From the Unité INSERM 377, Place de Verdun,
59045 Lille Cedex, France and Laboratoires ** de Biochimie et de
Biologie Moléculaire and ¶ d'Endocrinologie de
l'Hôpital C. Huriez, Centre Hospitalier Régional et
Universitaire, 59037 Lille Cedex, France, and
Faculté de
Médecine, Université de Lille II,
59045 Lille France
Received for publication, November 21, 2000, and in revised form, January 29, 2001
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ABSTRACT |
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In gastric cancer, altered expression
of MUC1, MUC2, MUC5AC, and MUC6 mucin genes has
already been described. We show in this report by the means of in
situ hybridization, reverse transcriptase-polymerase chain
reaction, and transfection assays that MUC5B is also
abnormally expressed in gastric carcinomatous tissues and cell lines.
We thus undertook to elucidate the molecular mechanisms that regulate the transcription of MUC5B in gastric cancer cells. To this
end, high expressing (KATO-III) and low expressing (AGS) gastric cancer cell lines were chosen to study human mucin gene MUC5B
expression and promoter activity. Sequencing of the promoter region
revealed a distal TATA box located 1 kilobase upstream of the proximal TATA box. Functional activity of the promoter was addressed by using
deletion mutants covering 2044 nucleotides upstream of the MUC5B transcription start site. We identified a distal
promoter 10 times more active than the proximal promoter in KATO-III
cells. In AGS cells, both promoters, much less active, showed the same range of activity. Binding assays allowed us to show that the transcription factor ATF-1 binds to a cis-element present
in the distal promoter. Sp1, which binds to both promoters specifically transactivates the proximal promoter. Treatment of transfected cells
with PMA, cholera toxin A subunit, and calcium ionophore A23187 showed
that only PMA led to a substantial activation of the distal promoter.
MUC5B 5'-flanking region having a high GC content,
influence of methylation on the MUC5B expression was assessed. Our results indicate that repression of MUC5B
expression visualized in AGS cells is due in part to the presence of
numerous methylated cytosine residues throughout the 5'-flanking
region. Altogether these results demonstrate that MUC5B
expression in gastric cancer cells is governed by a highly active
distal promoter that is up-regulated by protein kinase C and that
repression is under the influence of methylation.
Mucins are high molecular weight O-glycoproteins
synthesized by epithelial cells as large secreted or membrane-bound
glycoproteins (1). So far, eight mucin genes have been well
characterized (MUC1, MUC2, MUC3,
MUC4, MUC5AC, MUC5B, MUC6,
and MUC7) (1), and cDNAs have been proposed for
MUC8, MUC9, MUC11, and
MUC12 (1, 2). Numerous studies have now demonstrated that
the expression of mucin genes is tissue- and cell-specific and that their expression is altered during the pathogenesis of several diseases, which suggests that human mucin gene expression is tightly regulated and that they may play important roles during cell
differentiation and carcinogenesis (3-8).
In normal stomach, MUC5AC is expressed at the
surface/foveolar epithelium and MUC6 in the mucous neck
cells and in the antral glands (9-11). Other mucin genes expressed in
normal gastric mucosae are MUC1 and to a lesser extent
MUC2, MUC3, and MUC4. In gastric carcinomas, a decrease of MUC1, MUC5AC and MUC6
expression and an increase of MUC2, MUC3, and
MUC4 expression has already been demonstrated (11-15).
Human mucin gene MUC5B has been extensively studied in our
laboratory. Studies of MUC5B genomic sequence (39.1 kb)1 showed that it encodes a
high molecular weight polypeptide (627,000) (16-20). MUC5B
mucin gene is localized on chromosome 11 (band p15) and is clustered
with three other mucin genes: MUC2, MUC6, and MUC5AC (21). In normal adult, MUC5B is
essentially expressed in trachea, bronchi, submaxillary glands,
pancreas, gallbladder, and endocervix (4, 22-24). In cancer,
MUC5B has been shown to be highly expressed in colon
carcinoma (5), in HT-29 treated with methotrexate (19, 20, 25), and
LS174T (20, 24, 26) mucus-secreting colon cancer cell lines.
We recently characterized the first 956 nucleotides located upstream of
MUC5B transcription start site and studied the promoter functional activity in colon cancer cells (20). The region is characterized by the presence of a TATA box and numerous putative binding sites for ubiquitous (Sp1) and specific transcription factors
(NF- In this report, we show that MUC5B is abnormally expressed
in gastric carcinomatous tissues and cell lines. Computer analysis of
the genomic sequence upstream of the MUC5B transcription
start site revealed the presence of a distal TATA box. Numerous
putative binding sites for Sp1, CREB, ATF-1, and AP-1 transcription
factors were found adjacent to this TATA-box. Binding assays showed
that the nuclear factors Sp1 and ATF-1 bind to the distal promoter. The
functional activity of the two promoters of MUC5B was
studied in two gastric cancer cell lines that either show a high
(KATO-III) or a low (AGS) level of MUC5B mRNA
expression. From these studies, a distal region highly active in
KATO-III cells was identified. Moreover, this region was shown to be
highly methylated in AGS cells in accordance to the low level of
MUC5B mRNA transcripts found in these cells.
In Situ Hybridization--
Specimens of tumoral and normal
gastric mucosae were obtained from six patients undergoing gastrectomy
for gastric carcinoma (cardia, well differentiated (n = 2); fundus, mucous type (n = 1); fundus, well
differentiated (n = 1); antrum, well differentiated (n = 1); and antrum, moderately differentiated
(n = 1)). Each specimen was immediately immersed in 4%
paraformaldehyde in phosphate buffer and further embedded in paraffin.
Sections 3-µm-thick were cut and mounted onto gelatin covered slides.
Adjacent sections from the same blocks were systematically stained with
hematoxylin-eosin-safran and Astra blue for a histological control.
In situ hybridization was performed using a specific
MUC5B 35S-labeled oligonucleotide probe. The
48-mer oligonucleotide antisense probe
(5'-TGTGGTCAGCTCTGTGAGGATCCAGGTCGTCCCCGAGTGGAGAGGG-3') was chosen in
the tandem repeat domain of MUC5B (27). The labeling of the
probe and the hybridization steps were as described in Ref. 4. Controls
consisted in a treatment of tissue sections with a large excess of
unlabeled oligonucleotide identical or distinct from the
MUC5B radiolabeled probe.
