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J. Biol. Chem., Vol. 277, Issue 50, 48270-48275, December 13, 2002
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§,,,
, and
From the Second Department of Biochemistry and
¶ First Department of Internal Medicine, Hirosaki University
School of Medicine, Zaifucho 5, Hirosaki, Aomori 036-8562, Japan and
Center for Education and Research of Lifelong Learning,
Hirosaki University, Bunkyo-cho 1, Hirosaki,
Aomori 036-8560, Japan
Received for publication, August 6, 2002, and in revised form, September 19, 2002
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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MUC2 is one of the major components of
mucins that provide a protective barrier between epithelial
surfaces and the gut lumen. We investigated possible alterations of
MUC2 gene expression by p53 and
p21Sdi1/Waf1/Cip1 in a human colon cancer cell line, DLD-1,
establishing subclones in which a tetracycline-regulatable promoter
controls exogenous p53 and p21 expression. MUC2 mRNA
more significantly increased in response to p53 than to p21.
Unexpectedly, MUC2 expression was also induced in human
osteosarcoma cells, U-2OS and Saos-2, by exogenous p53. We next
performed a reporter assay to test the direct regulation of
MUC2 gene expression by p53. Deletion and mutagenesis of
the MUC2 promoter region showed that it contains two sites
for transactivation by p53. Furthermore, an electrophoretic mobility
shift assay indicated that p53 binds to those elements. We analyzed
MUC2 expression in other cell types possessing a functional p53 after exposure to various forms of stress. In MCF7 breast cancer
and A427 lung cancer cells, MUC2 expression was increased along with the endogenous p53 level by actinomycin D, UVC, and x-ray,
but not in RERF-LC-MS lung cancer cells carrying a mutated p53. These
results suggest that p53 directly activates the MUC2 gene
in many cell types.
The tumor suppressor p53 gene is frequently mutated in
a wide variety of human malignant tumors (1). p53 protein is a
transcription factor and regulates the expression of a number of growth
control genes involved in cell cycle progression, DNA repair,
apoptosis, and angiogenesis (2-4). p53 is also suggested to induce
differentiation in human tumor cells (5, 6). In such cases, although
p21Sdi1/WAF1/Cip1, one of the downstream target genes of
p53 (7, 8), might mediate regulation of a cell type-specific phenotype
through functional control of cyclin-dependent kinase and
retinoblastoma family proteins, p53 would directly regulate
tissue-specific genes related to differentiation. Furthermore, it was
suggested that p53 regulates production of the extracellular matrix,
cytoskeleton, and secreted proteins (9, 10), although the machinery for
the regulation has not yet been identified. Activation of these kinds
of genes might be closely related with cellular differentiation and
cell type-specific. It is possible that part of the genes regulated by
p53 are not always the same among cell types because of differences in
components of the transcription machinery, since the degree of
differentiation and/or genetic and epigenetic alterations could be
different. Therefore, to advance our understanding of the functions of
p53, results obtained using different cell types need to be accumulated.
Mucins are the major components of mucus, which coats the epithelia of
the intestines, airways, and other mucous membrane-containing organs.
They are thought to provide a protective, lubricating barrier against
particles and infectious agents on mucosal surfaces. MUC2 is one
of the major secreted mucins in human large and small intestine (11,
12). Several studies on MUC2 expression of colorectal carcinomas
(CRCs)1 have revealed that it
is strongly expressed in the mucinous carcinomas but decreased in
nonmucinous carcinomas compared with normal mucosa (13-16).
Furthermore, in a colon cancer cell line, its expression was increased
along with cellular differentiation (17). Regulation of MUC2 by p53
and/or p21 has not yet been demonstrated, although the p53 gene is
frequently mutated during the development of CRCs as in other cancers
(1).
To investigate the biological significance of inactivation of the p53-
and RB-signaling pathways, we have established subsets of cell lines in
which a tetracycline-regulatable promoter controls the induction of
tumor suppressor genes, and we are analyzing phenotypic alterations of
cancer cell lines by these genes (18-20). We applied our system to a
colorectal carcinoma cell line and screened alterations in the
expression of several genes related to differentiation of the colonic
mucosa, including the MUC2 gene. Here, we demonstrate that
transcription of the MUC2 gene is directly stimulated by p53
and that the activation occurs not only in colonic epithelial cells but
also in other cell types.
