From the Department of Anatomy and Cell Biology and the Shands Cancer Center, College of Medicine, The University of Florida, Gainesville, Florida 32610
Received for publication, February 9, 2001
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ABSTRACT |
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The APC
(adenomatous polyposis coli)
gene product is involved in cell cycle arrest and in apoptosis. The
loss of APC function is associated with the development of colorectal
carcinogenesis. In previous studies, we have shown that the
APC gene is inducible and that the DNA damage-induced level
of APC mRNA requires p53. In the present study, we
examined the role of p53 in the transcriptional regulation of
APC promoter and characterized two p53-binding sites on the
cloned APC promoter (pAPCP). Results of electrophoretic mobility shift assay showed specific interactions of p53 protein with p53-binding site oligonucleotides. The DNA-protein complex formed
in electrophoretic mobility shift assay was competed with unlabeled
excess of p53-binding site oligonucleotide, unaffected with p53-binding
site mutant or Sp1-binding site oligonucleotides, and supershifted with
anti-p53 antibodies. In a transient transfection assay, the pAPCP
promoter activity was lower in HCT-116(p53+/+) cells
versus HCT-116(p53 The development and progression of colon cancer is a multistep
process in which growth control is progressively impaired. Mutations of
the APC1
(adenomatous polyposis coli),
Ki-ras, deleted in colorectal cancer (DCC), DNA
mismatch repair (MMR), and p53 genes play important roles at
different stages of colorectal carcinogenesis (for review, see
Ref. 1). Mutation of the APC gene is an early event in familial adenomatous polyposis, a syndrome of inherited predisposition to colon cancer (2, 3).
Mutations in the APC gene are also found in 60-80% of
sporadic colorectal cancers and adenomas (1). Patients with
APC mutations are more prone to hundreds to thousands of
colorectal adenomas and early onset carcinoma. Familial adenomatous
polyposis patients are also prone to small intestinal adenomas (and
carcinomas), intra-abdominal desmoids and osteomas tumors (Gardener
syndrome), congenital hypertrophy of retinal pigment epithelium, fundic
gland polyps in the stomach, pancreas, and thyroid, dental
abnormalities, and epidermal cysts (for reviews, see Refs. 1 and 4).
Mutations in APC are also associated with malignant brain
tumors (Turcot's syndrome; Ref. 5), and the APC locus on
chromosoyme 5q21 shows loss of heterozygosity in ~25% of breast
cancers (6). Approximately 18% of somatic breast cancers carry
APC gene mutations (7). Furthermore, loss of heterozygosity
at the APC gene locus is prominent in the early stages of
non-small cell lung cancers (8). An animal model for carcinogen-induced
lung tumors was used to show association with a decrease in the
APC gene expression instead of an increase in APC
mutations (9).
Although APC is expressed constitutively within the normal colonic
epithelium, little is known about how mutations of (or abnormal
expression of) APC contribute to the development of colon cancer. The
APC gene product is a 310-kDa homodimeric protein localized
both in the cytoplasm and in the nucleus (Refs. 10-13; for reviews,
see Refs. 1 and 4). Previous studies indicate that the cellular level
of wild-type APC is critical to cytoskeletal integrity, cellular
adhesion, and Wingless/Wnt signaling (for reviews, see Refs. 1 and 4).
In addition, APC may act as a negative regulator of In a simple model, Wingless/Wnt signaling regulates the assembly of a
complex consisting of Axin (and its homolog Axil and conductin), APC,
Tumor suppressor p53, which plays a critical role in the cellular
response to DNA damage, oxidative stress, and hypoxia, is also
frequently mutated in colon cancer cells (for reviews, see Refs. 28 and
29). The target genes regulated by p53 include p21WAF1/Cip1, GADD45,
mdm2, bax, and cyclin G (29). The
transcriptional activation function of p53 requires post-translational
modification of the p53 protein via phosphorylation,
acetylation, or glycosylation (for reviews, see Refs. 29 and 30).
Several reports show that p53 becomes phosphorylated at the serine and
threonine residues after cells are treated with DNA-damaging agents
(31-36). In recent studies, we and others have reported that p53 is
phosphorylated on Ser15 and Ser392 in HCT-116
cells treated with DNA alkylating agents
N-methyl-N-nitrosourea, N-methyl-N'-nitro-N-nitrosoguanidine
(MNNG), and methylmethane sulfonate (MMS)
(37).2
In our earlier studies, a link between APC and p53 was established in
which APC mRNA levels were increased in a
p53-dependent manner by DNA alkylating agents (38, 39). To
further understand the mechanisms of transcriptional regulation of APC,
we recently cloned the APC promoter and identified
transcriptional regulatory elements (40). The APC gene has a
TATA-less promoter and contains consensus binding sites for octamer,
AP2, Sp1, a CAAT-box, and three nucleotide sequences for E-box A, B,
and M. In those previous studies, we showed that the APC
gene is transcriptionally regulated by upstream stimulating factors 1 and 2 (USF1 and USF2; Ref. 40). In addition, we have identified two
p53-binding elements on the APC promoter. The objective of
the present study is to characterize the p53-binding element of the
APC promoter and to examine the mechanisms by which p53
regulates DNA alkylation-induced APC gene transcription.