Cell Lines and Cell Culture--
The KATO-III and AGS gastric
adenocarcinoma cell lines were purchased from European Collection of
Cell Culture (Salisbury, UK) (28, 29). KATO-III cells were
cultured in RPMI 1640 medium supplemented with 20% fetal calf serum
(Roche Diagnostics, Meylan, France). AGS cells were cultured in Ham's
F-12 medium supplemented with 10% fetal calf serum. Both cell lines
were maintained in a 37 °C incubator with 5% CO2. The
inhibitor of methylation, 5-aza-2'-deoxycytidine (5 µM)
(Sigma Saint-Quetin Fallavier, France), was added to confluent cells,
and cells were cultured in the presence of the chemical reagent for 3 more days before being harvested in appropriate buffer to prepare total RNA.
Cloning--
Inserts were prepared using the restriction map of
the cosmid clone called ELO9 (30), which covers the 5'-flanking region of MUC5B. Gel purified fragments (QIAquick gel extraction
kit, Qiagen, Courtaboeuf, France) were subcloned into the promoterless pGL3 Basic vector (Promega, Charbonnières, France). Internal deletion mutants were generated by PCR using pairs of primers bearing
specific restriction sites at their 5' and 3' ends (see Table I). PCR
products were digested, gel purified and subcloned into the pGL3 vector
that had been previously cut with the same restriction enzymes. All
clones were sequenced on both strands on an automatic LI-COR sequencer
(ScienceTec, Les Ulis, France) using infrared labeled RV3 and GL2
primers (Promega). Plasmids used for transfection studies were prepared
using the Endofree plasmid Mega kit (Qiagen).
RT-PCR--
Total RNAs from gastric cancer cells were prepared
using the RNeasy midi-kit from Qiagen. Cells were harvested at 70% of
confluence, and 1.5 µg of total RNA was used to prepare cDNA
(AdvantageTM RT-for-PCR kit, Clontech,
Ozyme, France). PCR was performed on 5 µl of cDNA using specific
pairs of primers for MUC5B mucin gene (MUC5B
forward primer: 5'-CTGCGAGACCGAGGTCAACATC-3'; MUC5B reverse primer: 5'-TGGGCAGCAGGAGCACGGAG-3' (nucleotides 9057-9078 and nucleotides 10108-10127; accession number Y09788) (17). The PCR
product expected size is 415 bp. Single-stranded oligonucleotides were
synthesized by MWG-Biotech, Germany. Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control. PCR reactions and PCR
product analyses were carried out as previously described (20).
Oligonucleotides and DNA Probes--
The oligonucleotides used
for gel shift assays are indicated in Table II. They were synthesized
by MWG-Biotech (Ebersberg, Germany). Equimolar amounts of
single-stranded oligonucleotides were annealed and radiolabeled using
T4 polynucleotide kinase (Promega) and [ Primer Extension--
Primer extension reactions were performed
using 25 µg of total RNAs prepared from KATO-III and AGS cells as
above and from human trachea (Clontech). Annealing
and labeling of the exon 1 (5BOAS): 5'-TGCCTGCGGCACCACGAGCATG-3' and
NAU 647: 5'-TCCCTGGTCACCAGCGTCCTG-3' reverse primers and extension
reactions were performed as previously described (20). Transfections--
Transfections were performed using
Effectene® reagent (Qiagen). Cells were passed the day before the
transfection. Transfection conditions were optimized to 1 µg of DNA,
5 µl of Effectene®, and 0.5 × 106 cells/well in a
6-well plate. Transfected cells were then incubated for 48 h at
37 °C. Total cell extracts were prepared using 1× reagent lysis
buffer (Promega) as described in the manufacturer's instruction
manual. Results were corrected for transfection efficiency by
cotransfecting 0.1 µg of pSV- Nuclear Extract Preparation--
Nuclear extracts from cell
lines of interest were prepared as described in (31) and kept at
Electrophoretic Mobility Shift Assays--
Nuclear proteins (5 µg) were preincubated for 20 min on ice in 20 µl of binding buffer
with 2 µg of poly(dI-dC) (Sigma) and 1 µg of sonicated salmon sperm
DNA. Radiolabeled DNA probe was added (120,000 cpm/reaction), and the
reaction was left for another 20 min on ice. For super-shift analyses,
1 µl of the antibody of interest (anti-Sp1, anti-Sp2, anti-ATF-1,
anti-CREB-1, and anti-HoxD9; TEBU, Le Perray en Yvelines, France) was
added to the proteins and left for 1 h on ice before adding the
radiolabeled probe. Cold competition were performed by preincubating
the nuclear proteins with an excess (×50) of the cold oligonucleotide
for 20 min before adding the radiolabeled probe. Negative controls were
carried out using 1 µl of irrelevant antibody in the reaction mixture. Reactions were stopped by adding 2 µl of loading buffer and
loaded onto a 4% nondenaturing polyacrylamide gel, and electrophoresis conditions were as described in Ref. 20. Gels were vacuum dried and
autoradiographed overnight at Preparation of Genomic DNA for Methylation Studies--
Genomic
DNA was prepared using a blood and cell culture DNA mini kit (Qiagen).
20 µg of genomic DNA was submitted to an overnight digestion with
BamHI (50 units) at 37 °C. To study methylation, BamHI-digested DNA was ethanol precipitated and submitted to
either a HpaII (methylation-sensitive, 40 units) or a
MspI (methylation insensitive, 40 units) digestion overnight
at 37 °C. Digested DNA was then loaded on a 2% agarose gel.