Cell Lines and Their Treatment--
The human colon cancer cell
line DLD-1 and human lung cancer cell line RERF-LC-MS were obtained
from the Japanese Cancer Resource Bank Cell Bank Center. The human lung
cancer cell line A427 and human breast cancer cell line MCF7 were
purchased from the American Type Culture Collection. All cells were
grown in Dulbecco's modified Eagle's medium (Nissui) supplemented
with 10% fetal calf serum at 37 °C in a humidified 5%
CO2 atmosphere. From DLD-1 cells, we established subclones
in which a tetracycline-regulatable promoter controls the induction of
p53 and p21 in the same manner as described previously (18-20). Up53-1
and Sp53-3 cells are human osteosarcoma-derived subclones in which a
tetracycline-regulatable promoter controls the induction of p53 (18,
19). These transfectants were maintained in culture medium containing
hygromycin B (0.3 mg/ml; Invitrogen) and G418 (0.5 mg/ml;
Invitrogen) with 1 µg/ml of tetracycline.
Actinomycin D, solubilized in H2O, was added to the culture
medium at a final concentration of 5 nM. Prior to UVC
irradiation, the culture medium was removed, and the cell layer was
then irradiated at 12 mJ/cm2 with a Stratalinker
(Stratagene) and further cultured in the original conditional medium.
X-ray irradiation was performed in the culture medium with 60 grays
from an MBR-1505R2 x-ray source (Hitachi Medical Corp.) at a dose rate
of 3 grays/min with settings at 5 mA and 150 kV.
Western Blotting--
Cells were lysed with lysis buffer as
described elsewhere (19). First, 50 µg of cellular protein was
separated by 8% SDS-PAGE and electroblotted to a Hybond-ECL
nitrocellulose membrane (Amersham Biosciences). Equal loading of
protein was confirmed by staining the membrane after detection. After
blocking with 4% nonfat dry milk in Tris-buffered saline, the
membranes were incubated with anti-p53 monoclonal antibody (mAb)
PAb1801 (Oncogene Science). The blots were then probed using an ECL
Western blotting detection system (Amersham Biosciences).
For detection of MUC2 protein, 50 µg of cellular protein was
separated by 3.5% agarose gel containing 0.1% SDS and 0.375 M Tris-HCl (pH 8.8) and electroblotted to a Hybond ECL
nitrocellulose membrane without methanol. After blocking, the membranes
were incubated with anti-MUC2 antibody (ccp58; BD Pharmingen). The blots were then probed using an ECL PLUS Western blotting detection system (Amersham Biosciences).
RT-PCR Analysis--
Total RNA was isolated from the cells using
a RNeasyTM Total RNA Kit (Qiagen) with DNase I. The RT-PCR
was performed in 20 µl of a reaction mixture consisting of 45 nM forward and reverse primers, 1× EZ buffer, 300 µM each dNTP, 2 units of rTth DNA polymerase (PerkinElmer
Life Sciences), and 0.1 µg of template total RNA. The primers for the
amplification of MUC2 were 5'-GAC CTC CAG CAC AGT TTT
ATC AAC A-3' and 5'-GCC AGC AAC AAT TGA CAC GTA TCT-3'. The primers for
p21 were 5'-GTG AGC GAT GGA ACT TCG ACT T-3' and 5'-GGC GTT
TGG AGT GGT AGA AAT C-3'. The primers for Cloning and Analysis of the Human MUC2 Promoter--
For the
reporter analysis of the MUC2 promoter, DNA fragments
containing MUC2 genomic sequences were amplified from normal human genomic DNA using the PCR and primers based on the DNA sequence of human genomic MUC2 (GenBankTM accession
number U67167). The amplified DNA fragment was subcloned into the
luciferase reporter vector pGL3-Basic (Promega). Deletion mutants were
generated by PCR (22). The p53 expression vector was constructed by
ligating p53 cDNA from pT2p53neo (19) into the pCMV3.1+
vector (Invitrogen). Cells were transfected using EffectenTM transfection reagent (Qiagen) in the presence of
trace amounts of phRL-TK. Luciferase assays were performed using the
Dual Luciferase Assay System (Promega), and all activity was normalized
to Renilla luciferase activity.
