From the Division of Tumor Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands
Received for publication, October 17, 2000
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ABSTRACT |
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The mucin-like glycoprotein episialin (MUC1) is
highly overproduced by a number of human carcinomas. We have shown
previously in a variety of mammalian cell lines that overexpression of
this very large transmembrane molecule diminishes cellular adhesion, suggesting that episialin/MUC1 overexpression may play an important role in tumor invasion and metastasis. By using in situ
hybridization, we show here that episialin/MUC1 mRNA expression can
be increased more than 10-fold in breast carcinoma cells relative to
the expression in adjacent normal breast epithelium. In search of the
molecular mechanism of this overexpression, we observed that the
episialin/MUC1 promoter contains a candidate binding site
for transcription factors of the STAT family ~500 base pairs upstream
of the transcription start site. Cytokines and/or growth factors such
as interleukin-6 or interferon- Episialin/MUC11 (also
known as MUC1, PEM, CA 15-3 antigen, or EMA) is a transmembrane
molecule with a large extracellular mucin-like domain. In normal cells
episialin/MUC1 is exclusively present at the apical side of the cell,
but in carcinoma cells normal polarization is lost and episialin/MUC1
co-localizes with adhesion molecules such as integrins and cadherins.
The long and relatively rigid extracellular domain of episialin/MUC1
can shield these adhesion molecules and diminish cellular adhesion, if
present at a sufficiently high density on the cell surface (1-3).
Overexpression of episialin/MUC1 in carcinoma cells has been frequently
reported (4-6) and is expected to have a similar effect on cellular
behavior as loss of E-cadherin, the major epithelial cell-cell adhesion molecule, which has been shown to promote invasion and metastasis of
carcinoma cells (for review see Refs. 7-9). Therefore, we have
proposed that episialin/MUC1 also plays an important role in invasion
and metastasis in vivo (10). Indeed, transgenic mice
overexpressing episialin/MUC1 develop more aggressive lung tumors than
nontransgenic mice,2 whereas
episialin/MUC1 null mice show a slower rate of tumor progression
(11).
Recent reports have shown that episialin overexpression in various
types of neoplasia correlates with poor survival (12-14). Episialin/MUC1 is also the antigen that is measured in the CA 15-3 assay (the main blood marker to detect recurrence of breast cancer),
and it is a molecule that is widely considered as one of the most
promising molecules to be used for vaccination against breast cancer.
Therefore, knowledge about the regulation of episialin/MUC1 overexpression in tumor cells is of utmost clinical importance. In B
cell lymphomas it has been shown that episialin/MUC1 overexpression is
frequently the result of a t(1;14) (q21;q32) translocation, which
brings the episialin/MUC1 gene under the control of an
immunoglobulin We hypothesized that overexpression of episialin/MUC1 in carcinoma
cells is the result of constitutive activation of a transcription factor or inactivation of a repressor. Constitutively activated signaling pathways are common in carcinoma cells and strongly contribute to the malignant properties of these cells. As an initial step to elucidate such a putative factor or pathway, we studied the
normal transcriptional regulation of the episialin/MUC1 gene first. As a lead we considered exogenous factors that have been shown
to up-regulate episialin biosynthesis.
IFN- From the considerations presented above, we concluded that STAT
proteins might be one of the important regulators of
episialin/MUC1 transcription. Further evidence that STATs
might be involved in episialin/MUC1 (over)expression is as follows. (i)
A putative STAT-binding element, which is conserved in human and mouse,
is present around 500 bp upstream of the transcription start site (28-30) and is located well within the 750-bp proximal region that has
been identified to contain the regulatory sequences for episialin/MUC1 expression in various cancer cell lines (30,
31).4 (ii) STAT proteins are
known to be constitutively activated in a number of cancer types
including breast carcinoma (32, 33). In addition, a recent publication
(34) shows that STAT3 can act as an oncogene. Our results unambiguously
show for the first time that the expression of the
episialin/MUC1 gene indeed is highly up-regulated in breast
cancer and that STAT transcription factors contribute to this
overexpression of episialin/MUC1.
Cell Lines--
T47D cells (human breast carcinoma cell line)
were a kind gift from Dr. J. Taylor-Papadimitriou, Imperial
Cancer Research Fund, London, UK. A549 (human lung carcinoma cells),
HBL-100 (human SV40 immortalized normal breast epithelial cells), and
HPAF (human pancreatic carcinoma cells) cell lines were obtained from
the American Type Culture Collection. ZR-75-1 and MCF-7 cells (both human breast carcinoma cell lines) were obtained from Centocor Inc.,
Malvern, PA. HL-60 cells (human promyelocytic leukemia) were obtained
from Dr. C. Figdor, Netherlands Cancer Institute, Amsterdam,
Netherlands. All cell lines were maintained in Dulbecco's modified
Eagle's medium (DMEM) supplemented with penicillin, streptomycin, and
10% fetal bovine serum (FBS).
Antibodies--
Murine monoclonal antibody C-111, directed
against amino acids 613-739 of human STAT1, and rabbit polyclonal
serum C-20, raised against amino acids 750-769 of mouse STAT3, both
from Santa Cruz Biotechnology, were used. The latter antiserum
cross-reacts with human STAT3. A rabbit polyclonal antibody specific
for the phosphorylated tyrosine (Tyr-705) of STAT3 was
obtained from New England Biolabs. Monoclonal anti-STAT2, -3, -5, and
-6 from Transduction Laboratories were used to supershift STAT2, -5, and -6. Monoclonal antibodies 139H2 (35) and 214D4 (36) are directed
against a peptide epitope in the repeat domain of episialin. Monoclonal
antibody 232A1 is directed against a peptide epitope in the non-mucin
domain of episialin (36).
In Situ Hybridization--
In situ hybridization
experiments on breast carcinoma samples were performed according to
Wilkinson and Nieto (37). 35S-Labeled sense and antisense
riboprobes containing the 3' part of the human episialin/MUC1 cDNA
(downstream from the repeats) were generated by transcription of a
pSP64 plasmid containing this fragment with either SP6 or T7 RNA
polymerase. Fresh breast carcinoma samples were obtained during routine
examination of surgically removed tissue. The specimens were fixed in
fresh 4% paraformaldehyde. Following overnight incubation, the
sections were dehydrated and stored in 96% ethanol until further use.