Electrophoresis was run in 1× Tris-borate-EDTA buffer. After
electrophoresis, denatured DNA was transferred onto a nylon membrane
(Biotrans +, 0.45 µm; ICN, Orsay, France) in 20× SSC buffer
overnight and UV cross-linked for 4 min. The membrane was first
incubated in prehybridization buffer (6× SSC, 5× Denhardt's
solution, 0.5% SDS) for 3 h at 65 °C followed by a 3-h
incubation at 65 °C with 1450 and 1896 DNA probes (1 × 106 cpm/lane) in hybridization buffer (6× SSC, 5×
Denhardt's solution, 0.1% SDS, 10% dextran sulfate (w/v), 0.25 mg/ml
herring sperm DNA). Excess of the probe was washed off with 10 ml of
0.1× SSC, 0.1% SDS for 15 min at 65 °C, and the wash was repeated
once. The blot was then rinsed with 3× SSC and autoradiographed for a
few days at DNA Sequence and Transcription Factor Binding Site
Analyses--
DNA sequences were analyzed using PC-GENE software, and
the TRANSFAC 4.0 data base was used to define potential transcription factor binding sites within the clones of interest. The search was
conducted using MatInspector V2.2 software (32).
Expression of MUC5B in Gastric Adenocarcinoma and Gastric Cancer
Cell Lines--
Expression of MUC5B was analyzed in gastric
adenocarcinoma and normal resection margins from six patients
undergoing gastric resection using in situ hybridization.
MUC5B was not detected in the specimens of normal gastric
mucosa (6/6) (Fig. 1A). In contrast, a signal was detected with the MUC5B probe in four
of six specimens of gastric carcinomas (all well differentiated). The
labeling was distributed heterogeneously throughout the tumoral glands
(Fig. 1B). The hybridization procedure was repeated several times. Competition studies checked the validity of the signal. The
labeling disappeared when a large excess of unlabeled MUC5B oligonucleotide was added to the 35S-labeled
MUC5B probe (Fig. 1C).
The expression of MUC5B mRNA was also
studied by RT-PCR in two gastric cancer cell lines (Fig.
2A). A high expressing
(KATO-III) and a low expressing (AGS) cell line were chosen. As shown
in Fig. 2A, the expected 415-bp PCR product was found in a
large amount in KATO-III cells (lane 3, KATO-III), whereas a
very faint band was observed in AGS cells (lane 3, AGS).
DNA Sequence and Characterization of MUC5B 5'-Flanking
Region--
Having shown that MUC5B is abnormally expressed
in gastric adenocarcinoma tissues and cancer cell lines, further
investigation of MUC5B 5'-flanking region DNA sequence was
conducted to identify new regulatory regions. The first 956 nucleotides
upstream of the transcription start site were previously described
(20). Numerous Sp1 binding sites were found clustered in the close
vicinity of the TATA box. In this report, further sequencing of the
region located upstream of DNA fragment 1896 over 1.1 kb was conducted, and analysis of the DNA sequence using PC-GENE software revealed the
presence of a second TATA-box like sequence (TAAATAAAA). The distal
TATA box is located 1.1 kb upstream of the proximal TATA box (Fig.
3). Using the TRANSFAC 4.0 data base, we
found out that the region adjacent to this second TATA box is
characterized by the presence of two clustered putative binding sites
for CREB/ATF and AP-1 transcription factors. Further upstream were
located potential binding sites for Sp1, glucocorticoid receptor,
thyroid transcription factor-1, retinoid orphan receptor- Identification of a Distal Transcription Unit in MUC5B 5'-Flanking
Region--
The presence of a distal putative TATA box suggests that a
distal transcription start site may exist in this region. To address this question, a reverse primer called NAU 647 was designed and chosen
131 bp downstream of the putative distal TATA box. Primer extension
experiments were also performed using the reverse primer located in
exon 1 (5BOAS) (20). The extension product obtained with the 5BOAS
oligonucleotide is 124 bp long as expected (Fig. 2B). The
intensity of the band is about the same in KATO-III (Fig. 2B, lane 5) and AGS cells (Fig. 2B,
lane 6). The positive control with RNA from human trachea, a
tissue in which MUC5B is expressed, also produced a 124-bp
extension product (Fig. 2B, lane 7). No extension
product was observed in the negative control (Fig. 2B, lane 8). The extension with NAU 647 confirmed the presence
of a transcription start site in the distal part of the 5'-flanking region of MUC5B in both cell lines. The extension product is
109 bp long (Fig. 2B, lanes 2-4) and starts at a
cytosine residue located 23 bp downstream of the distal TATA box (Fig.
3). One can note that the intensity of the band is far more intense in KATO-III (lane 4) than in AGS cells (Fig. 2B,
lane 3) or human trachea (Fig. 2B, lane
2). This result confirms the data obtained by RT-PCR (Fig.
2A) in which a high amount of MUC5B mRNAs was found in KATO-III cells and indicates that the highly active distal transcription unit may thus be responsible for the high expression of
MUC5B in KATO-III cells. RT-PCR experiments on cDNA
prepared from KATO-III total RNA were conducted with pair of primers
covering the MUC5B Promoter Activity in KATO-III and AGS Gastric Cancer
Cells--
To identify the DNA sequences involved in MUC5B
transcriptional activity, constructs were generated in the promoterless
pGL3 Basic vector and analyzed for transcriptional activity after cell transfection. Insert sequences were confirmed by infrared sequencing of
both strands and aligned with ELO9 cosmid sequence, which covers the
5'-flanking region of MUC5B.
The 11 deletion mutants used in the transfection experiments cover
2044 nucleotides upstream of the proximal
transcription start site (Fig. 4A and Table
I). The fragments located upstream of the
proximal TATA box are 1916, 1896, 1597, 1596, 1895, 1595 and 1598. They
cover 956 nucleotides upstream of the proximal transcription start site
and represent the proximal promoter of MUC5B that was
previously characterized in our laboratory (20). The fragments 1916, 1896, 1597 and 1596 contain the TATA box-like sequence (TACATAA), the
three Sp1 binding sites and the CACCC box. Fragments 1916 and 1597 contain the 5'-untranslated region segment long of 56 bp. The
luciferase activity diagram indicates that the active transcription
region in AGS cells is included in fragments 1916, 1896, 1597 and 1596 (Fig. 4B). In these cells, the luciferase activity is four
times greater than the control vector (pGL3 basic). On the other hand,
in KATO-III cells the luciferase activity was only present in fragment
1596 (2-fold activation). Thus, these results indicate that the first
223 bp (fragment 1596) adjoining the proximal transcription start site suffice to drive basal promoter activity of the luciferase reporter gene both in AGS and KATO-III cells. Fragments 1895, 1595, and 1598, which cover the upstream 734 nucleotides do not possess any luciferase
activity and act as inhibitory domains in both cell lines.