Cell Lines and Treatments--
Human colon cancer cell lines
HCT-116(p53 Oligonucleotides and Plasmids--
For use in electrophoretic
mobility shift assay (EMSA), complimentary single-stranded
oligonucleotides of p53-binding sites (indicated as P1 and P2) of the
cloned APC promoter (pAPCP) were annealed to produce
double-stranded oligonucleotides with the indicated sequence (see Fig.
1). Several other oligonucleotides were synthesized with
XbaI and KpnI restriction endonuclease sites on
the 5' and 3' ends of the P1 and P2 sites. Recently, we have shown that
the APC promoter contains multiple transcriptional initiation sites, i.e. +1, +52, +95, +121, +123, and +134
(40). Three major initiation sites at +1, +52, and +95 were identified by in vitro run-off transcription assay and by primer
extension analysis (40). In the present study, the P1 and P2
oligonucleotides were joined with +95 transcriptional initiation site
oligonucleotides for cloning and gene expression studies. After
annealing with the complementary oligonucleotides, DNA was digested
with XbaI and KpnI restriction endonucleases and
then subcloned into CAT reporter plasmids. The cloned plasmids and
their oligonucleotide sequences are given in Fig. 1.
Electrophoretic Mobility Shift Assay--
For EMSAs, the
recombinant human p53 protein was an overexpressed and purified from
baculovirus-infected insect cell. DNA-protein binding reactions were
carried out in 20 µl of final volume containing 20 mM
HEPES, pH 7.9, 1 mM dithiothreitol, 3.5 mM
MgCl2, 100 mM KCl, 0.03% (v/v) Nonidet P-40,
10% (v/v) glycerol, 1 µg of poly(dI·dC), and different
concentrations of purified p53 protein. Reactions were carried out for
10 min, followed by the addition of 1 ng of 32P-labeled
p53-binding oligonucleotide of the pAPCP or p21P promoter and incubated
further for 20 min at 22 °C. The entire reaction mixture was loaded
directly on a native 4% polyacrylamide gel. The electrophoresis
conditions were same as described by Gu and Roeder (41). After
electrophoresis, the DNA-protein complexes were visualized by
autoradiography. For competition experiments, a molar excess of
unlabeled oligonucleotide was added to the reaction mixture 10 min
before the addition of 32P-labeled probe, as indicated in
the figure legends. For supershift analysis, 1 µg of anti-p53
antibody Ab-1 or DO-1 was added to the reaction mixture and incubated
for 20 min before the addition of 32P-labeled oligonucleotide.
Transient Transfection of
Cells--
HCT-116(p53 Characterization of p53-binding Sites of APC Promoter Using
EMSA--
p53 binds to DNA in a sequence-specific manner and modulates
the expression of target genes (28, 29). To examine the role of p53 in
transcriptional regulation of APC, we first characterized the p53-binding element of the pAPCP promoter. Based on sequence comparisons, two potential p53-binding sites P1 and P2 of 10 base pairs each were identified at
To further examine the specificity of p53 protein binding to p21P and
P1+2 oligonucleotides, we performed a similar EMSA in the presence of
anti-p53 antibody Ab-1 or DO-1. A supershifted DNA-p53 protein complex
was formed with these oligonucleotides (Fig. 3B, lanes
2 and 3 and lanes 5 and 6,
respectively). These results indicate that p53 protein binds to the
pAPCP promoter at the P1+2 sites whose binding affinity is similar to
the p53-binding site of the p21P promoter. These results also indicate
that p53 may influence pAPCP promoter activity after binding with the
P1 and P2 sites.