Electrophoretic Mobility Shift Assay--
Complementary
single-stranded oligonucleotides were labeled with
[ Fluorescent Immunostain--
Cells were seeded onto sterile
glass coverslips in six-well plates 36-48 h before fixation and
cultured in the presence or absence of 5 nM actinomycin D
for 24 h. The cells were washed in phosphate-buffered saline,
fixed in 4% paraformaldehyde for 20 min, and permeabilized with 100%
methanol for 1 min. The coverslips were washed with phosphate-buffered
saline, blocked with 8% bovine serum albumin in Tris-buffered saline
for 20 min, washed with phosphate-buffered saline, and then incubated
with the anti-MUC2 antibody (ccp58; BD Pharmingen) for 2 h.
After a wash with Tris-buffered saline three times, the coverslips were
incubated with secondary antibody (fluorescein
isothiocyanate-conjugated goat anti mouse Ig; Dako) for 1 h.
After another wash in Tris-buffered saline, the coverslips were mounted
and examined under a fluorescent microscope (Olympus).
MUC2 mRNA Levels Are Elevated in Response to p53
Induction--
To investigate possible alterations in gene expression
related to differentiation of the colonic mucosa by p53 and
p21Sdi1/Waf1/Cip1, we established subclones, in which a
tetracycline-regulatable promoter controls the induction of p53 and
p21, from a human colon cancer cell line, DLD-1, carrying mutated
p53 genes (24) (Fig. 1A). In Dp53-1 and Dp53-7
cells, exogenous p53 protein was significantly induced by the removal
of Tc, the amount produced differing between them, and a slight leakage
of expression of the introduced gene was observed. In Dp21-1 cells, the
introduced p21 gene was induced to express by the withdrawal
of Tc, whereas the amount of protein was less than that for p53
induction in Dp53-1 and Dp53-7 cells. As shown in Fig. 1B,
MUC2 mRNA was significantly more increased in response
to p53 than to p21, and its elevation was correlated with the amount of
p53 protein. In the time course experiment using Dp53-1 cells,
induction of exogenous p53 protein was observed from 6 h after the
removal of Tc (Fig. 1C). MUC2 was also detected at this time point as well as p21, an authentic p53-inducible gene
product (Fig. 1D).
To test whether the elevation of MUC2 mRNA caused by p53
was specific to the colon cancer cell line, we evaluated the expression in human osteosarcoma cell lines in which a tetracycline-regulatable promoter controls the expression of exogenous p53 (Fig.
2A). Unexpectedly, MUC2 gene expression was also increased by p53 in these
cells (Fig. 2B). In the time course experiment, Up53-1 cells
showed induction of p53 from 3 h after withdrawal of Tc, and the
p21 mRNA level was elevated as well, whereas the
increase in MUC2 mRNA was delayed and observed from
6 h. On the other hand, in another osteosarcoma cell line, Sp53-3,
alterations in p53, MUC2, and p21 expression
almost paralleled each other and were similar to the colon cancer
case.
p53 Transactivates the MUC2 Promoter at Two Sites--
Since the
increase in MUC2 mRNA expression was related to the
induction of p53 protein, we searched for candidate elements in the
MUC2 gene that may interact with p53. This gene was found to
possess two sites with convincing homology to the published p53
consensus sequence (25) in its 5' promoter region. One sequence, designated M1, is located 1100 bp and the other sequence, M2, 650 bp
upstream of the first exon (Fig.
3A). Both putative p53-binding sites in the MUC2 gene matched the consensus sequence in 18 of 20 bp. Furthermore, one more 10-bp motif, that matched the consensus sequence in 7 of 10 bp, was next to the upstream site at a distance of 1 bp in M1.
To investigate the functional significance of these sites, we cloned
the 5' promoter region of the MUC2 gene, comprising two putative p53-binding sites, into pGL3-Basic (WT MUC2 Promoter Contains Two p53 Binding Elements--
To
investigate whether p53 could in fact bind to those p53-responsive
elements identified in the MUC2 promoter, we performed an
EMSA using nuclear extract from Dp53-1 or Sp53-3 cells that were
cultured without or with tetracycline for 24 h. As shown in Fig.
4A, two oligonucleotides, M1
and M2, were bound more by the nuclear extracts from p53-induced cells
than from uninduced cells. These bindings were competed by an excess of
unlabeled M1 or M2, as well as an excess of p53-binding
oligonucleotides from the GADD45 gene, but not by the
mutated GADD45 sequence (Fig. 4B). The
anti-p53 mAb PAb1801 caused a supershift of M1 and M2 complexes
efficiently (Fig. 4C, a and c). On the
other hand, another anti-p53 mAb PAb421 efficiently supershifted M1
oligonucleotide and enhanced the binding (Fig. 4C,
upper panel, b), whereas it appeared
to inhibit the binding to M2 oligonucleotide and supershifted what
little DNA-binding complex was detected only slightly (Fig. 4C, lower panel, d). These
supershift experiments demonstrated the presence of p53 in both
complexes.