The fragments located upstream of the distal TATA box are 1599, 1600, 1634 and 2140 and they cover 0.9 kb of DNA sequence. The fragments 1599 and 1600, 212 and 209 bp in length, respectively, contain the TATA
box-like sequence (TAAATAAAA) that was characterized using the TRANSFAC
4.0 data base. In AGS cells, both fragments are active (five times more
than the pGL3 basic vector) and are slightly more active (20%) than
fragment 1596 of the proximal promoter. In KATO-III cells, these two
fragments show a very strong activity (10 times more than the pGL3
basic vector) that is 2-fold higher than in AGS cells. The fragment
further upstream of 1600, that is fragment 1634, which covers 714 nucleotides is inhibitory in both cell lines. Fragment 2140, which
covers the 1599 + 1634 region of 927 nucleotides possesses luciferase
activity but is not as active as 1599. This latter result suggests that
inhibitory cis-elements are present in the DNA fragment 1634.
From these studies, it can be stressed that MUC5B promoter
activity in gastric cancer cells is driven by two different DNA segments of the 5'-flanking region. In KATO-III cells, the fragment that contains the distal TATA box is by far the most active region, whereas in AGS cells the two regions containing a TATA box have about
the same range of transcriptional activity. In AGS cells, the activity
of the distal region is much less important than in KATO-III cells. In
conclusion, a distal region in MUC5B 5'-flanking region was
identified that showed high transcription activity in KATO-III cells
that may account for the high amount of MUC5B mRNA found
in these cells.
Binding Studies of MUC5B 5'-Flanking Region with Nuclear
Proteins--
To characterize cis-elements and
trans-nuclear factors that could account for the
cell-specific activity of the promoter of MUC5B in gastric
cancer cells, DNA-protein binding studies were carried out using the
EMSA technique. In Fig. 5 is shown the
autoradiogram of the gel shifts performed with nuclear proteins
prepared from KATO-III and AGS cells incubated with different DNA
probes. Two double-stranded oligonucleotides located in the proximal
region (T20 and T33) were chosen from our computer studies with the
TRANSFAC 4.0 data base. T20 covers the bases
From our computational studies, two other probes (T23 and T17) were
designed from sequences located in the distal region of the promoter.
T23 probe is located at Role of Sp1 in MUC5B Promoter Activity--
Based on the above
EMSA results and previously published data (20), we hypothesized that
Sp1 may play a regulatory role on MUC5B promoter activity in
gastric cancer cells. To test this hypothesis, cotransfection
experiments were carried out in the presence of pCMV4, pCMV-Sp1, or
pCMV-Sp3 expression vectors (Fig. 6). The
luciferase diagram indicates that Sp1 strongly transactivates the
proximal region (1896) in both cell lines (3.0- and 2.6-fold activation
in KATO-III and AGS, respectively), whereas it has no effect on the
distal region (2140). On the other hand Sp3, another member of the Sp
family known to compete with Sp1 for the same binding sites did not
have any effect on the proximal promoter but strongly inhibited the
distal promoter in both cell lines. One can conclude from these results
that Sp1 strongly transactivates the proximal promoter of
MUC5B, whereas Sp3 inhibits the activity of the distal
promoter.
Signaling Pathways Involved in MUC5B Promoter
Regulation--
ATF-1 is a transcription factor that binds to cAMP
response elements and that is known to be activated through the cAMP
protein kinase cascade. Because it binds to the distal region of
MUC5B promoter (Fig. 5), we looked whether this signaling
pathway was able to transactivate this region. To this end, transfected
cells were treated for 24 h with CTA, a cAMP-dependent
protein kinase activator, before measuring luciferase activity (Fig.
7). The luciferase diagram clearly
indicates that CTA activates transcription of both promoters
(approximately 2.5-fold) in KATO-III cells, whereas in AGS cells,
cholera toxin A subunit effect was very weak.
Although each of the ATF/CREB proteins appears capable of binding cAMP
response elements in its homodimeric form, certain of these proteins
also bind as heterodimers with members of the AP-1 transcription factor
family to induce gene transcription (33). We thus hypothesized that
ATF-1 may heterodimerize with AP-1 in the distal promoter of
MUC5B and that PKC would then be the signaling pathway used
to transactivate this region. To test this hypothesis, transfected
cells were treated for 24 h with PMA, a strong PKC activator. As
it is shown in Fig. 7, one can see that PMA indeed strongly induced the
transcription activity of the fragment 2140, which contains the distal
TATA box (2.7- and 3.8-fold activation in KATO-III and AGS cells,
respectively). The same PMA treatment was much less effective on the
proximal promoter (1896) (2-fold activation in AGS cells). Finally, as it had already been described in the literature that increase of
intracellular calcium induces mucin secretion and transcription, we
tested whether that signaling pathway had an effect on MUC5B promoter activity. As shown in Fig. 7, calcium ionophore A23187 (250 nM for 1 h) effect on MUC5B transcription
was mild (1.8-fold activation) and restricted to the proximal promoter
in AGS cells and to the distal promoter in KATO-III cells. Altogether
these results show that cAMP-dependent protein kinase
signaling pathway leads to the activation of both promoters in KATO-III
cells and that PKC induces a strong activation of the distal promoter
in both cell lines.
Role of Methylation in MUC5B Transcription--
Having shown that
Sp1 binds and modulates the activity of the promoter region of
MUC5B and knowing that Sp1 elements in promoters hampers
methylation of mammalian genes and thus modulate transcription activity
(34), we undertook to study the level of methylation of
MUC5B promoter and the effect of methylation on
MUC5B transcriptional rate in both cell lines.