Immunohistochemistry--
Sections of the paraformaldehyde fixed
tissues were stained using an indirect immunoperoxidase staining
procedure as described before (38). Immunostaining was performed using
monoclonal antibody 139H2, 214D4, and 232A1 (35, 36). All sections were
counterstained with hematoxylin.
Immunofluorescence Assay--
Cells were detached with PBS
containing 5.10 RNA Stability Studies--
The stability of the
episialin/MUC1mRNA was determined in HBL100 cells. Cells were
incubated for the indicated times with actinomycin D (final
concentration 10 µg/ml). RNA was subsequently isolated according to
the method of Chomczynski and Sacchi (39). 10 µg of total RNA was
loaded on all RNA gels. The episialin/MUC1 mRNA was detected using
the K5 probe representing the repeat domain of episialin (18). c-Myc
and Construction of Plasmids--
An XmnI-XmnI
fragment spanning the region from Intracellular Protein Levels, mRNA Levels, and mRNA
Stability Experiments--
To determine the effects of IL-6
on several variables, T47D cells were cultured for 2 days in DMEM with
0.25% FBS in the presence or absence of 400 units/ml IL-6 (Roche
Molecular Biochemicals). Intracellular episialin levels were determined
after lysis of the cells, followed by SDS-polyacrylamide gel
electrophoresis, Western blotting, and detection of the protein with
the monoclonal antibody 214D4, which is specific for the repetitive
domain of episialin. The effect of IL-6 on mRNA stability was
measured by culturing the cells for 1 day in medium with or without
IL-6. Subsequently, actinomycin D (10 µg/ml) was added. Total RNA was prepared from cells, and mRNA levels were determined in a standard Northern blot experiment. Additional details are described above. The
Northern blot was exposed either to Kodak X-Omat AR films or to a
PhosphorImager screen to quantify the signal strengths (Fujix 2000 BAS
PhosphorImager and Tina 2.09 software).
Transfection of Cells--
Mammary carcinoma cell lines T47D and
ZR-75-1, pancreatic carcinoma cell line HPAF, and lung carcinoma cell
line A549 were cultured in 6-well plates and transfected using the
DEAE-dextran method 1 day after they had been seeded at 300,000 cells
per well. Cells were washed with PBS and TBS-D (137 mM
NaCl; 2.7 mM KCl; 25 mM Tris-HCl, pH 7.4; 0.1%
w/v glucose) and subsequently incubated with a mixture of DNA and
DEAE-dextran (1 mg/ml in TBS-D). The DNA mixture consisted of 0.5 µg
of W3B or M3B and 0.01 µg of SV40 Renilla luciferase
construct (Promega, control for transfection efficiency) and salmon
sperm DNA to a total of 2.5 µg of DNA. The cells were incubated with
the DNA/DEAE-dextran mixture until the cells started to round off.
Exact incubation conditions varied for each cell line. T47D and A549
cells were preincubated for 2-4 h with 100 µM
chloroquine and were subsequently incubated for 8-12 min with 375 µl
of DNA/DEAE-dextran mixture and were subsequently cultured in DMEM
without FBS. For the ZR-75-1 cells, the chloroquine treatment was
omitted, and incubation with the 375-µl DNA/DEAE-dextran mixture was
for 15-20 min, which was followed by a 90-s glycerol shock (15%
glycerol in PBS). Subsequently, cells were cultured in DMEM
supplemented with 0.25% FBS. The HPAF cells were incubated with 500 µl of DNA/DEAE-dextran mixture for 10-15 min, and a 45-s glycerol
shock was applied. Cells were subsequently grown in DMEM with no FBS or
0.25% FBS. Recombinant IFN- Luciferase Assay--
Cells were washed with PBS and
subsequently lysed at room temperature with 200 µl of Passive Lysis
Buffer (Promega) for 20 min. The lysates were collected, and the wells
were rinsed with 200 µl of Passive Lysis Buffer. Lysates and wash
were combined, and cellular debris was removed by spinning at 14000 rpm
in a microcentrifuge for 2 min. The supernatant was stored at
The firefly luciferase reporter and Renilla luciferase
(internal control) activities of 15 µl of lysate were measured
employing the Dual Luciferase Reagent Assay Kit (Promega) using 75 µl
of LARII and 75 µl of Stop and Glo buffers. Luminescence was measured using a luminometer (Berthold).
Electrophoretic Mobility Shift Assays (EMSAs)--
DNA-binding
proteins were extracted basically according to the method of Andrews
and Faller (40) with minor modifications. Since the activated
(phosphorylated) form of the STATs is predominantly nuclear, additional
wash and centrifugation steps were included after the swelling and
rupture of the cells to separate the nuclei from the cytoplasmic
proteins. Protein yield was determined using the Bio-Rad protein kit.
The following double-stranded oligonucleotides were synthesized:
wt-MUC1-oligonucleotide, 5'-GGCTATTCCGGGAAGTGGT-3';
mutant-MUC1-oligonucleotide, 5'-GGCTACTCGAGAAGTGGT-3'; and
ICAM1-oligonucleotide, 5'-GAGGTTTCCGGGAAAGCAG-3'. The
core-binding site of the STATs is underlined. 6 pmol of the double-stranded oligonucleotide was labeled in a final volume of 10 µl using T4 polynucleotide kinase. The oligonucleotide was ethanol-precipitated and redissolved in 100 µl of TE. 7.5 µg of nuclear extract (in 1-5 µl depending on the protein concentration) was mixed with 4 µl of 5× binding buffer (20% glycerol, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM DTT, 250 mM NaCl, 50 mM
Tris-HCl, pH 7.5), 1 µg of poly(dI-dC)·poly(dI-dC), and 1 µg of
salmon sperm DNA in a final volume of 17 µl (bandshift assay) or 16 µl (supershift assay). This mixture was incubated for 10 min on ice
and subsequently 10 min at room temperature. 2 µl of labeled
oligonucleotide was added, and the incubation was continued for 30 min
at room temperature. In the case of a supershift assay, 1 µl of the
antibody preparation was added at the end of this incubation, and the
mixture was either left at room temperature for 20 min or on ice for
1 h. In the case of competition experiments, unlabeled
oligonucleotide was added during the incubation of the nuclear extract
in binding buffer on ice, and the volume of the added water was
corrected to keep the final volume constant.