MUC2 mRNA and Protein Levels Are Elevated in Response to p53
Activation by Cell Stress--
We next determined whether
MUC2 expression was induced by the activation of endogenous
p53 through cellular stress such as actinomycin D treatment, UVC, and
x-ray irradiation (26, 27). In MCF7, in which the p53 gene
is a wild type (28), endogenous p53 protein increased in response to
these stresses to different degrees (Fig.
5A). The MUC2 and
p21 transcripts were increased along with p53
alteration, although relative amounts of MUC2 mRNA were
not always correlated with those of p21 (Fig. 5B). In A427, another cell line carrying the wild-type p53 gene (29),
endogenous p53 was also increased by stress, and the MUC2
transcripts were expressed except in the case of x-ray irradiation.
p21 transcripts increased in proportion to p53 protein. On
the other hand, in RERF-LC-MS, carrying a mutated p53 gene
(30), although p53 protein was detected and its amount changed little,
MUC2 mRNA was not detected and not increased by the
stresses. Induction of MUC2 expression along with an
increase in p53 protein caused by actinomycin D was also observed in
other cell lines, U-2OS osteosarcoma and HepG2 hepatoma carrying
wild-type p53 (31, 32), but not in Saos-2 osteosarcoma cells
lacking the suppressor gene (31) (data not shown). In MCF7 and A427
cells, an increase in endogenous p53 was observed from 3 h after
the addition of actinomycin D, with the alteration being more evident
in MCF7 than in A427 (Fig. 5C). The enhanced MUC2
and p21 expression was well correlated with p53, although
the expression of MUC2 was delayed in comparison with that
of p21 (Fig. 5D). Induction of MUC2 and
p21 expression was not observed in RERF-LC-MS up to 12 h after actinomycin D treatment.
To demonstrate the up-regulation of MUC2 protein expression in MCF7
cells caused by an increase in p53 protein following exposure to
stress, we performed immunofluorescence analysis, since it is hard to
detect MUC2 protein of more than 550 kDa (11, 12) by conventional
Western blot analysis. A representative result is shown in Fig.
6. The fluorescence was observed in some
cells at 24 h after actinomycin D treatment (Fig. 6a)
but not in the nontreated MCF7 cells (Fig. 6c). In positive
cells, fine granules were detected in the cytoplasm, particularly in
the perinuclear region (Fig. 6e). A similar positive result
was obtained in A427 cells after actinomycin D treatment but not in
RERF-LC-MS cells (data not shown).
To further confirm the increase of MUC2 protein, we carried out Western
blot analysis by using an agarose gel instead of the polyacrylamide
gel. In MCF7 and A427 cells, a smeary band was observed in the samples
treated with actinomycin D, UVC, and x-ray but not in the control (Fig.
7A). In RERF-LC-MS cells, it
was never detected (data not shown). The intensity of the smeary band was extremely increased by UVC treatment in MCF7 and was not so much
increased by actinomycin D treatment in MCF7 and A427 cells. Finally,
the smeary band was also detected in Dp53-1 cells at 12 h after
induction of wild-type p53 but not in the absence of wild-type p53
(Fig. 7B).
In this study, we demonstrated that MUC2
expression was increased along with the induction of exogenous
wild-type p53 in some carcinoma cell lines. There are two putative
p53-binding sites in the promoter region of the MUC2 gene.
Our results showed that each of them contributed to stimulation of the
promoter activity of the MUC2 gene. EMSA has revealed that
p53 binds to those p53-responsive elements. Interestingly, in EMSA
using mAbs, the binding of p53 to the distal element was enhanced by
PAb421, whereas that to the proximal element was reduced. Although the
difference would be partly due to the length and/or number of 10-bp
motifs, our result was similar to the case for the p21 gene
(33). However, the increase in MUC2 expression did not
always parallel that of p21 in the cell lines examined. To
date, many genes have been reported to be regulated by p53, and the
time course of mRNA expression differs among them (9, 34). This
would be due to differences in the stability of the mRNA and in the
context of the p53-binding sequence in the regulatory region. Moreover,
indirect effects of p53 might play an important role. Recently, it was
suggested that modification of the p53 protein such as phosphorylation
and/or acetylation determines its transcriptional properties including the selection of target genes (2-4). It would be interesting to study
the above issues to explain the differences in the regulation of
MUC2 and p21 by p53 as well as the responses by
cell lines.