In the first set of experiments (Fig. 2A), cells were
treated with the methylation inhibitor 5-aza-2'-deoxycytidine for
72 h after cells became confluent. Total RNA was prepared and
RT-PCR performed on untreated and 5-aza-treated cells. The result
presented in Fig. 2A shows that in KATO-III cells,
expression of MUC5B is not affected by the treatment of
cells with the methylation inhibitor agent (KATO-III, lanes 3 and 4). On the contrary, in AGS cells, where
MUC5B is expressed at a low level in untreated cells (AGS, lane 3), its expression is increased by 4-fold after the
treatment with 5-aza-2'-deoxycytidine (AGS, lane 4). Thus,
this experiment confirms the fact that methylation of the promoter of
MUC5B is one of the mechanisms used to repress
MUC5B expression in AGS gastric cancer cells.
Having shown that methylation of MUC5B promoter occurs in
AGS cells, we then undertook to map the cytosine residues potentially methylated within the promoter region. Potential
HpaII-methylation sites (C*CGG) were mapped after analysis
of the DNA sequence of the 5'-flanking region of MUC5B.
Methylation pattern of the promoter was obtained using two DNA probes,
1450 and 1896, that cover the 5'-flanking region of MUC5B
(Fig. 8A). Nine putative
methylation sites were found throughout the sequence covering the
With the 1896 DNA probe, which covers the proximal region of the
promoter, the presence of the 425-bp band when DNA was digested either
with BamHI-HpaII (KATO-III, lane 2) or
BamHI-MspI (KATO-III, lane 3) in
KATO-III cells, indicates that the cytosine at Mucins are expressed in a cell- and tissue-specific manner in
normal human tissues (1, 3-5, 11) and in normal stomach mucosae,
MUC1, MUC5AC, and MUC6 are the main
mucin genes (9, 10). Altered expression of mucin genes in carcinomas
have extensively been described in the literature (3, 5, 11-15), and
in gastric cancers, a decrease of the expression of MUC5AC
and MUC6 mRNAs and increased levels of MUC2,
MUC3 and MUC4 mRNAs have been demonstrated (10,
11). Other studies have focused on MUC1 and MUC2
mucin gene expression in gastric carcinomas because MUC1 is
often overexpressed in various carcinomas and MUC2
expression is correlated with the intestinal metaplasia observed during
the development of gastric carcinoma (13, 15, 35). All these studies
suggest that carbohydrate and peptide moieties modification on mucins
may be valuable markers of gastric neoplastic and preneoplastic states
(11, 36-38).
However, control of gene expression in gastric cells remains poorly
understood, and an understanding of the regulatory network of nuclear
proteins that direct transcriptional initiation of mucin genes is
mandatory to decipher the mechanisms of normal development and
differentiation as well as disease processes such as neoplasia. These
molecules, which can either be secreted to form the mucus or be
included in the membrane architecture, play roles of receptors and are
involved in cell-cell, cell-substratum, and cell-immune system
interactions (1, 36-38). In cancers, such interactions can be altered
to allow the tumor cells to migrate and induce metastasis. It is thus
clear that any change in their expression will affect all these
functions and modify the behavior of cancer cells.
In our laboratory, mucin gene expression has been extensively studied
for many years using in situ hybridization, and a method to
detect all mucin genes from the same sample by RT-PCR was recently developed (20, 39). The results have pointed out that it is important
to look at mucin genes that are not or weakly expressed in normal
tissues. Buisine et al. (6), for example, showed that
MUC5AC transcripts absent in normal adult colon are
re-expressed in rectovillous adenocarcinoma. MUC5AC being
also expressed in fetal colon, it was thus concluded that it
corresponds to a typical oncofetal expression pattern (7). Another
study from Balagué et al. (40) on MUC4
showed the same pattern of expression in pancreatic adenocarcinomas.
Very recently, it was shown in our laboratory that MUC5B is
expressed in embryonic and fetal gastric tissues (41). Thus, to define
whether this expression during the early stages of development could
correspond to an oncofetal pattern of expression of MUC5B in
gastric mucosae, we studied MUC5B expression in adult normal and carcinomatous gastric mucosae. In this report, we show that MUC5B is indeed expressed in both gastric carcinomatous
tissues and cell lines. Thus, for a better understanding of the
molecular mechanisms that prevail to this abnormal expression, the
regulation of the transcriptional activity of MUC5B promoter
was studied in KATO-III (high expression of MUC5B) and AGS
(low expression of MUC5B) gastric cancer cells.
In a previous work, MUC5B promoter activity was studied in
colon cancer cell lines with different phenotypes and was shown to be
regulated, in part, by the ubiquitous transcription factor Sp1 (20). It
was then suggested that Sp1 would be the transcription factor
responsible for the basal activity of MUC5B in the cells expressing that gene. This hypothesis was confirmed in this report as
we showed that Sp1 binds and transactivates the proximal promoter of
MUC5B in both gastric cancer cell lines. To explain the high abundance of MUC5B transcripts in KATO-III cells, it was
thus hypothesized that another highly active DNA segment, yet to be characterized, was present in the 5'-flanking region. We thus undertook
to further sequence and analyze the DNA region located upstream of
1896. Interestingly enough, a highly active transcription unit
containing a TATA box like sequence flanked by two clustered AP-1/ATF/CREB putative binding sites was characterized. The presence of
a distal transcription unit in MUC5B 5'-flanking region is not unique in mucin genes. Recently, such a regulatory region was
described for MUC1 and was demonstrated to be responsible for the high expression of this gene in breast cancers (42).
Cell transfections with a panel of pGL3 deletion mutants and gel
retardation assays confirmed that a highly active distal promoter is
present within the 5'-flanking region of MUC5B. It contains
an active TATA box, binds ATF-1 and Sp1 transcription factors, and is
activated by cAMP-dependent protein kinase and PKC
signaling pathways. In this report we showed that CTA, which has
already been shown to activate mucin secretion in colon cancer cell
(43), is capable of specifically inducing MUC5B promoter activity in KATO-III but not in AGS cells. Thus, we can postulate that
MUC5B promoter activation via cAMP signaling in KATO-III cells involves activation of adenyl cyclase through activation of
Gs regulatory proteins. Both the fact that ATF-1 can
heterodimerize with AP-1 transcription factor family and the fact that
Gs regulatory proteins can induce the PKC signaling pathway
suggest that PKC may also be involved in MUC5B regulation.