Down-regulation of the pAPCP Promoter Activity in Untreated Cells
containing Wild-type versus Knock-out p53 Gene--
To investigate the
possible role of p53 in p53-mediated transcriptional regulation of
APC promoter, we used the HCT-116 cell line with the
wild-type p53 gene (p53+/+) or a homozygous cell
line with the p53 gene knock-out (p53
To further examine whether the lower activity of the pAPCP promoter in
HCT-116(p53+/+) cells was caused by p53, we overexpressed
wild-type p53 in the HCT-116(p53 Up-regulation of the pAPCP Promoter Activity in
HCT-116(p53 Role of p53-binding Sites in MNNG-induced Transcriptional
Up-regulation of the pAPCP Promoter--
Once we established that DNA
alkylation-induced regulation of the APC promoter in
HCT-116(p53+/+) cells is dependent upon p53, we examined
whether p53 and other cis-elements play a role in
transcriptional up-regulation of the APC gene. To test the
function of p53-binding sites and the role of other elements, if any,
in DNA alkylation-induced regulation of APC gene, we created
nine deletion mutants of the pAPCP plasmid. These mutants were
transfected into HCT-116(p53+/+) cells and tested for MNNG
response in a CAT reporter assay (Fig. 7). Results showed a significant increase
in the promoter activity of several deletion mutants including
pAPCP(542), pAPCP(569), pAPCP(592), pAPCP(622), pAPCP(668), and
pAPCP(737) plasmids. The pAPCP(737) plasmid containing E-box B and
E-box M sites was the minimum promoter that responded significantly to
MNNG-induced promoter activity. Further deletion of the E-box B site
(i.e. pAPCP(752)) abolished the MNNG-induced transcriptional
activity of the promoter that now contained only the E-box M site. This result suggested that MNNG treatment might activate APC gene
expression through the APC promoter E-box B site. The
maximum response of the MNNG treatment on the pAPCP promoter activity
was found with pAPCP(592) plasmid, a construct containing the
Sp1-binding site but lacking the p53-binding sites (Fig. 7). Because
further deletion of the pAPCP plasmid retained similar promoter
up-regulation until the E-box B site was deleted, the results indicated
that the E-box A and the CAAT-box might not be important for
MNNG-induced transcriptional up-regulation of the pAPCP promoter. From
these results it can be concluded that DNA alkylation-induced
expression of APC gene transcription may involve p53- and
Sp1-binding sites and the E-box B site.
Because the deletion mutation search did not firmly assign the role of
p53-binding sites in DNA alkylation-induced APC gene expression, additional experimentation will be required to determine whether p53 was involved in this regulatory process. Given that DNA
alkylation-induced pAPCP activity was higher in
HCT-116(p53+/+) cells than in HCT-116(p53
To examine whether the plasmids containing the p53-binding site respond
to MNNG treatment, we transfected pAPCP, p(P1+2)P, p(P1)P, and p(P2)P
plasmids into HCT-116(p53+/+) cells followed by treatment
with MNNG. The results showed a significant increase in the activity of
pAPCP, p(P1+2), and p(P1)P promoters; however, the activity of the
p(P2)P promoter was lower than that of p(P1)P (Fig. 8B).
Also, when both P1 and P2 sites were together, the p(P1+2)P promoter
activity was lower than p(P1)P. These results suggest that the P2 site
does play a role in p53-mediated up-regulation of the pAPCP promoter
activity in response to DNA alkylation damage.
Colon cells can operate normally if they retain one normal
APC allele and make sufficient functional APC protein (for
review, see Ref. 1). However, once both alleles are damaged, the
important brake on tumor suppression mediated by APC is lost. Previous
findings suggest that the phenotype of a cell expressing a mutated
APC gene can be reverted by increased expression of the
remaining wild-type APC allele and provide evidence that
overexpression of the APC wild-type gene alone can suppress
tumorigenicity (46-48). Thus, understanding the mechanisms by which
APC gene expression can be induced in colon cancer cells may
lead to the design of more effective chemopreventive drugs that block
tumor cell progression. In previous studies, we have shown that the
APC gene was transcriptionally activated in response to DNA
alkylation damage and that this increase was dependent upon wild-type
p53 (38, 39). In the present study, we characterized the APC
promoter for the p53-binding element and examined whether
p53-dependent transcriptional up-regulation of the
APC gene in response to DNA alkylation damage was mediated by p53 involving the p53-binding site of the APC promoter.
The structure of the APC promoter has been previously
described (40, 49, 50). Recently, we reported that APC gene
could be regulated by USF1 and USF2 through E-box sites (40). Using
electrophoretic mobility shift and supershift analysis, we found a
specific binding of p53 protein with p53-binding sites of the
APC promoter and showed that these sites are
transcriptionally functional and respond to DNA alkylation damage.
Currently, the mechanisms are unknown by which p53 interacts with the
transcriptional machinery and regulates the rate of APC gene
transcription in the presence or the absence of signals generated by
DNA alkylating agents.
p53 activates transcription from specific DNA-binding sites and
represses transcription in a binding site-independent manner. Treatment
of cells with DNA-damaging agents induces nuclear accumulation of p53,
which trans-activates cell cycle- and apoptosis-related genes (for reviews, see Refs. 28-30). p53 is post-translationally modified by phosphorylation, acetylation, or glycosylation, which could
be important in understanding how cells detect and respond to DNA
damage (for reviews, see Refs. 28-30). Because p53 is activated by a
variety of DNA-damaging agents, it appears to be a universal signaling
molecule for the DNA damage response (51). p53 is phosphorylated at
multiple sites in both the amino- and carboxyl-terminal regions of the
molecule (Refs. 31-36; for reviews, see Refs. 28-30 and 52).
The significance of the p53 phosphorylation is associated with
sequence-specific DNA binding. Recently, we reported that the p53 of
HCT-116 cells is phosphorylated at Ser15 and
Ser392 after treatment with MMS.2 Duckett
et al. (37) reported a similar phosphorylation of p53 in
HCT-116 cells after treatment with MNNG and
N-methyl-N-nitrosourea.