In addition to exogenous p53, MUC2 expression was induced
along with the increase in endogenous wild-type p53 after cellular stress. In the immunofluorescence analysis, the staining pattern and
the percentage of MUC2-positive cells were consistent with those for
colonic goblet cells (13, 14, 16, 35). This result supports our
findings at the protein level. Overall, an increase in MUC2
expression accompanied by induction of wild-type p53 protein was
observed in colon, lung, breast cancer, osteosarcoma, and hepatoma cell
lines. Although it would not be entirely ruled out that MUC2
was induced by indirect effects of p53 or by some other process
rather than by the effects of p53 in the case of cellular stress, our
results indicate that the MUC2 gene is one of the
p53-inducible genes. The MUC2 gene was reported to be
regulated by DNA methylation of the promoter region and the Sp family
of transcription factors (15, 36, 37). However, little is known about
its regulation by cellular stress. In light of the protective function
of mucin, it is possible that cellular stress promotes the construction
of a protective barrier around damaged cells by the production of MUC2
via p53. The physical barrier constructed by MUC2 would be readily
permeable to ions and low molecular weight solutes but obstructive to
larger molecules such as damaging proteases. Therefore, the barrier
would not be effective against cellular stress but would protect the
surroundings from damaged cells.
Several clinical studies have been performed on MUC2 expression. In
CRCs, it was reported that there was an inverse association between the
immunoreactivity of MUC2 and alteration of p53 (16). Furthermore, in contrast to nonmucinous CRCs, in mucinous carcinomas, MUC2 was expressed at relatively high levels, but alterations of p53
were not so frequently detected (38). These correlations between MUC2
and p53 in CRCs would be partly due to the regulation of the MUC2 gene
by p53. On the other hand, it was shown that detection of MUC2
expression was a favorable prognostic indicator in gastric carcinomas,
intrahepatic bile duct tumors, and pancreas tumors (39-42). Although
MUC2 was suggested to be involved in the suppression of tumor formation
in mice (43), in those cases, tumors with MUC2 expression might retain
wild-type p53.
In conclusion, our results showed that MUC2 expression is
transcriptionally regulated by p53 protein in several cell lines. MUC2
protein contains more than 5100 amino acid residues and has a complex
structure with heavy glycosylation (12, 44, 45). Therefore, it is
difficult to study by conventional techniques. The biological function
of MUC2 protein remains to be clarified. It would be interesting to
determine whether MUC2 contributes to the survival of damaged cells
and/or surrounding cells and to clarify the mechanisms of another
biological function that was recently suggested by Velcich et
al. (43). Further investigation is necessary to reveal the
biological significance of the stimulation of the MUC2 gene
by p53.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin were described
previously (21). RT-PCR products were electrophoresed in a 2.5%
agarose gel and visualized by ethidium bromide staining. The integrity
of RT-PCR was confirmed by sequencing a part of the RT-PCR products.
-32P]dATP and annealed to produce the following
double-strand oligonucleotides: M1 (5'-CTG CCA TCA TGC TGC CGG CAT GTC
CCT GGC ATG TTC AGG C-3') and M2 (5'-TCC GTA ACA TGT CCC CTG GCT GGG
CAT GTA CTC CC-3'). Three micrograms of nuclear extract and 0.18 pmol
of labeled double-stranded oligonucleotide were incubated in a total
volume of 20 µl of DNA binding buffer containing 10 mM
Tris-HCl (pH 7.5), 10% glycerol, 0.05% Nonidet P-40, 1 mM
EDTA, 0.5 mM dithiothreitol, 50 mM KCl, 5 mM MgCl2, and 50 of µg/ml
poly(dI-dC)·(dI-dC) for 20 min at room temperature. For competition
experiments, a 15- or 60-fold excess of the unlabeled double-stranded
oligonucleotides was added to the binding reaction before the labeled
oligonucleotide. These competitors were M1 oligonucleotide, M2
oligonucleotide, a p53 consensus oligonucleotide from the human
GADD45 promoter 5'-GTA CAG AAC ATG TCT AAG CAT GCT GGG
GAC-3', and its mutant oligonucleotide 5'-GTA CAG AAT CGC TCT AAG AGC
TCT GGG GAC-3' (23). For a supershift assay, 0.25 µg of the
monoclonal antibodies PAb1801 and PAb421 (Oncogene Science) and normal
mouse IgGs (Santa Cruz Biotechnology) were added to the reaction
mixture at 20 min after starting the incubation, and the mixture was
incubated for a further 20 min. Samples were loaded on a native 4%
acrylamide gel in 0.5× TBE and electrophoresed at 4 °C and 150 V
for 90 min. The gel was dried and visualized using the Molecular
Analyst phosphorimaging system (Bio-Rad).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Increase of MUC2 gene
expression by p53 in a human colon cancer cell line, DLD-1.