Following incubation, 1 µl of 10× loading buffer (250 mM
Tris-HCl, pH 7.5, 0.2% bromphenol blue, 0.2% xylene,cyanol, 40%
glycerol) was added to the sample. Samples were analyzed by
electrophoresis in 5% polyacrylamide gels for 3 h at 200 V at
4 °C. Running buffer concentrations varied from 0.1× TBE to 1× TBE
(1× TBE is 50 mM Tris, 50 mM boric acid, 1 mM Na2EDTA). 2 µl of the same samples were
run separately on similar gels for a shorter period to detect unbound
oligonucleotide. After electrophoresis the gels were dried and exposed
to Kodak X-Omat R films at Overexpression of Episialin/MUC1 in Breast Carcinomas Is Caused by
Increased mRNA Expression--
The first objective of this study
was to obtain unequivocal evidence for an increased expression of
episialin/MUC1 mRNA in carcinomas at the single cell level. To this
end, we performed immunohistochemical staining and in situ
hybridization of serial sections of breast carcinoma specimens using
the monoclonal antibody 139H2 directed against the repeat domain of
episialin/MUC1 and episialin/MUC1 antisense and sense RNA probes.
Subsequently, we compared the expression of episialin/MUC1 RNA and
protein in normal and carcinoma tissues in those areas where both cell
types were sufficiently close together to be directly comparable in the
same microscopic field. Fig. 1 shows a
representative picture of the increased protein and RNA levels in two
adjacent sections of a mammary carcinoma. The immunohistochemical
staining shows only a thin line of apical staining in the normal duct,
whereas it is obvious that the apical localization of episialin/MUC1 is
lost in the carcinoma cells (Fig. 1A). Another monoclonal
antibody, 232A1, which reacts with the nonrepeat domain of
episialin/MUC1 and is insensitive to glycosylation, gave very similar
results (results not shown). In Fig. 1B the difference
between the mRNA levels in normal and carcinoma cells was estimated
to be at least 10-fold, by counting the grains over the individual
cells. Similar numbers were found in other breast carcinomas. Our
in situ hybridization experiments constitute the most direct
proof that the overexpression of episialin/MUC1 can indeed be mediated
by increased mRNA levels.
A possible explanation for increased episialin/MUC1 mRNA levels may
be an increased stability of episialin/MUC1 mRNA in carcinoma cells. Since the mRNA stability cannot be monitored in in
vivo or ex vivo samples, we decided to compare
previously published data on the stability of episialin/MUC1 mRNA
in the mammary carcinoma cell line MCF-7 (41) with the mRNA
stability in HBL-100 cells. HBL-100 cells are immortalized breast
epithelial cells that produce low levels of episialin/MUC1 mRNA and
protein, whereas episialin/MUC1 mRNA and protein levels in MCF-7
are high. As shown in Fig. 2, episialin/MUC1 mRNA is very stable in HBL-100 cells. After
quantification using a PhosphorImager, we estimate the mRNA
half-life by extrapolation of the data to be ~24 h. This is entirely
comparable with the half-life in MCF-7 carcinoma cells. Thus a major
role for mRNA stability to explain the increased levels of
episialin/MUC1 mRNA in carcinoma cells seems unlikely.
The Promoter of Episialin/MUC1 Contains an Active STAT
Site--
The findings reported above prompted us to study the
transcriptional regulation of the episialin/MUC1 gene by
some of the known inducers of episialin/MUC1 expression, in particular
those that activate STATs. A candidate STAT-binding site
(5'-TTCCGGGAA-3'), which conforms to the consensus TTCNNNGAA
(42), has been identified at positions
The human mammary carcinoma cell line T47D, a cell line with a high
expression level of episialin/MUC1, and the lung carcinoma cell line
A549, which shows a low episialin/MUC1 expression, were transiently
transfected with both constructs. Both cell lines can be transfected
with an acceptable efficiency (~1% of the T47D cells were
transfected, which is quite normal for this kind of transfection
method). Incubation of transfected T47D cells with IL-6 for 24 h
stimulated the wild-type promoter 6-10-fold, whereas the activity of
the mutant promoter was hardly affected (Fig. 3B). The
activity of the wild-type promoter in A549 cells could also be
stimulated by IL-6, although the stimulation was less (Fig.
3C). The response to IL-6 was dose-dependent;
optimal stimulation was reached with 800-1600 units/ml (results not
shown). For practical reasons all further experiments were carried out
with 400 units/ml, which leads to 70% of the maximal response in T47D
cells. FBS had little or no effect on the transcriptional activation
through this site, because in the presence of 10% FBS the magnitude of induction by IL-6 was the same or slightly lower than in the absence FBS.
In ZR-75-1 breast carcinoma cells and HPAF pancreatic carcinoma cells,
both which express high levels of episialin/MUC1, the luciferase
activity of the W3B construct was 3-5-fold stimulated, which is
similar to the response of the W3B reporter in T47D cells (results not
shown). The absolute luciferase activity in these cells, however, was
lower than in T47D cells, which may be due to a lower transfection
efficiency. When the M3B construct was transfected into ZR-75-1 or HPAF
cells, IL-6 had no effect on the luciferase activity.
Interestingly, the wild-type promoter of W3B in T47D cells was
consistently about 3-fold more active than the mutant construct in the
absence of any growth factor or serum (Fig. 3B), suggesting that one or more STAT factors are constitutively activated in this cell
line. As expected, we could not observe a difference between the basal
activity of the W3B and M3B constructs in A549 cells, which have a very
low episialin/MUC1 expression in contrast to T47D cells (Fig.
3C).