In the present studies, a down-regulation of APC promoter
activity might be a result of unphosphorylated form of p53, which is
present in untreated (unstressed) cells. However, once the p53 was
phosphorylated at Ser15 and Ser392 in response
to DNA alkylating agents, it activated APC gene expression. Currently, it is unclear how unphosphorylated or phosphorylated p53 is
involved in the transcriptional regulation of APC gene expression. In previous studies, it has been suggested that
phosphorylation of p53 enhances its binding activity for the DNA
consensus sequence that could strongly activate gene expression (53).
On the other hand, phosphorylation of p53 did not affect its binding
activity to DNA consensus sequence in the ribosomal gene cluster or
muscle creatine kinase (54, 55). Our recent studies showed that
phosphorylation of p53 at Ser15 and Ser392
enhanced consensus DNA binding activity but decreased the
p21 promoter activity. However, unphosphorylated p53
enhanced p21 promoter activity, but the opposite effect was
seen with APC promoter, where the phosphorylated form of p53
up-regulated activity and the unphosphorylated form of p53
down-regulated APC gene expression. One of the differences
in p21 and APC promoters is that the
p21 promoter contains a TATA-box and the APC gene
contains a TATA-less promoter (40, 42). These studies indicate that
differential phosphorylation of p53 may have differential effects on
the regulation of target genes by modulating DNA binding activities and
interacting with other transcription factors. For example, p53
represses the transcriptional activity of TATA-box-containing promoters
but either has no effect or up-regulates TATA-less promoters (56).
The interaction of p53 with the transcriptional preinitiation complex
of the TATA-less promoter of the APC gene could be through either p53-binding site or through protein-protein interaction involving the Sp1-binding site or the TFIIH complex. The results presented in this study showed that the APC promoter has
functional p53-binding sites that respond to DNA alkylation damage.
However, after DNA alkylation damage not only the wild-type or
p53-binding site-containing plasmids, but also pAPCP(592), pAPCP(622),
pAPCP(668), and pAPCP(737) plasmids showed increased promoter
activities. These results indicate that p53 may regulate APC
promoter activity through protein-protein interactions as well. The
increased promoter activity observed with the pAPCP(592) plasmid in
response to DNA alkylation damage can be attributed through one or more
than one of the following elements: the Sp1-binding site, the E-box A, B, and M sites, and the CAAT-box site. The maximum response of MNNG
treatment, observed with the Sp1-binding site-containing plasmid
pAPCP(592), was decreased with the pAPCP(622) plasmid that lacks
Sp1-binding site. Furthermore, a significantly increased promoter
activity was seen with pAPCP(737) plasmid, which did not contain p53-
and Sp1-binding sites and E-box A and CAAT-box sites; instead it
contained E-box B and E-box M sites. Further deletion of the E-box B
site from the pAPCP(737) plasmid caused the MNNG response to be lost,
indicating that E-box B site was important for MNNG-induced
APC promoter activity. Thus, the Sp1-binding site and E-box
B site may be involved in p53-mediated expression of APC
gene transcription. Furthermore, from these results, we cannot rule out
that the Sp1-binding site or the E-box B site is functioning
independently of p53 in MNNG-induced expression of APC gene
transcription. In fact, we have recently shown that the E-box B site
and the USF1 and USF2 factors binding to this site are important for
transcriptional regulation of the pAPCP promoter (40). Currently, we do
not know how p53 interacts with the Sp1-binding site or the E-box B
site of the APC promoter. However, from other studies we
know that the interaction of Sp1 and p53 can occur through a GC-rich
region (Sp1-binding site) as found with SV40 virus (57, 58) or through
protein-protein interaction as described with insulin-like growth
factor-1 receptor gene (59). Our future studies will be focused to
examine these possibilities.
TFIIH is another possible factor through which p53 can be involved in
p53-binding site-independent transcriptional up-regulation of
APC gene expression. In our preliminary studies, an
increased level of phosphorylated p53 has been detected in the TFIIH
complex of HCT-116(p53+/+) cells after treatment with
MNNG,2 which suggests a role of TFIIH complex in
p53-dependent activation of APC gene expression.