A, a representative Western blot showing induction of p53 or
p21 expression under tetracycline control. Dp53-1 and Dp53-7 are
subclones that express p53 under the control of a Tc-regulatable
promoter. Dp21-1 is a subclone that expresses p21 in the same way.
DTA-neo is a control clone, which was isolated following transfection
of pT2p21/neo and is negative for exogenous p21. Cells were harvested
after culture for 48 h with or without 1 µg/ml Tc. The blots
were probed with antibodies against p53 and p21. B, a
representative RT-PCR analysis of MUC2 and p21.
RT-PCR was performed using mRNA isolated from the same samples as
described for A. -Actin mRNA was assayed as a
control. C, Western blot analysis of the time course of p53
expression. Dp53-1 cells were harvested after culture for 0, 3, 6, and
12 h with or without 1 µg/ml of Tc. The blots were probed with
anti-p53 mAb. D, RT-PCR analysis of the time course of
MUC2 and p21 mRNA expression. RT-PCR was
performed using mRNA isolated from the same samples as described
for C.
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Fig. 2.
Increase of MUC2 gene
expression induced by p53 in human osteosarcoma cell lines.
A, a representative Western blot showing induction of p53
expression under Tc control. Up53-1 and Sp53-3 are osteosarcoma-derived
subclones that express p53 under the control of a Tc-regulatable
promoter. Cells were harvested after culture for 48 h with or
without 1 µg/ml of Tc. The blot was probed with anti-p53 mAb.
B, a representative RT-PCR analysis of MUC2 and
p21. RT-PCR was performed using mRNA isolated from the
same samples as described for A. C, Western blot
analysis of the time course of p53 expression. Protein lysates were
prepared from cells cultured with or without 1 µg/ml of Tc at the
indicated time points. The blots were probed with anti-p53 mAb.
D, RT-PCR analysis of the time course of MUC2 and
p21 mRNA expression. RT-PCR was performed using mRNA
isolated from the same samples as described for C.
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Fig. 3.
Functional analysis of the MUC2
promoter. A, comparison of candidate DNA elements
in the MUC2 promoter with the p53 consensus binding
sequence. R, G or A; W, A or T; Y, C
or T. B, PCR-based deletions were introduced into the 1.4-kb
MUC2 promoter (WT 
1411/+27) cloned upstream of the
luciferase gene. The positions of the deletions are shown on the
left. The constructs were transfected into DLD-1 cells with
mock plasmid, pCMV, or the p53 expression construct, pCMVp53.
Luciferase activities were assayed at 48 h after transfection.
Promoter activity is indicated relative to the activity of pGL3-Basic
vector and shown on the right. Relative increase (-fold)
caused by p53 expression is indicated beside the fixed
bar. The experiments were repeated three times, and the
results are presented as the mean ± S.D.
1411/+27 construct in
Fig. 3B) and compared its response to p53 with that of
reporter vectors harboring deletions of these sites (Fig.
3B). The WT1411/+27 construct showed a 2.7-fold increase
of activity due to p53 expression. In contrast, the WT636/+27
construct, which lacked the sequence from 637 to 1411 of the
WT1411/+27 construct, displayed no remarkable increase in response to
p53 (1.1-fold). The 
1146/1097 construct, which had only the
proximal p53-binding site (M2), showed little increase of activity
(1.2-fold). The 676/642 construct, which had only the distal
site (M1), displayed a 2.0-fold increase. Finally, a slight decrease of
activity was observed in the 1146/1097 676/642 construct
(0.8-fold), which lacked both M1 and M2 due to a small deletion around
each site.
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Fig. 4.