Since IFN- STAT3 and STAT1 Bind to the 5'-TTCCGGGAA-3' Element in the
Episialin/MUC1 Promoter--
To prove further that the element
targeted in this study is a genuine STAT-binding site and to identify
the binding proteins, we performed EMSAs or bandshift experiments,
using nuclear extracts from T47D cells. A 19-nt double-stranded
oligonucleotide representing the putative STAT site in the
episialin/MUC1 promoter was incubated with nuclear extracts
from non-induced cells and from cells that had been exposed to IL-6 for
15 min. Pilot experiments had shown that after this incubation time
binding to the oligonucleotide was maximal, which is in agreement with
data from the literature (43). Nuclear extracts of IL-6-stimulated T47D
cells, incubated with the wild-type oligonucleotide, induced the
formation of a protein-DNA complex. This complex was neither formed
with nuclear extracts from nonstimulated cells nor with an
oligonucleotide containing a mutated STAT site (Fig.
5), indicating that the IL-6-induced complex is specific for the putative STAT site.
The protein binding to the putative STAT element after stimulation of
T47D cells with IL-6 was positively identified as STAT3 by using a
specific antiserum in a supershift experiment (Fig. 5). Bandshift and
supershift experiments with IL-6-stimulated ZR-75-1 breast carcinoma
cells also revealed binding of STAT3 to the STAT element in the
episialin/MUC1 promoter (results not shown). The specificity
of STAT3 binding to the STAT element in the episialin/MUC1
promoter was shown by competition experiments with excess cold
oligonucleotides. The cold episialin/MUC1 wild-type STAT
oligonucleotide as well as an oligonucleotide representing the
STAT-binding site in the human ICAM1 (CD54) promoter competed the
IL-6-induced STAT3 complex formed with the episialin/MUC1 wild-type
STAT oligonucleotide (Fig. 5). The "ICAM" STAT-binding site has a
core binding site identical to the episialin/MUC1' STAT-binding site
(but different flanking nucleotides) and has been proven to be capable
of binding both STAT1 and STAT3 (44, 45). An excess of the mutated
episialin/MUC1 STAT oligonucleotide did not compete the IL-6-induced
STAT3 complex (Fig. 5C). Moreover, the IL-6-induced protein
complex indeed bound to the 32P-labeled ICAM STAT
oligonucleotide (results not shown). These results confirm our notion
that the episialin/MUC1 promoter contains a genuine
STAT-binding site and show that the binding proteins are specific for
the core sequence.
In addition to the IL-6-induced STAT3 complex, we observed an
additional band in EMSAs using nuclear extracts from either induced or
non-induced T47D (Fig. 5) or ZR-75-1 cells (not shown) in all lanes in
which the wild-type episialin/MUC1 STAT or ICAM STAT oligonucleotides
were used. We did not observe this complex when the mutated STAT
oligonucleotide was used. Furthermore, this complex could be competed
by either cold wild-type episialin/MUC1 STAT or ICAM STAT
oligonucleotide (Fig. 5) and not by cold mutated episialin/MUC1 STAT
oligonucleotide, which shows that the binding of this complex is also
specific. However, the identities of the protein(s) in this complex
have not been established yet. None of the tested antibodies against
STAT1, -2, -3, -5, and -6 were reactive with this protein complex in
supershift experiments. Also antibodies against c-Rel, which has been
reported to bind to a STAT5-binding site (46), did not
supershift.5 We therefore
refer to this band as "non-identified". EMSAs with A549 extracts
did not show the non-identified complex (results not shown).
Similar bandshift and supershift experiments were performed with
IFN- Induction of Endogenous Episialin/MUC1 by Cytokines in Cell
Lines--
Next, we investigated whether IFN-
Next, we tested whether cytokines could induce episialin/MUC1
expression in an episialin/MUC1 negative non-epithelial cell line.
HL-60 promyelocytic cells do not synthesize episialin/MUC1, but
expression of several genes can be induced by IFN-
The lack of an IL-6 response on the basal expression level of
episialin/MUC1 in T47D and ZR-75 cells cannot be attributed to factors
missing in the IL-6 transduction pathway as the reporter gene did
respond. We therefore investigated whether a presumptively increased
transcription of the episialin/MUC1 gene by IL-6 was counteracted at some later step in the biosynthesis of episialin, for
instance by a decreased stability of the mRNA. In accordance with
the results described above, we did not observe a significant difference in episialin/MUC1 protein levels (Fig.
8A) or in the steady-state
levels of episialin/MUC1 mRNA in T47D cells in the absence or
presence of IL-6 (Fig. 8B). When the cells were cultured in
the presence of the mRNA synthesis inhibitor actinomycin D, we also
could not find a difference in the amount of episialin/MUC1 mRNA
levels whether or not IL-6 was present in the culture medium. This
means that the half-life/stability of episialin/MUC1 mRNA also is
not changed by IL-6 (Fig. 8B). This clearly confirms our notion that the explanation for the remarkable differences between the
luciferase and episialin protein levels as a result of IL-6 induction
in these cells resides at the level of episialin/MUC1 promoter regulation.
Immunohistological studies of tissue sections with monoclonal
antibodies against peptide epitopes in the mucin domain of episialin suggested that this molecule is strongly overexpressed in breast cancer
(4, 5). However, the mucin domain of episialin on normal breast
epithelial cells carries numerous branched O-linked carbohydrates that will block or hamper binding of the antibodies, whereas episialin molecules derived from carcinomas mainly contain shorter nonbranched glycans, more easily allowing the access of the
antibodies to their epitopes (49-52). Thus, the increased reactivity of most monoclonal antibodies against episialin with tumor cells as
reported in the literature could well be explained by differences in
post-translational modifications in normal and tumor cells and not
necessarily by overexpression of the episialin/MUC1 gene. In
this report, our immunohistological studies, employing monoclonal antibodies that are hardly, if at all, affected by
O-glycosylation, indicate that overexpression of
episialin/MUC1 protein in breast carcinoma cells indeed occurs. The
reports of Zaretsky et al. (6) and Bièche and Lidereau
(17) indicate that increased episialin mRNA levels are responsible
for the overexpression of episialin/MUC1 protein in breast carcinoma
cells. However, their studies were performed on whole tissue
homogenates and therefore are difficult to interpret. Our in
situ hybridization results unequivocally show for the first time
that the episialin/MUC1 gene is indeed strongly
overexpressed in breast carcinomas relative to normal breast epithelium.