Among the general transcription factors, TFIIH is a versatile,
multifunctional protein complex with transcriptional properties coupled
to those of DNA repair (for review, see Ref. 60). If p53 of the TFIIH
complex plays a role in DNA alkylation-induced transcriptional
up-regulation of APC, then it is likely that increased expression of
APC might be associated with cell cycle arrest and/or apoptosis of
cancer cells. In fact, in previous studies a role for APC has been
implicated in cell cycle arrest (61) and apoptosis (62). Thus, p53 is
transcriptionally competent to stimulate APC gene expression
by direct interaction through p53-binding sites or by indirect
interaction through Sp1-binding site, E-box B site, or the TFIIH
complex. Nonetheless, the collaboration between p53 and APC after DNA
alkylation damage may indicate an important role for APC in the cell
cycle arrest and/or apoptosis of cancer cells. Our results further
suggest an alternative pathway for p53 to function as a tumor
suppressor involving APC, rather than p21, which is generally
associated with p53-induced G1 arrest (42, 63, 64). In
support of this hypothesis, our results suggest that p21
gene expression in HCT-116(p53+/+) cells is down-regulated
in response to MMS,2 indicating that the p53 Based on our present study, we suggest an alternative pathway for p53
function involving APC. The wild-type APC down-regulates the level of
/
) cells.
p53-dependent down-regulation was further confirmed after co-transfection of pAPCP plasmid with pCMV-p53 into
HCT-116(p53
/
) and SAOS-2 (p53-negative) cells. However,
the treatment of cells with DNA alkylating agents methylmethane
sulfonate and
N-methyl-N'-nitro-N-nitrosoguanidine, which cause phosphorylation of p53 at Ser15 and
Ser392, induced pAPCP promoter activity in
HCT-116(p53+/+) cells. Other than p53-binding sites, using
deletion mutation constructs, we have shown that
N-methyl-N'-nitro-N-nitrosoguanidine-induced transcriptional activation of the pAPCP promoter in
HCT-116(p53+/+) cells depended upon the Sp1-binding site
and the E-box B site. From these results, we conclude that
unphosphorylated p53 can down-regulate and phosphorylated p53 can
up-regulate the pAPCP promoter activity involving the p53, Sp1, or
E-box B elements. These studies are important to understanding the role
of p53 and APC in DNA damage-induced cell cycle arrest and/or apoptosis
of cancer cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-catenin
signaling in the transformation of colonic epithelial cells (14, 15),
and in melanoma progression (16).
-catenin, and glycogen synthase-3
kinase (GSK3
). Axin
(Axil/conductin) binds to APC,
-catenin, and GSK3
and thereby promotes
-catenin phosphorylation and subsequent ubiquitination and
degradation in the proteasome (17, 18). GSK3
regulates this process
by phosphorylating components of the complex (19, 20). Activation of
the Wingless/Wnt signaling pathway inhibits GSK3
and stabilizes
-catenin (21-23). Stabilizing mutations in
-catenin or
truncation in APC also occur both in colon cancer and melanoma cells
and increase the stability of
-catenin (16, 23). The stabilized pool
of
-catenin associates with members of the Tcf-Lef family of
transcription factors and regulates transcriptional expression of
proto-oncogene and cell cycle regulator c-myc (24), the
G1/S-regulating cyclin D1 (25), the gene
encoding the matrix-degrading metalloproteinase, matrysin
(26), the AP-1 transcription factors c-jun and
fra-1, and the urokinase-type plasminogen activator receptor
(27). Thus, the regulation by APC of the level of
-catenin plays a
role in
-catenin/Tcf-Lef-mediated transcriptional regulation of
genes. However, the mechanisms by which these transcriptional changes
contribute to early stage colorectal carcinogenesis are still unclear.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
) and HCT-116(p53+/+) and
SAOS-2 cell lines were grown in McCoy's 5a and Dulbecco's modified
Eagle's medium, respectively, and supplemented with 10% fetal bovine
serum (Life Technologies, Inc.) at 37 °C under a humidified
atmosphere of 5% CO2. After cells reached 70% confluence, fresh medium without fetal bovine serum was added to each dish and then
further incubated for an additional 20 h. Treatment regimens with
DNA alkylating agents MNNG and MMS (Aldrich) are given in the figure legends.
/
),
HCT- 116(p53+/+), and SAOS-2 cell lines were transfected
with the indicated plasmids using LipofectAMINE reagent (Life
Technologies, Inc.) as described earlier (39). We used p21 promoter
(p21P, cloned in pGL2 reporter plasmid), which is well characterized
for the mechanism of p53-dependent transcriptional regulation (42). For comparative purposes, the pAPCP wild-type promoter
was also cloned into a pGL2 reporter plasmid while other constructs
were cloned into the CAT reporter plasmid. Therefore, both luciferase
and CAT reporter assays were performed to determine the promoter
activity. Briefly, cells were grown to 60% confluence in 60-mm tissue
culture dishes and transfected with 1.5 µg/ml of APC promoter
constructs, 1.5 µg/ml of the p21P plasmid, 0.25 µg/ml of the
pCMV-
-gal plasmid, and 14 µg/ml of the LipofectAMINE reagent.
pCMV-
-gal served as an internal control to correct the differences
in the transfection efficiency. A DNA-lipid complex was then added to
the cells according to the recommended protocol of the manufacturer.