EMSA with the p53-responsive elements and
nuclear extracts from p53-regulatable cells. A, nuclear
extracts prepared from Dp53-1 and Sp53-3 cells cultured for 24 h
with or without 1 µg/ml of Tc were incubated with labeled M1 or M2
oligonucleotides and then electrophoresed in a native acrylamide gel.
B, nuclear extract prepared from Sp53-3 cells at 24 h
after p53 induction was incubated with labeled M1 or M2
oligonucleotides in the absence or presence of a 15- or 60-fold excess
of each unlabeled competitor as indicated. A p53 consensus
oligonucleotide from the human GADD45 promoter
(wt.) and its mutant oligonucleotide (mut.) were
also used as controls. C, nuclear extract from Sp53-3 cells
with induction of p53 was incubated with labeled M1 or M2
oligonucleotides in the presence of control normal mouse IgGs, mAb
PAb1801 or mAb PAb421. The upper panels show the
results obtained using labeled M1 oligonucleotide, and lower
panels show results for the labeled M2 oligonucleotide. The
arrows at the left indicate the DNA-protein
complex. The positions of the supershifted DNA-protein-antibody complex
are indicated at the right in C.
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Fig. 5.
Increase of MUC2 expression
in cell lines with wild-type p53 in response to cellular stress.
MCF7 breast cancer cells and A427 lung cancer cells contain wild-type
p53, whereas RERF-LC-MS lung cancer cells harbor mutated
p53. A, a representative Western blot showing an
increase in p53 protein in MCF7 and A427 cells but not in RERF-LC-MS
cells due to cellular stress. Cells were harvested at 24 h after
exposure to 5 nM actinomycin D (Act. D), 12 mJ/cm2 UVC, or 60 grays of x-ray. The blot was probed with
anti-p53 mAb. B, a representative RT-PCR analysis of
MUC2 and p21. RT-PCR was performed using mRNA
isolated from the same samples as described for A. C, Western blot analysis of the time course of p53
expression after exposure to actinomycin D. Protein lysates were
prepared from cells treated with 5 nM actinomycin D at the
indicated time points. The blot was probed with anti-p53 mAb.
D, RT-PCR analysis of the time course of MUC2 and
p21 mRNA expression after exposure to actinomycin D. RT-PCR was performed using mRNA isolated from the same samples as
described for C.
View larger version (104K):
[in a new window]
Fig. 6.
Expression of MUC2 protein in MCF7 cells by
actinomycin D. After 24 h of incubation in the presence
(a, b, and e) or absence (c
and d) of 5 nM of actinomycin D, cells were
fixed and immunofluorescently stained for MUC2 protein. Cells were
photographed using fluorescence (a and c) or
bright field microscopies (b and d) of the same
field (a and b, c and d).
Fluorescence exposure time was the same in each field (a and
c). Photographs in a-d were taken at ×200. The
photograph in e was taken using a fluorescence microscope
with original magnification of ×400.
View larger version (39K):
[in a new window]
Fig. 7.
Increase of MUC2 protein in MCF7 and A427
cells in response to cellular stress and in Dp53-1 cells by induction
of wild-type p53. A, a representative Western blot
showing an increase in MUC2 protein in MCF7 and A427 cells due to
cellular stress. Protein samples were the same as described in the
legend to Fig. 5A. B, a representative Western
blot showing an induction of MUC2 protein in Dp53-1 cells. Cells were
harvested at 12 h after culture with or without 1 µg/ml Tc, and
their protein samples were the same as described in Fig. 1C.
The cell lysates were separated by 3.5% agarose gel containing 0.1%
SDS and 0.375 M Tris-HCl (pH 8.8) and transferred to
nitrocellulose membrane. The membranes were incubated with the
anti-MUC2 antibody.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
| |
FOOTNOTES |
|---|
* This work was supported by grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan and by a grant-in-aid from Aomori Medical Foundation.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. Tel.: 81-172-39-5019; Fax: 81-172-39-5205; E-mail: kookawa@cc.hirosaki-u.ac.jp.
Published, JBC Papers in Press, October 8, 2002, DOI 10.1074/jbc.M207986200
| |
ABBREVIATIONS |
|---|
The abbreviations used are: CRC, colorectal carcinoma; Tc, tetracycline; RT-PCR, reverse transcription-PCR; mAb, monoclonal antibody; EMSA, electrophoretic mobility shift assay.
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REFERENCES |
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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