One possible explanation for the increased episialin/MUC1 mRNA
levels in carcinoma cells is stabilization of the mRNA. However, the half-life in the normal breast epithelial cell line HBL-100 (Fig.
2) and in the tumor cell lines MCF-7 (41) and T47D (Fig. 8) is not
significantly different. In fact, episialin/MUC1 mRNA is very
stable, which is compatible with a role of episialin as a structural
protein. Other ways to overproduce an mRNA are amplification of the
gene and, more rarely, up-regulation of the promoter by mutation. The
first mechanism is indeed operative in the case of episialin/MUC1, as
shown by Bièche and Lidereau (17), but it is neither able to
explain all cases of overexpression nor it can explain the extent of
mRNA overexpression in carcinomas and cell lines. A promoter
mutation is expected to affect only one of the two alleles. Northern
data provided by Ligtenberg et al. (18) and Bièche and
Lidereau (17) clearly indicate that the expression of both alleles,
which can be easily distinguished from one another on the basis of
differences in the number of repeats, is directly proportional to the
number of gene copies as determined on Southern blots. These
observations exclude mutation(s) in the promoter as a mechanism of
overexpression and leave changes in transcriptional regulation of the
MUC1 gene as the most important mechanism of episialin/MUC1
mRNA overexpression.
Our study shows that the episialin/MUC1 promoter can be
stimulated by STAT transcription factors via a STAT-binding site in its
promoter. The position and sequence of this site is completely conserved in the promoters of mouse (29) and gibbon (28), and the
9-nucleotide core sequence is identical to that of the proven binding
site for STAT1 and STAT3 in the ICAM1 promoter (44, 45). We
identified the proteins that bind to the STAT site in the
episialin/MUC1 promoter as STAT1 in the case of induction by
IFN- Even without the addition of any growth factor or serum the W3B
construct, which contains a firefly luciferase reporter gene driven by
720 bp of the episialin/MUC1 promoter (including the STAT-responsive element), already showed a significant activity in
reporter assays in the mammary carcinoma cell line T47D, which decreases 3-fold upon mutation of the STAT site. This observation indicates that a constitutively activated (STAT) factor is present in
this cell line, which may contribute to overexpression of
episialin/MUC1. Indeed constitutively activated STATs have been
reported in breast cancers (32, 33). Moreover, our bandshift
experiments do show a specific, STAT response element binding
complex that is unaffected by treatment of the cells with IL-6 or
IFN- Although a strong induction of the promoter activity by IL-6 occurs in
reporter assays with T47D and ZR-75-1 cells, episialin/MUC1 protein
levels do not increase to the same extent when the same cells are
treated with IL-6. Since we have found no evidence for post-transcriptional regulatory mechanisms affecting the episialin/MUC1 levels, as for instance has been reported for the rat sialomucin complex (54), this discrepancy must be attributed to either the
inability of the promoter to be further stimulated, because it is
already fully activated in these cells, or to the presence of the
promoter in a truncated form in a plasmid instead of its normal genomic
surroundings. Indeed, the level of episialin/MUC1 in the two cell lines
showing the highest expression, T47D and ZR-75-1, can barely be induced
any further, whereas in cell lines with an intermediate or low
expression level, episialin/MUC1 can be up-regulated by either IL-6 or
IFN- Another explanation for the unresponsiveness of the episialin protein
levels to induction by IL-6 in some of the IL-6 receptor-positive carcinoma cells may be the inability of the STAT3 protein to bind to
its binding site in the episialin/MUC1 promoter, because the site is already occupied, for instance by the previously mentioned unidentified protein in the bandshifts. Preliminary experiments suggest
that this band has a similar or even higher affinity for the
oligonucleotide than STAT3. The fact that a STAT3-specific band can be
observed in the EMSAs should then be attributed to the presence of
excess target oligonucleotide. Induction of the promoter activity in
the reporter construct by IL-6 can be similarly explained by the
abundance of reporter plasmid in the transiently transfected cells.
Finally, it can be envisaged that by transfecting multiple copies of
the promoter plasmid, a repressor of the STAT site is titered out,
resulting in an IL-6-induced expression of the MUC1 promoter
in transfected cells only. However, a concentration series of W3B,
while keeping the total amount of DNA constant, did not show any
evidence for such an effect. Induction of the promoter could be
observed with a broad range of W3B DNA concentrations (0.05-2.0 µg
of added plasmid, results not shown).
Several other regulatory elements have been identified in the
episialin/MUC1 promoter. For instance, Kovarik et
al. (30, 55) identified an E box, termed E-MUC1, at position Episialin is expressed in activated T cells (56) and plasma cells and
not or at very low levels during earlier stages of B cell
development.7 In addition,
STATs are known to regulate various stages of lymphocyte development.
Therefore, STAT-dependent regulation of episialin/MUC1 during development of hematopoietic cells can be expected. In this
respect it is important to note that IL-6 is an autocrine growth factor
in various leukemia cells, including myeloma cells and other plasma
cell malignancies, which frequently express high levels of
episialin/MUC1 (57-59).
Induction of episialin expression has important consequences for
cellular adhesion in cancer cells and certain normal cells. Tight
regulation of the expression levels seems therefore of utmost importance. Regulation of episialin/MUC1 via cytokines and the JAK-STAT
pathway seems functional in several cell types. Constitutive activation
of STATs, as has been reported in breast carcinomas and in various
lymphomas and leukemias, is probably one of the factors responsible for
the overexpression of episialin in these cells.