After 24 h of post-transfection cells were starved in a serum-free
medium for an additional 18 h and then treated with DNA alkylating
agents for 50 h. After the treatment, cellular lysates were
prepared for luciferase or CAT reporter assays. Quantitative analysis
of the data was done by electronic autoradiography using Instant Imager
from Packard Instrument Company (Meriden, CT). Luciferase activity was
measured using MonolightTM 3010 illuminometer from
Pharmingen (San Diego, CA)
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
144 to
135 nucleotides
(5'-GGGCATACCC-3') and
122 to
113 nucleotides (5'-GGGCTAGGGC-3'),
respectively. These sites are separated by 12 base pairs and have 90 and 80% homology to known consensus p53-binding sites (43, 44). The consensus DNA-binding site for p53 is defined to contain two copies of
a 10-base pair motif that are separated by 0-13 base pairs. Based on
this criterion, the P1 and P2 sites of the pAPCP plasmid appear to be
genuine p53-binding sites. Because the purpose of this study is to
elucidate the mechanisms by which the APC gene is
transcriptionally regulated by p53, it was necessary to first determine
whether the P1 and P2 sites of the pAPCP plasmid are functional
cis-regulatory elements. To begin understanding the role of
the P1 and P2 sites in the regulation of APC gene
expression, the DNA binding activity of p53 was measured in EMSA with
purified p53 and double-stranded P1 and P2 oligonucleotides from the
pAPCP (Fig. 1). In these experiments, we
also used p21P promoter oligonucleotide to serve as a positive control
(44). The p53 protein formed a complex in a
concentration-dependent manner with p21P, P1+2, P1, and P2
oligonucleotides (Fig. 2). The migration
of the DNA-p53 protein complex with p21P oligonucleotide was faster
than P-oligonucleotides because of a difference in the size of their
oligonucleotides. The DNA-p53 protein complexes formed with
32P-labeled P1+2 and p21P oligonucleotides were eliminated
by competition with an excess of unlabeled P1+2 and p21P
oligonucleotides (Fig. 3A,
lanes 2-4 and 5-7, respectively). On the other
hand, the DNA-p53 protein complex formed with 32P-labeled
P1+2 oligonucleotide was unaffected with a similar concentration of
unlabeled Sp1 or P1+2 mutant (P-mut) oligonucleotides (Fig. 3A, lanes 8-10 and 11-13,
respectively).
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Fig. 1.
Structure of the oligonucleotides used in gel
shift analysis and for cloning of p-53-binding sites of the pAPCP
promoter into the CAT reporter plasmid. Plasmid p(P1+2)P contains
both p53-binding sites ( 150 to
109 nucleotides) joined with
transcriptional initiation site at +95 (+88 to +105 nucleotides).
Plasmids p(P1)P and p(P2)P contain distal and proximal p53-binding
sites, respectively, with transcriptional initiation site at +95 (+88
to +105 nucleotides). The bold letters of the
oligonucleotide sequences indicate the p53-binding sites P1 and P2, and
the lowercase letters indicate the mutations in these sites.
The underlined letter indicates the transcriptional
initiation site of the cloned promoters.
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Fig. 2.
Complex formation of p53 protein with the
p53-binding site oligonucleotide of the APC
promoter. A gel shift analysis of purified p53 protein and
32P-labeled p53-binding site oligonucleotides (P1+2, P1,
and P2) of the APC promoter DNA was performed to determine
protein-DNA complex formation. We used consensus p53-binding site
oligonucleotide of the p21 promoter as a control
(CON, lanes 1-4). The arrows show
DNA-protein complexes and free probes. The photograph of the
autoradiograms is representative of three independent
experiments.
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Fig. 3.
Characterization of p53 binding of the
APC promoter. A shows the specificity of
p53 protein binding to the p53-binding site oligonucleotide of the
APC promoter. The p53 protein binding with
32P-labeled P1+2 oligonucleotide was competed with 50-, 100-, and 150-fold excess of unlabeled oligonucleotides. B
depicts the supershift analysis of p53 protein-P1+2 (lanes
1-3) and p53 protein-p21P DNA (lanes 4-6) complex
with p53 antibodies Ab-1 and DO-1. The arrows indicate the
position of free, shifted, and supershifted bands. The photograph of
the autoradiograms is representative of three independent experiments.
CON, control.
/
), and
the SAOS-2 cell line with a deleted p53 gene. As a control, we always performed parallel experiments with p21 promoter,
which is well known to exhibit p53-dependent
transcriptional regulation (42). First, we determined the effect of
pAPCP and p21P plasmid concentration on luciferase reporter activity in
the HCT-116(p53
/
) and HCT-116(p53+/+) cell
lines. Results showed a significantly lower pAPCP promoter activity in
HCT-116(p53+/+) cells versus
HCT-116(p53
/
) cells. On the other hand, the p21P
promoter activity was significantly higher in
HCT-116(p53+/+) cells versus
HCT-116(p53
/
) cells (Fig.
4). Results with the p21P promoter are
similar to those reported earlier (42).
View larger version (22K):
[in a new window]
Fig. 4.
