloprotease (21
, 22)
. NO has been found
to increase intracellular calcium as well (23, 24, 25)
. Our
data, as demonstrated in Fig. 3
and 4
in particular, suggest that NO
may be able to facilitate such dissociation and thereby increase free
ß-catenin in the cytoplasm and nucleus. The morphological changes of
NOR-1 treated cells, namely the enlargement as well as the swelling and
blebbing of the cells (9)
, could be attributable to the
dissociation of the ß-catenin and E-cadherin complex at the membrane
and the subsequent disruption of the adherens junctions at the
cell-cell border (17, 18, 19)
. Our demonstration that NO may
cause the possible dissociation of membrane-bound ß-catenin and the
formation of nuclear ß-catenin/LEF-1 DNA binding complex may be
important events in the early stage of colonic carcinogenesis. We are
currently investigating whether the effect of NO on intracellular
calcium mobilization and activation of matrix metalloprotease is
causally related to its effect on the dissociation of ß-catenin from
membrane-bound E-cadherins.
Another possible mechanism for NO to exert its effect on cytoplasmic ß-catenin accumulation is through the disruption of the ß-catenin degradation apparatus, namely the connections among APC, GSK-3ß, Axin, and ß-catenin, the "tetrameric" interactions. For this degradation process to be effective and efficient, regulated interactions are essential among the proteins involved. For example, the recruitment of GSK-3ß and ß-catenin by Axin must take place prior to the phosphorylation of ß-catenin, which is crucial for its subsequent degradation by APC (16) . This complex interaction presumably is redox-sensitive and, therefore, could be affected by NO. In addition, the mechanism by which ß-catenin is transported from the cytoplasm to nucleus remains unclear. In the context of nuclear protein trafficking, it is plausible that there is a specific transporter protein that facilitates the crossing of the nuclear membrane by ß-catenin. Whether NO plays any role in this translocation remains an intriguing question.
Although the exact role of NO in tumor biology is not completely understood, NO is undoubtedly a potent mediator in the processes of inflammation, infection, and cancer. NO and NOS II have been suggested to play important roles in the early events of colonic carcinogenesis (26 , 27) . Furthermore, NO has been found to have important interactions with ECM metabolism (28 , 29) . In addition to its effect on matrix-associated metalloprotease mentioned earlier (21) , NO, produced by inflammatory cells such as macrophages, has been reported to affect the turnover of ECM of neighboring cells and to influence cellular attachment, migration, and proliferation through its interactions with ECM (28 , 29) . The finding that NO can trigger the accumulation of free soluble cytoplasmic ß-catenin and the formation of ß-catenin/LEF-1 DNA binding complex, as presented in this study, supports the hypothesis that NO may be involved in the early stages of colorectal carcinogenesis. This may be especially important when genetic predisposition, such as ApcMin/+ mutation, is concurrently present.
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Metabolism and Cancer Susceptibility Section, Basic
Research Laboratory, Division of Basic Sciences, National Cancer
Institute, Room 12-90, Building 560, Frederick, MD 21702. Phone: (301)
846-5368; Fax: (301) 846-6093; E-mail: Mei{at}mail.ncifcrf.gov 
2 Present address: Department of Food Science and
Human Nutrition, Michigan State University, 2100 Anthony Hall, East
Lansing, MI 48824-1225.
3 The abbreviations used are: Apc,
adenomatous polyposis coli; NO, nitric oxide; NOS II, NO synthase II;
LEF, lymphoid enhancer factor; Tcf, T-cell factor; GSK-3ß, glycogen
synthase kinase 3ß; YAMC, young adult mouse colon; IMCE, immortal
mouse colon epithelium; NOR-1,
(E)-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexeneamide;
SNAP, S-nitroso-N-acetylpenicillamine;
EMSA, electrophoretic mobility shift assay; ECM, extracellular
matrix.
Received 3/20/00. Accepted 5/17/00.
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