can activate STATs. In the human
breast carcinoma cell line T47D, both compounds are able to stimulate
transcription of a luciferase reporter gene under the control of a
750-base pair MUC1 promoter fragment proximal to the
transcription start site. The observed increase is entirely mediated by
the single STAT-binding site, since mutation of this site abolishes
stimulation of the reporter by interleukin-6 and interferon-
. In
addition, mutation of the STAT site also decreased the promoter
activity in nonstimulated T47D cells, suggesting that the STAT-binding site is among the elements that are involved in the overexpression of
MUC1 in tumor cells.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
heavy chain enhancer (15, 16) and is consistent with
a role in tumorigenesis of these lymphomas. However, the cause(s) of episialin/MUC1 overexpression in carcinoma cells have not been established yet. Previous studies (6, 17) have shown that the
episialin/MUC1 mRNA level is severalfold increased in primary breast carcinoma specimens relative to adjacent normal epithelium. However, these results, which were obtained using mRNA blots of tissue homogenates, are inherently imprecise, since both breast carcinoma and normal breast tissue specimens usually contain
significant amounts of other nonepithelial cell types. Even so, these
results indicate that overexpression of episialin/MUC1 coincides with increased mRNA levels. Episialin/MUC1 mRNA levels in breast
carcinoma cell lines also are increased in comparison to immortalized
nontransformed epithelial cells (18). The study of Bièche and
Lidereau (17) showed that the higher episialin/MUC1 mRNA
levels to some degree may be caused by amplification of the
MUC1 gene, but additional mechanisms of overexpression
clearly are operative as well, since the level of overexpression often
is much higher than the level of amplification.
is known to up-regulate episialin/MUC1 expression in cell lines
derived from breast carcinomas
(19).3 Similarly, mouse
episialin/MUC1 expression is positively regulated by prolactin, when
cells are grown on matrigel-coated membrane filters (20). Induction of
expression by prolactin is in line with the increased expression of
human episialin during lactation (4), when prolactin levels are
strongly increased. Notably, breast carcinoma cells may synthesize
prolactin (21, 22) that could lead to autocrine stimulation of
episialin expression. Both IFN-
and prolactin act via signaling
transducers and activators of transcription (STATs). Different members
of this protein family function during inflammatory processes and
during mammary gland development and lactation to up-regulate the
transcription of a large number of genes (23). In addition to
interferons and prolactin, STATs may also be activated by cytokines of
the interleukin family, such as interleukin-6 (IL-6). IL-6, which
signals via STAT3, is produced by a variety of cells among which are
epithelial cells (for a review see Klein (24)). It is noteworthy that
enhanced levels of IL-6 are found in many solid tumors (25), whereas in
the breast carcinoma cell lines T47D and ZR-75-1 IL-6 cause an enhanced
motility and decreased adhesion (26, 27), properties that could involve
overexpression of episialin/MUC1.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
4 M disodium EDTA,
washed once in DMEM plus 10% FBS. Episialin was detected in an
indirect immunofluorescence assay using monoclonal 214D4 directed against the protein backbone in the repeat domain of episialin/MUC1 and
fluorescein isothiocyanate-labeled secondary antibody. The fluorescence
intensity of the cells was determined by FACScan analysis.
-actin mRNAs were used as controls.
1642 to +33 of the human
episialin/MUC1 gene (numbering according to Kovarik et
al. (30)) was initially cloned in the SmaI site of
pGL2-basic (Promega), a firefly luciferase vector. This construct was
used for the mutagenesis of the presumptive STAT-binding site, which is
located at positions
503 to
495 and has the sequence
5'-TTCCGGGAA-3'. In the mutant construct this sequence was changed to
5'-TTACGTAAA-3', thereby creating a
BsaAI site. Mutagenesis was performed using the Transformer
site-directed mutagenesis kit (CLONTECH). Mutated plasmids were detected by screening for the BsaAI site. A
SacI-SacI fragment of both the wild-type and
mutant pGL2 constructs was subsequently subcloned into the
SacI site of pGL3, which has a much higher luciferase
activity in most cell lines. The final constructs contained a 755-bp
promoter fragment, from the SacI site at
722 to the
XmnI site at +33 in the untranslated part of exon 1 of the
episialin/MUC1 gene, followed by a small part of the
pGL2-basic polylinker (from the SmaI site to the
SacI site). The resulting plasmids were named W3B (wild-type
pGL3-basic) and M3B (mutant pGL3-basic), respectively.
(Roche Molecular Biochemicals; 200 units/ml) or recombinant IL-6 (Roche Molecular Biochemicals; 400 units/ml) were added 24 h after transfection, and the cells were
incubated for an additional 24 h.
80 °C.
80 °C.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Overexpression of episialin/MUC1 mRNA in
a mammary carcinoma as demonstrated by in situ
hybridization. A, immunostaining of a mammary
carcinoma section with an anti-episialin antibody 139H2 (directed
against the repeats). B, for comparison, in situ
hybridization was performed on an adjacent section according to
Wilkinson and Nieto (37), using an antisense RNA probe that was
complementary to the unique region in the episialin/MUC1 mRNA
downstream from the repetitive domain. A normal duct, showing a low
mRNA signal and a thin, but clear, apical staining
(arrows), is surrounded by carcinoma cells that have a very
high hybridization signal and an intense immunostaining. Another
section was used for in situ hybridization with the sense
probe and showed a very low and evenly distributed background staining
(not shown).
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Fig. 2.
Stability of the episialin/MUC1 mRNA in
HBL100 cells. HBL100 cells were treated for increasing times with
actinomycin D, and the decay of the mRNA was determined by Northern
hybridization with a 32P-labeled episialin/MUC1 cDNA
probe (K5) and with c-Myc and actin probes as controls representing an
unstable and a stable mRNA, respectively. Note, in HBL100 cells
both MUC1 alleles have the same size. The lane marked eth
shows a control in which the same amount of ethanol was added to the
cells as was used to dissolve the actinomycin D.
503 to
495 in the
human episialin/MUC1 promoter (30) (numbering also according
to this reference). This putative STAT site might well mediate the
effects of IFN-
and/or prolactin, both proven inducers of MUC1
expression, and IL-6 which is often produced by solid tumors, including
breast carcinomas (see Introduction). To obtain evidence that this
element indeed represents a STAT-binding site, a
SacI-XmnI promoter fragment spanning nucleotide
722 to +33 and ending in exon 1, 36 nt upstream from the start codon,
was cloned in front of the firefly luciferase gene of pGL3-basic
resulting in reporter plasmid W3B. In addition, a control construct,
M3B, in which the putative STAT site was mutated into 5'-TTACGTAAA-3',
was also created (Fig.
3A).
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Fig. 3.