In this report we showed that PMA induced a strong activation of
MUC5B distal promoter in gastric cancer cells. This
mechanism may be specific to gastric cancer cells because it was
recently shown that PMA did not induce MUC5B expression in
T84 and HT-29/A1 colon cancer cells (44). Increase of intracellular
calcium content within mucus-secreting cells is also a pathway that
induces mucin secretion (45). In this work, treatment of cells with
calcium ionophore A23187 did not have a significant effect on
MUC5B promoter activity. Thus, PKC signaling pathway seems
to be the pathway of choice to induce MUC5B promoter
activity in gastric cancer cells.
MUC5B is located on chromosome 11p15.5 and is part of a
mucin gene cluster comprising MUC6-MUC2-MUC5AC-MUC5B (21).
One of the aims in this work was to provide a better understanding of MUC5B regulation and promoter activity as a member of this
cluster. The cluster is 400 kb long and is rich in CpG islands. Among
the four genes, promoter sequence is known for MUC2,
MUC5AC, and MUC5B but not for MUC6.
The first three genes are transcribed in the same orientation (46),
whereas MUC6 is transcribed in the opposite way (30). The
human 11p15 region displays a high density of CpG islands and contains
a cluster of 9-10 genes, such as imprinted H19 and
IGFII (insulin growth
factor II) genes and Wilm's tumor 1 tumor
suppressor gene that have already been shown to be regulated by
methylation (47-49). Methylation is an epigenetic mechanism that is
commonly used by cells to shut off the expression of a gene (50, 51)
and that has profound effects in cancers (51-53). As a central event
in the evolution of cancers, along with genetics events, methylation
changes constitute potentially sensitive molecular markers to define
risk states, monitor prevention strategies, achieve early diagnosis,
and track the prognosis of cancer (53). In gastric carcinomas,
association between aberrant methylation and CpG island methylator
phenotype was recently published (54). Interestingly enough
MUC2, the major mucin expressed in normal colon, was also
shown to be repressed by methylation in colon cancer cells (55). From
our results, methylation appears now to control the expression of
another gene of the 11p15 mucin gene cluster in gastric cancer cells,
that is MUC5B. Investigations about the methylation status
of the four 11p15 mucin genes are now in progress in our laboratory and
tend to show that these genes are indeed regulated by methylation in
various cancer cell lines. In the two cell lines studied in this
report, MUC2 and MUC6 were found to be repressed
by methylation in KATO-III cells but not
MUC5AC.2 In AGS
cells, MUC2 and MUC5B (this report) were found to
be repressed by such mechanism. From these studies, it is clear that
methylation is a common mechanism used to control the transcription of
these four mucin genes and that it could participate besides to the transcription factors to the specific pattern of mucin gene expression in cancer cell lines and tissues. Finally, it is known that Sp1 elements interfere with methylation of promoters and thus affects their
activity (34). Our laboratory and others (56) have already suggested
that the regulation of the 11p15 mucin genes is complex and most likely
involves components or genetic mechanisms that are responsible for the
tissue- and cell-specific expression of the four genes. Sp1 seems a
good candidate because it has now been shown to be involved in the
regulation of the first three genes so far described, that is
MUC2 (57), MUC5AC (data not shown), and
MUC5B (20). Studies of the relationship between the binding
activity of Sp1 to the promoters and the methylation status of the
cluster will certainly help into the understanding of how this region
is regulated in cancers.
In conclusion, this work demonstrates that abnormal expression of
MUC5B visualized in well differentiated gastric carcinoma is
due to the presence of a highly active distal transcription unit that
is up-regulated by PKC. The transcription factor Sp1, on the other
hand, would be responsible for the basal expression of MUC5B
by transactivating the proximal promoter. Besides this regulation by
transcription factors, MUC5B also appears to be regulated in
gastric cancer cells by methylation. The deciphering of the molecular
mechanisms that control the transcription of mucin genes in
gastro-intestinal diseases is mandatory to identify transcription
factors that target mucin genes during cell differentiation and
proliferation and consider mucin genes as potential molecular markers
in carcinogenesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B, c-Myc). The high expression of MUC5B was
correlated with the mucus-secreting phenotype of the LS174T colon
cancer cell line. Introns 1 and 37 of MUC5B have also been
studied in our laboratory because they contain tandemly repeated GA-
and GC-rich sequences that bind the transcription factors Sp1 (20) and
NF1-MUC5B, respectively (19).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]dATP. DNA
fragments 1450 and 1896 cloned into pGL3 vector used as probes were
first digested with SacI-MluI (1450)
and KpnI-MluI (1896) to obtain the insert. The
fragments were then gel purified (QIAquick gel extraction kit, Qiagen)
and labeled with [
-32P]dCTP at the 3' end by a fill-in
reaction using the Klenow fragment (Roche Diagnostics).
Oligonucleotides and DNA fragments radiolabeled probes were separated
from free nucleotides on Bio-Gel P-6 and Bio-Gel P-30 columns,
respectively (Bio-Rad Marnes la Coquette, France).
X174
DNA/HinfI dephosphorylated markers (Promega) were
radiolabeled with [
-32P]dATP just before use. Manual
sequencing of DNA fragment 1429 was performed using the T7 Sequenase
version 2.0 kit (Amersham Pharmacia Biotech, Orsay, France). Samples
were denatured for 10 min at 90 °C before loading on a 6%
sequencing gel (Sequagel-6, National diagnostic, Prolabo, France). The
gel was then vacuum dried and autoradiographed for 3-4 days at
80 °C.
Gal vector (Promega).
-Galactosidase activity was measured in 96-well plates as described
in the manufacturer's instruction manual using 10 µl of cell
extracts (Promega). Luciferase activity was measured on a Berthold 9501 luminometer on 20 µl of cell extracts using luciferase assay reagent
(Promega). The luciferase activity is expressed as fold of induction of
the test plasmid activity compared with that of the corresponding
control vector (pGL3 control vector, Promega) after correction for
transfection efficiency by dividing by
-galactosidase activity. Each
plasmid was assayed in duplicate in at least three separate
experiments. Cotransfection studies with pCMV-Sp1 and pCMV-Sp3
expression vectors were performed as previously described (20). PMA
(100 nM) or CTA (1 µg/ml) were added to the cells 24 h after cell transfection and left for another 24 h before
harvesting cells to measure luciferase activity. Calcium ionophore
A23187 (250 nM) was added to the cells 1 h prior to
harvesting cells. This corresponds to the optimized conditions to
obtain the maximum effect of each reagent on MUC5B promoter
activity. All reagents were from Sigma unless otherwise indicated.