Concentration effect of cloned pAPCP and p21P
plasmids after transfection into HCT-116(p53 /
) and
HCT-116(p53+/+) cells. Different concentrations of
promoter DNA were co-transfected with 0.25 µg/ml of pCMV-
-gal
expressing plasmid for 50 h. The CAT reporter activity for each
promoter construct is shown. The data were normalized to
-gal
activity in the same experiment and are the means ± S.E. of three
different experiments. *, significantly different as compared with
HCT-116(p53
/
) and HCT-116(p53+/+) cells
(p < 0.05).
/
) and SAOS-2 cell
lines to mimic the results of HCT-116(p53+/+) cells. As
expected, the overexpression of p53 into these cells significantly
decreased the pAPCP promoter activity (Fig.
5). The promoter activities of p53 target
genes p21 and PCNA (42, 45) were significantly
increased in HCT-116(p53
/
) and SAOS-2 cell lines,
respectively, after overexpression of p53 (Fig. 5). We conclude that
under normal (unstressed) conditions, p53 is involved in the
down-regulation of pAPCP promoter activity.
View larger version (51K):
[in a new window]
Fig. 5.
p53-dependent transcriptional
activity of pAPCP promoter. p53 overexpression plasmid, pCMV-p53,
was co-transfected with 1.5 µg/ml of pAPCP, p21P, or PCNA plasmids
and 0.25 µg/ml of -gal plasmid into HCT-116(p53
/
)
(A) or SAOS-2 (B) cells for 50 h. CAT
reporter activity for each promoter construct is shown. The data were
normalized to
-gal activity in the same experiment and are the
means ± S.E. of three (A) and the averages of two
(B) different experiments. *, significantly different from
controls (untransfected with pCMV-p53 plasmid) (p < 0.05).
/
) and HCT-116(p53+/+) Cell
Lines Treated with DNA Alkylating Agents MNNG and MMS--
In earlier
studies, we found a p53-dependent increase of
APC mRNA in cells treated with DNA alkylating agents
(38, 39). Nuclear run-on assays showed that nascent APC
mRNA synthesis was higher in nuclei isolated from MNNG-treated
versus untreated (control) cells, indicating that
transcriptional mechanisms were involved in the regulation of
APC after DNA alkylation damage (38). Therefore, to test
that p53 can up-regulate APC promoter activity after stress, such as DNA alkylation damage, we transfected pAPCP plasmid into HCT-116(p53
/
) and HCT-116(p53+/+) cell
lines. CAT reporter activity of the pAPCP promoter was determined after
treatment of cells with different concentrations of MMS or MNNG.
Results showed a concentration-dependent increase in the pAPCP
promoter activity in both HCT-116(p53
/
) and
HCT-116(p53+/+) cell lines. However, the extent of increase
was higher in HCT-116(p53+/+) cells than in
HCT-116(p53
/
) cells (Fig.
6). These results indicate
that in response to DNA alkylation damage the pAPCP
promoter activity is up-regulated in HCT-116 by both
p53-dependent and independent mechanisms, although a higher
increase in the pAPCP promoter activity was seen in the presence of
p53.
View larger version (42K):
[in a new window]
Fig. 6.
Treatment of HCT-116 cells with DNA
alkylating agents MMS and MNNG cause p53-dependent and
-independent activation of pAPCP promoter. 1.5 µg/ml of pAPCP
plasmid was co-transfected with 0.25 µg/ml of -gal plasmid into
HCT-116(p53
/
) and HCT-116(p53+/+) cells.
After 20 h of transfection, cells were grown in a serum-free
medium for 18 h and then treated with different concentrations of
MMS or MNNG for additional 25 h. Cells were harvested and
processed for determining the CAT reporter activity. The data were
normalized to
-gal activity in the same experiment and are the
means ± S.E. of three different experiments. *, significantly
different from untreated controls (p < 0.05).
View larger version (34K):
[in a new window]
Fig. 7.
Analysis of MNNG-responsive
cis-element(s) of the pAPCP promoter.
A describes the structure of the deletion mutant constructs
of the pAPCP promoter. B shows the CAT reporter activity.
HCT-116(p53+/+) cells were co-transfected with 0.25 µg/ml
of -gal and 1.5 µg/ml of different deletion mutant plasmids of
pAPCP promoter as indicated. After 20 h of transfection, cells
were grown in a serum-free medium for 18 h and then treated with
50 µM of MNNG for additional 25 h. Cells were
harvested and processed for determining the CAT reporter activity. The
data were normalized to
-gal activity in the same experiment and are
the means ± S.E. of three different experiments. *, significantly
different from untreated controls (p < 0.05).