Stimulation of the episialin/MUC1
promoter in T47D breast carcinoma cells and A549 lung carcinoma
cells by IL-6. A, the W3B (wild-type promoter in
pGL3-Basic) and the M3B (mutant promoter in pGL3-Basic) firefly
luciferase reporter constructs and the exact nature of the nucleotide
changes in M3B are shown. B and C, the relative
luciferase activities of the W3B and M3B constructs in T47D cells
(B) and A549 cells (C) in the presence and
absence of IL-6 are presented. The assay was carried out with cells
that were transiently transfected with 0.5 µg of the W3B or M3B
constructs (firefly luciferase), 0.01 µg of the SV40
Renilla luciferase construct, and 1 µg of salmon sperm
DNA. Both luciferase activities were measured, and the firefly
luciferase activities were normalized using the Renilla
luciferase results. All assays were performed in triplicate.
Bars indicate the S.D. STAT-RE, STAT response element.
has been shown to stimulate episialin/MUC1 expression in
ovarian carcinoma cells (19) and is known to stimulate gene expression
through STAT1, we investigated whether this cytokine could also
activate the episialin/MUC1 promoter via the putative STAT-binding site. To this end, T47D cells, transiently transfected with the W3B and M3B constructs, were treated with IFN-
for 24 h. Fig. 4 shows that IFN-
can
stimulate the luciferase activity from the W3B construct ~8-10-fold
but not from the construct with the mutant STAT site (M3B), suggesting
that in addition to IL-6-activated STAT3, STAT1 can also bind to the
episialin/MUC1 promoter.
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Fig. 4.
Stimulation of the episialin/MUC1
promoter in T47D breast carcinoma cells by
IFN- . T47D cells were transiently
transfected with the W3B or M3B reporter constructs and stimulated with
IFN-
. For details of the assay see legend Fig. 3. The firefly
luciferase results were normalized using the Renilla
luciferase activities. The assays were performed in triplicate.
Bars indicate the S.D.
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Fig. 5.
Nuclear extracts from IL-6-stimulated T47D
cells induce a specific bandshift with an oligonucleotide representing
the putative STAT-binding site in the promoter of episialin/MUC1.
EMSA using a 32P-labeled 19-nt double-stranded
oligonucleotide (WT = 5'-GGCTATTCCGGGAAGTGGT-3') containing the centrally located
episialin/MUC1 STAT-binding site and its flanking sequences
(3rd to 10th lanes) or a mutant
oligonucleotide (MT = 5'-GGCTACTCGAGAAGTGGT-3', mutant nucleotides are
shown in bold, 1st and 2nd
lanes), and nuclear extracts from untreated T47D cells
(1st and 3rd lanes) or from cells
treated with IL-6 (400 units/ml) for 15 min (2nd and
4th to 10th lanes). The
oligonucleotide-protein complexes were separated on a 5%
polyacrylamide gel in 1× TBE. The IL-6-induced STAT-RE binding complex
is indicated, whereas an additional, non-identified but specific
STAT-RE binding protein complex is designated as non-identified. *,
nonspecific band; the intensity of this band varied between and even
within experiments. The IL-6-induced STAT-RE binding complex could be
supershifted with a polyclonal antiserum against STAT3 (5th
lane). An excess of either cold wild-type oligonucleotide
(10th lane) or cold ICAM-1 oligonucleotide
(representing the proven STAT1/STAT3-binding site in the human
ICAM-1 promoter, which has the identical core sequence as
the episialin/MUC1 STAT-binding site;
5'-GAGGTTTCCGGGAAAGCAG-3', 8th and
9th lanes) could compete for binding of both the
IL-6-induced STAT-RE binding complex as well as the non-identified but
specific, STAT-RE binding protein complex. An excess of cold mutant
oligonucleotide did not compete for binding of these protein complexes
(6th and 7th lane). 2 µl of the
samples were run separately on similar gels for a shorter time to
detect unbound oligonucleotide; this is shown at the bottom
of the figure. STAT-RE, STAT response element,
5'-TTCCGGGAA-3'.
-stimulated T47D cells. Clear bandshift was observed and a
monoclonal antibody against STAT1 revealed a supershifted complex of a
slightly higher mobility than the supershifted STAT3-containing complex
(Fig. 6). EMSAs with HL-60 cell extracts
did not show the non-identified band, and the STAT1 bandshift was
clearly observed with extracts of the latter cells stimulated with
IFN-
(Fig. 7). No supershifted complex
was detected with anti-STAT1 in EMSAs using IL-6-stimulated T47D
extracts. Similarly, no supershift complex was detected with anti-STAT3
in EMSAs using IFN-
-stimulated T47D or HL-60 extracts (only the
results with HL-60 cells are shown in Fig. 7). In conclusion, STAT1 and
-3 are capable of binding to the TTCCGGGAA element in the
episialin/MUC1 promoter. Thus, in the context of the W3B
construct, STAT1 and STAT3 binding most likely leads to the observed
up-regulation of the transcription of the luciferase reporter gene upon
stimulation with IFN-
and IL-6, respectively.
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Fig. 6.
IFN- induces binding
of STAT1 to the episialin/MUC1 promoter. EMSA
using 32P-labeled WT episialin/MUC1 STAT-binding oligo and
nuclear extracts from either untreated or IFN-
stimulated T47D cells
(200 units/ml, 30 min). The IFN-
-induced STAT-RE binding complex is
indicated, while an additional, nonidentified but specific, STAT-RE
binding protein complex is designated as `non-identified'. A
supershift with a monoclonal antibody against STAT1 could be observed.
Addition of 100-fold excess cold WT-oligo was used as a control for
specificity. STAT-RE, STAT response element.
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Fig. 7.