80 °C until use. Protein content (5 µl of cell extracts) was
measured using the bicinchoninic acid method in 96-well plates as
described in the manufacturer's instruction manual (PERBIO Science,
Bezons, France).
80 °C.
80 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (94K):
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Fig. 1.
In situ hybridization for
MUC5B mRNAs in normal stomach and gastric
carcinoma. A, normal stomach (antrum) with the
MUC5B probe with methyl green pyronin counterstain. The
hybridization signal is absent. Magnification, ×200. B and
C, gastric carcinoma (antrum, well differentiated) with the
MUC5B probe. B, with the
35S-labeled MUC5B probe, the signal is
distributed throughout tumoral glands. C, with the
35S-labeled MUC5B probe and a large excess of
unlabeled MUC5B probe, the hybridization signal is absent.
Methyl green pyronin counterstain was used. Magnification, ×200.
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Fig. 2.
Expression of MUC5B in
KATO-III and AGS cells. A, RT-PCR on 1.5 µg of RNA
used for cDNA synthesis. Glyceraldehyde-3-phosphate dehydrogenase
(lanes 1 and 2) and MUC5B (lanes
3 and 4) PCR products are 980 and 415 bp long,
respectively. PCR products were separated on a 2% agarose gel.
Untreated (lanes 1 and 3) and
5-aza-2'-deoxycytidine-treated (lanes 2 and 4)
KATO-III and AGS cells. B, primer extension on 25 µg of
total RNA from human trachea (lanes 2 and 7), AGS
(lanes 3 and 6), and KATO-III (lanes 4 and 5) cells. Lanes 1 and 8, no RNA.
Two extension products of 109 bp (lanes 2-4) and 124 bp
(lanes 6-8) were produced when using reverse primers
located downstream of the distal TATA box (NAU 647) and in exon 1 of
MUC5B (5BOAS), respectively. X174 DNA/HinfI
dephosphorylated markers previously radiolabeled and denatured are
indicated on each side of the gel. The sequence of the fragment 1429 is
shown.
1, TGT3,
Wilm's tumor-1 transcription factor (KTS), and insulin receptor
factor-2.
View larger version (75K):
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Fig. 3.
DNA sequence of the human MUC5B
promoter. The proximal TATA box (TACATAA) at 32/
26 is
double-underlined, and potential binding sites for known
transcription factors are shaded. The distal TATA-like
sequence at
1142/
1134 (TAAATAAAA) is also
double-underlined. The proximal transcription start site is
designated as +1, and the first ATG is bold and
italicized. The distal transcription start site at
1120 is
bold and underlined. HpaII (C*CGG)
potential methylation sites and sequences of the oligonucleotides used
in gel shift assay experiments are boxed.
1117/
1 region and showed that the whole region is
transcribed (not shown). Altogether these results indicate that two
active transcription units are present in the 5'-flanking region of
MUC5B.
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Fig. 4.
Position and transcriptional activity of the
pGL3 deletion mutants in MUC5B promoter.
A, schematic representation of the localization of the
different pGL3 deletion mutants covering 2044 nucleotides upstream of
the first ATG. Numbering refers to the proximal
transcription start site designated +1. The TATA box locations are
indicated as well as the Sp1 and ATF-1 binding sites found in the
5'-flanking region of the gene. B, transcriptional activity
of the deletion mutants was studied in KATO-III (black bars)
and AGS (white bars) cell lines. Background activity of pGL3
basic promoterless vector used to subclone MUC5B fragments
is shown. The results are the means ± S.D. and represent more
than three different experiments in duplicate for each fragment.
Sequences of the pairs of oligonucleotides used in PCR to produce
deletion mutants in MUC5B 5'-flanking region
203/
180 and contains a
CACCC box and a Sp1 binding site. T33 covers the bases
137/
111 and contains a Sp1 putative binding site (Fig. 3 and Table
II). When incubated with nuclear
proteins, the probe T20 led to one low mobility shifted band in both
cell lines much more intense in KATO-III cells (compare lanes 2 and 5). The complex was totally supershifted when the
anti-Sp1 antibody was added in the reaction mixture (lanes 3 and 6). Anti-Sp2 antibody used as a negative control
did not produce any supershift (lanes 4 and 7).
With the T33 probe (lanes 8-12), a very strong band was
visualized in both cell lines (lanes 9 and 11),
which was totally supershifted upon the addition of the anti-Sp1
antibody in the reaction mixture (lanes 10 and
12).
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Fig. 5.
Binding of Sp1 and ATF-1 transcription
factors to the promoter of MUC5B. Autoradiogram
of the EMSA performed with 5 µg of nuclear proteins isolated from
KATO-III and AGS cell lines. Nuclear proteins were incubated with the
radiolabeled DNA probes as indicated (T20 and T33: proximal promoter;
T23, T17: distal promoter). Super-shift experiments were carried out by
adding 1 µl of the antibodies of interest (Sp1, Sp2, ATF-1, CREB-1,
and Hox D9). Radiolabeled probe alone were loaded in the first lane of
each series. Sp1 binding was visualized with T20, T33, and T23 and
ATF-1 with T17. On the left-hand side, the arrow
indicates the position of the DNA-protein complex engaging Sp1, and the
arrow on the right-hand side indicates the
position of the DNA-protein complex with ATF-1.
Sequences of the sense oligonucleotides used for gel shift assay
experiments
1230/
1207 and contains a CACC box and a
glucocorticoid receptor element. The T17 probe is located at
1153/
1133 and contains a putative ATF-1/CREB-1/Hox D9 binding site.