/
)
cells, the involvement of p53 cannot be ruled out (Fig. 6). Thus, to
further determine whether the p53-binding sites of the pAPCP promoter
can function independently in response to DNA alkylation damage or
whether the presence of other cis-elements is required, synthetic oligonucleotides corresponding to the P1+2, P1, or P2 sites
and an oligonucleotide containing the +95 transcriptional initiation
site were cloned into CAT reporter plasmids (Fig. 1). First, we
examined whether the p53-binding site mediated the down-regulation of
the pAPCP promoter activity in the untreated cells. The p(P1+2)P, p(P1)P, and p(P2)P plasmids were co-transfected with a pCMV-p53 overexpression plasmid into HCT-116(p53
/
) cells, and
then the promoter activity was determined by CAT reporter assay. We
found no effect of p53 overexpression on the promoter activity of
p(P1+2)P, p(P1)P, or p(P2)P plasmids, which indicates that the
p53-binding site is not involved in down-regulation of the pAPCP
promoter activity (Fig. 8A).
Therefore, the down-regulation of the pAPCP promoter activity seen in
the experiments described above (Figs. 4 and 5) must be mediated by
other uncharacterized cis-elements.
View larger version (31K):
[in a new window]
Fig. 8.
Examining the role of p53-binding sites of
the APC gene in MNNG-induced transcriptional activity
of the cloned pAPCP promoter. To study the role of p53-binding
sites in the transcriptional regulation of pAPCP promoter, different
oligonucleotides containing p53-binding sites (P1+2, P1, or P2) were
synthesized and cloned into a CAT reporter plasmid. In A,
p(P1+2)P, p(P1)P, and p(P2)P plasmids were co-transfected into
HCT-116(p53 /
) cells with pCMV-p53 and
-gal
overexpression plasmids for 48 h. In B, p(P1+2)P,
p(P1)P, and p(P2)P plasmids were co-transfected into
HCT-116(p53+/+) cells with
-gal overexpression plasmid.
After 20 h of transfection, cells were grown in a serum-free
medium for 18 h and then treated with 50 µM of MNNG
for additional 25 h. Cells from A and B were
harvested and processed for determining the CAT reporter activity. The
data were normalized to
-gal activity in the same experiment and are
the means ± S.E. of three different experiments. *, significantly
different from untreated controls (p < 0.05).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
p21
pathway might not play a significant role and suggesting the existence
of an alternative pathway for MMS-induced signaling in the above cells.
-catenin, which controls the expression of
-catenin/Tcf-Lef-dependent cell cycle-related genes
c-myc and cyclin D1 (24, 25). At least in the
cell lines, where
-catenin level is regulated by APC, the increase
in the p53-dependent transcription of APC gene in response to DNA alkylating agents might block
-catenin/Tcf-Lef complex signaling and inhibit c-myc and/or cyclin
D1 gene expression. The abnormal expression of c-myc
and cyclin D1 has been shown in neoplastic transformation of
cancer cells (24, 25). In a recent study, we have reported an increased
level of APC mRNA in the lung cancer cell lines with the
decreased level of c-myc mRNA, which further establishes
an inverse relationship between APC and c-Myc (65). Thus, the increased
levels of APC and decreased levels of
-catenin might ultimately
result in decreased levels of c-Myc and cyclin D1, resulting in cell
cycle arrest and/or apoptosis of cancer cells. In summary,
these studies suggest a possible link of p53
APC
-catenin/Tcf-Lef pathway in DNA alkylation-induced cell cycle arrest
and/or apoptosis of colon cancer or other cancer cells involving
dysregulation of APC.
![]() |
ACKNOWLEDGEMENTS |
---|
The human colon cancer cell lines
HCT-116(p53/
) and HCT-116(p53+/+) and p21P
plasmid were provided by Dr. Bert Vogelstein (Johns Hopkins Oncology
Center, Baltimore, MD). The experiment with Saos-2 cells was performed
in the laboratory of Dr. Gokul M. Das (University of Texas, San
Antonio, TX). The purified recombinant p53 protein was a gift from Dr.
Daiqing Liao (University of Florida, Gainesville, FL). We are thankful
to Dr. Michael Kilberg for critically reading the manuscript, to Dr.
Virendra Kumar for technical assistance, and to Nirupama Gupta for
editorial comments.
![]() |
FOOTNOTES |
---|
* This work was supported by NCI, National Institutes of Health Grant CA77721 (to S. N.).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: UF Shands Cancer
Center, College of Medicine, Academic Research Bldg., Rm. R4-216, P.O.
Box 100232, University of Florida, Gainesville, FL 32610. Tel.:
352-846-1148; Fax: 352-392-5802; E-mail: snarayan@ufscc.ufl.edu.
Published, JBC Papers in Press, March 14, 2001, DOI 10.1074/jbc.M101298200
2 A. S. Jaiswal and S. Narayan, submitted for publication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
APC, adenomatous
polyposis coli;
GSK3, glycogen synthase-3
kinase;
-gal,
-galactosidase;
CAT, chloramphenicol acetyltransferase;
MMS, methylmethane sulfonate;
MNNG, N-methyl-N'-nitro-N-nitrosoguanidine;
pAPCP, adenomatous polyposis coli gene promoter;
USF, upstream
stimulating factor..
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