STAT1 from IFN-
induced HL-60 cells binds to the STAT-binding site in the
promoter of episialin/MUC1. Nuclear extracts of untreated HL-60
cells and cells treated with 200 units/ml IFN-
for 15 min were used
in band and supershift assays. The oligonucleotide-protein complex was
separated on a 5% polyacrylamide gel in 0.1x TBE. Additions to the
incubation mixture were: none, STAT1 antibody, STAT3 antiserum and
100-fold excess unlabeled WT-oligonucleotide respectively. A supershift
using the antibody against STAT1 could be observed, whereas the
anti-STAT3 antibody could not supershift the IFN-
induced
STAT-RE-binding protein complex. STAT-RE, STAT response element.
or IL-6 could
increase episialin/MUC1 expression in various cell lines showing no,
intermediate, or high levels of episialin/MUC1. IL-6 increased
episialin/MUC1 expression ~2.5-fold in A549 cells as measured by FACS
experiments (Table I). This induction is
comparable with the induction of the reporter gene in the W3B construct
(Fig. 3C). However, the induction of the reporter by IL-6 in
T47D cells, which show a high basal expression level of episialin/MUC1,
was not matched by a comparable increase in the episialin/MUC1 protein
levels in these cells (Table I). IFN-
also hardly stimulated
episialin/MUC1 expression in these cells. Similarly, IL-6 or IFN-
could not induce episialin/MUC1 expression in ZR-75-1 cells, which also express a very high basal level of episialin/MUC1. The MCF-7 cells used
in our studies show an intermediate expression level of episialin/MUC1. Episialin expression in this cell line was only slightly enhanced by
IL-6 but was 2-fold increased by IFN-
. These results suggest that
the stimulation of episialin/MUC1 expression by the cytokines is
moderate and dependent on the expression level in the non-induced state.
Stimulation of episialin/MUC1 expression in various cell lines by
IL-6 and IFN-
, including the Fc
receptor and MHC molecules (47, 48). Although bandshift experiments
revealed that in these cells STAT1 is strongly activated and binds to
the STAT site in the episialin/MUC1 promoter upon IFN-
treatment (Fig. 7), IFN-
did not induce expression of
episialin/MUC1. This shows that the endogenous
episialin/MUC1 promoter was not activated in these cells.
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Fig. 8.
Effect of IL-6 on MUC1 mRNA and protein
levels in T47D cells. A, total episialin/MUC1 protein
levels in either untreated or IL-6-treated T47D cells were determined
on Western blot by ECL using the monoclonal anti-repeat antibody 214D4.
The position of the 200-kDa marker is indicated; episialin/MUC1 has an
apparent molecular mass of over 400 kDa (2 allelic forms).
B, Northern blot using mRNA of T47D cells treated with
or without IL-6 (400 units/ml, 24 h) and actinomycin D (10 µg/ml, times as indicated). The blot was hybridized with
32P-labeled episialin/MUC1, c-Myc and -actin cDNAs.
The two episialin/MUC1 alleles in T47D differ in size, leading to
mRNAs of approx. 4 and 7 kb. The sizes of c-Myc and
-actin
mRNAs are respectively 2.4 kb and 1.7 kb. The positions of the 28S
and 18S ribosomal bands are indicated. The Northern blot was exposed
either to Kodak X-Omat AR films or to a phospho-imager screen.
Quantification was done using a Fujix BAS 2000 phospho-imager and Tina
2.09 software. Quantification of the samples that were not treated with
actinomycin D (lanes 1 and 5) showed that IL-6
does not influence steady-state episialin/MUC1 mRNA levels;
quantification of the other samples showed that episialin/MUC1 mRNA
stability also is not changed by IL-6. The experiment shown is
representative for multiple experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and STAT3 in the case of IL-6. This is completely in line with
the current model of IFN-
and IL-6 signaling (42, 53).
. This complex also binds to the ICAM-1 oligonucleotide but is
absent in bandshift experiments using an oligonucleotide with a mutated
STAT site. The identity of this constitutively activated protein
(complex) has not been established yet, but it may affect the
overexpression of episialin/MUC1 in carcinoma cells. The latter notion
is in line with the observation that neither the non-identified protein complex is present in the low episialin expressing A549 cells nor is
there a difference between the basal activity of the W3B and M3B
constructs transfected into these cells.
, suggesting that the promoter activity in the former cells has
reached its limits. Alternatively, an additional regulatory element
located outside the promoter fragment used in the W3B construct may
counteract the STAT-induced expression in T47D and ZR-75-1 cells.
Preliminary results show that increasing the size of the 5' promoter
fragment in the reporter construct to 2.9 kb (i.e. into the
3'-untranslated sequence of the trombospondin 3 gene) does not diminish
the ratio between the noninduced and the IL-6-induced luciferase
levels.6
84
to
74 and a Sp1-like binding site at position
101 to
89. The
latter site can bind an inhibitory factor, SpA, or in the absence of SpA the stimulatory factor Sp-1. Since SpA and E-MUC1 are expected to
be expressed in a tissue-specific fashion, they may confer tissue-specific expression of episialin/MUC1. In line with these results, we found that the presence of activated STAT1, capable of
binding to the STAT-binding site in the episialin/MUC1
promoter, does not lead to episialin/MUC1 expression in the
promyelocytic HL-60 cells.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Bernd Groner and Dr. Fabrice Gouilleux for their hospitality and advice during the initial phase of the study. We also thank Mandy Boer, Ernst van Bemmel, Anita van der Wal, and Tamara Prinsenberg for performing some of the experiments.
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FOOTNOTES |
---|
* 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.
Both authors contributed equally to this work.
§ Supported by NCI Grant 1R01 CA79580-01 from the National Institutes of Health.
¶ Supported by Grant 93-523 of the Dutch Cancer Society.
To whom correspondence should be addressed. Tel.:
31-20-5122018; Fax: 31-20-5122029; E-mail: jhi@nki.nl.
Published, JBC Papers in Press, November 17, 2000, DOI 10.1074/jbc.M009449200
2 J. Wesseling and J. Hilkens, unpublished data.
3 C. F. M. Molthoff, personal communication.
4 H. L. Vos, S. Van der Valk, and J. Hilkens, unpublished data.
5 I. Gaemers, unpublished results.
6 I. C. Gaemers, H. H. Volders, and J. Hilkens, manuscript in preparation.
7 J. Hilkens, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: MUC1, mucin-1; EMSA, electrophoretic mobility shift assay; FBS, fetal bovine serum; ICAM-1, intercellular adhesion molecule-1; IFN, interferon; IL-6, interleukin-6; STAT, signal transducer and activator of transcription; DMEM, Dulbecco's modified Eagle's medium; bp, base pair; PBS, phosphate-buffered saline.
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