With T23, three major shifted bands were visualized with KATO-III
(lane 14) and AGS (lane 16). The most intense
shifted band that is also characterized by the lowest mobility was
totally supershifted when the anti-Sp1 antibody was added to the
reaction mixture, which demonstrates that Sp1 binds to that
cis-element (lanes 15 and 17). With
the probe T17, only one shifted band could be visualized in both cell
lines (lanes 19 and 23). When using specific
antibodies against the three transcription factors of interest, a total
supershift specifically occurred upon the addition of the anti-ATF-1
antibody (lanes 20 and 24). Anti-CREB-1
(lanes 21 and 25) and anti-Hox D9 (lanes 22 and 26) antibodies had no effect. Specificity of the
binding of the T17 probe to ATF-1 was confirmed by performing cold
competitions by preincubating nuclear proteins with 50-, 150-, and
300-fold excess of the cold T17 probe before adding the radioactive
probe. The shifted banded totally disappeared when X50 excess of T17
was used (not shown). Altogether these results show that Sp1 binds to
two sites in the proximal region of the promoter, whereas it engages
once in the distal region where the ATF-1 transcription factor was also
shown to bind to its cognate cis-element.
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Fig. 6.
Transactivation of MUC5B
promoter by Sp1. Cotransfection experiments were performed
with 1 µg of MUC5B deletion mutants (1896 and 2140) and
0.25 µg of pCMV4, pCMV-Sp1 or pCMV-Sp3 expression vectors in KATO-III
(black bars) and AGS (white bars) cells. The
results are the means ± S.D. and represent more than three
different experiments in duplicate for each fragment.
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Fig. 7.
Effects of the activation of different
signaling pathways on the activity of MUC5B
promoter. pGL3 mutant-transfected KATO-III (black
bars) and AGS (white bars) gastric cancer cells were
either treated with 100 nM PMA for 24 h, 1 µg/ml CTA
for 24 h, or 250 nM calcium ionophore A23187 for
1 h before harvesting cells. The results are the means ± S.D. and represent more than three different experiments in duplicate
for each fragment.
2044/+3 bases and are shown on the schematic representation of
MUC5B 5'-flanking region (Fig. 8A). The
methylation status of genomic DNA covering the 5'-flanking region of
MUC5B from the two cell lines is shown in Fig.
8B. In KATO-III cells, one major band of 700 bp in length was recognized by the 1450 probe when genomic DNA was digested with
BamHI-HpaII (KATO-III, lane 2). The
spot seen on the left part of that same lane 2 above 1.2 kb
is nonspecific. When the same genomic DNA was digested with
BamHI-MspI, the latter enzyme being insensitive
to methylation, another strong wide band appeared besides the 700-bp
band; this wide band most likely comprises the three expected fragments
long of 273, 252, and 244 bp (KATO-III, lane 3) that cover
the 3'-end region of the genomic region recognized by the probe 1450. This results indicates that, in KATO-III, the cytosines between
1095/
695 are methylated. When the same
BamHI-HpaII treatment was applied to genomic DNA
from AGS cells (AGS, lane 2), bands shifted toward the high
molecular weights when compared with KATO-III cells (KATO-III,
lane 2). The absence of the 700-bp band thus indicates that all
the cytosines in the
2044/
442 region are methylated. This was
confirmed when the DNA was digested with BamHI-MspI because the bands with molecular
weights higher than 1200 bp, visualized after digestion of the DNA with
BamHI-HpaII (AGS, lane 2), totally
disappeared and led to the appearance of the 700-bp band as well as the
wide band comprising the 244-, 252-, and 273-bp fragments when DNA was
digested with BamHI-MspI (AGS, lane
3).
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Fig. 8.
Mapping of methylation sites within
MUC5B promoter in gastric cancer cells.
A, schematic representation of MUC5B 5'-flanking
region. Black dots indicate the location of the nine
putative methylation sites. Lengths of fragments between two
methylation sites are shown. Fragment 1896 covers the 956/
1 region
of the 5'-flanking region and fragment 1450 covers the
2044/
442
region. B, Southern blotting of 20 µg of genomic DNA from
KATO-III and AGS cells probed with 1450 and 1896 DNA fragments. Genomic
DNA was digested with BamHI (lanes 1),
BamHI + HpaII (lanes 2), and
BamHI + MspI (lanes 3). Molecular
weight markers are indicated on the left side of the
autoradiograms. Arrows on the right sides
indicate the position and size of the visualized fragments.
422 is not methylated.
The presence of the 273- and 252-bp bands in lane 3 but not
in lane 2 indicates that cytosines between
1095/
695 are
methylated (Fig. 8A). In AGS cells, a series of high
molecular weight bands larger than 1.2 kb as well as a 778-bp band were visualized on genomic DNA digested with
BamHI-HpaII (AGS, lane 2), indicating
that the cytosine at
422 is methylated in AGS cells. After digestion
with BamHI-MspI enzymes (AGS, lane 3), all the high molecular weight bands disappeared to leave place to the
expected bands of 425, 273, and 252 bp. Altogether these results
indicate that methylation occurs in KATO-III cells at cytosines located
between
1095/
695, whereas in AGS cells methylation occurs
throughout the 5'-flanking region.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We are indebted to Danièle Petitprez, Pascal Mathon, Marie-Paule Ducourouble, and Isabelle Clavereau for excellent technical help and Claude Vandeperre for the photographs. We thank the members of our E.U. consortium CEEBMH4-3222 for stimulating discussions.
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FOOTNOTES |
---|
* This work was supported by l'Association de Recherche contre le Cancer Grant 5785 and a grant from le Comité du Nord de la Ligue Nationale contre le Cancer.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ012453.
§ Recipient of a CHRU de Lille-Région Nord-Pas de Calais Ph.D. fellowship.
To whom correspondence should be addressed. Tel.:
33-320-29-88-65; Fax: 33-320-53-85-62; E-mail:
vanseuni@lille.inserm.fr.
Published, JBC Papers in Press, January 30, 2001, DOI 10.1074/jbc.M010534200
2 I. Van Seuningen-Lempire, unpublished observation.
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ABBREVIATIONS |
---|
The abbreviations used are: kb, kilobase(s); AP-1, activating protein 1; ATF-1, activating transcription factor 1; CREB, cAMP-responsive element binding protein; CTA, cholera toxin A subunit; EMSA, electrophoretic mobility shift assay; PCR, polymerase chain reaction; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; RT, reverse transcriptase; SSC, saline-sodium citrate buffer; bp, base pair(s).
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