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Publication of the International Union Against Cancer
Publication de l’Union Internationale Contre le Cancer
Int. J. Cancer: 71, 741–749 (1997)
r 1997 Wiley-Liss, Inc.
Amos BARUCH1,2, Mor-li HARTMANN1,2, Sheila ZRIHAN-LICHT1, Shulamit GREENSTEIN1, Matti BURSTEIN1, Iafa KEYDAR1,
Mordechai WEISS1, Nechama SMORODINSKY1 and Daniel H. WRESCHNER1*
1Department of Cell Research and Immunology, The George S. Wise Faculty of Life Sciences, Tel Aviv University,
Ramat Aviv 69978, Israel
The human MUC1 gene expresses at least 2 type 1 membrane proteins: MUC1/REP, a polymorphic high m.w. MUC1
glycoprotein often highly expressed in breast cancer tissues
and containing a variable number of tandem 20 amino acid
repeat units, and the MUC1/Y protein, which lacks this repeat
array and, therefore, is not polymorphic. Despite their documented importance in signal transduction processes, the
relative expression of the 2 isoforms in epithelial tumors is
unknown. Using antibody reagents which recognize different
MUC1 domains, the expression of these isoforms in malignant epithelial cells has been evaluated. A comparison of the
amounts of the 2 isoforms revealed preferential expression of
the novel MUC1/Y protein in breast cancer tissue samples.
Furthermore, although the MUC1/REP protein is almost
undetectable in HeLa cervical adenocarcinoma epithelial
cells, the MUC1/Y isoform is extensively expressed in these
cells. The presence of the MUC1/Y sequence as well as that of
an additional tandem-repeat-array-lacking isoform, designated MUC1/X, were demonstrated by reverse transcriptase
PCR amplification of RNA extracted from HeLa and ovarian
carcinoma cells. It has been shown previously that the MUC1
cytoplasmic domain interacts with the SH2 domain containing GRB2 protein, which transduces signals to ras, a protein
which in its activated form can lead to cell transformation.
We present here data demonstrating that MUC1/Y isoform
expression increases the tumorigenic potential of DA3 mouse
mammary epithelial cells; in contrast, potentiation of tumorigenicity is not observed with MUC1/REP expression. Our
studies thus demonstrate that expression of the MUC1 gene
in epithelial tumors can give rise to substantial levels of
MUC1 proteins devoid of the tandem repeat array, which are
generated by alternative splicing mechanisms. Int. J. Cancer
71:741–749, 1997.
r 1997 Wiley-Liss, Inc.
The MUC1 gene is frequently expressed with high intensity in
human carcinomas, especially in breast cancer tissue (Ceriani et al.,
1977; Burchell et al., 1987). In these patients, augmented concentrations of MUC1 proteins (previously referred to as episialin,
H23Ag; ETA, epithelial tumor antigen; PEM, polymorphic epithelial mucin; EMA, epithelial membrane antigen; CA15-3; MCA,
mammary carcinoma antigen) may be one of the earliest signs of
disease progression, and, as such, the MUC1 gene has aroused
much interest. A major MUC1 gene product (designated MUC1/
REP) is a polymorphic type 1 transmembrane molecule which
consists of a large, heavily glycosylated extracellular domain, a
transmembrane domain and a 72 amino acid cytoplasmic tail
(Wreschner et al., 1990; Hareuveni et al., 1990; Ligtenberg et al.,
1990; Gendler et al., 1990). This isoform is proteolytically cleaved
early after translation (Ligtenberg et al., 1992a) and is shed from
the cell by an unknown mechanism. The MUC1 isoform, MUC1/Y,
generated by alternative splicing (Zrihan-Licht et al., 1994b), has a
molecular mass of 42–45 kDa and, in contrast to MUC1/REP, does
not undergo proteolytic cleavage (Zrihan-Licht et al., 1994b). Furthermore, this novel MUC1 isoform retains the N-terminal as well as the
transmembrane and cytoplasmic MUC1 domains but lacks the tandem
repeat array that has been considered the hallmark of MUC1.
To date, the relative extent of expression in epithelial tumors of
the MUC1 protein containing the tandem repeat array vs. that of the
novel isoform devoid of the tandem repeats has not been studied.
To address this issue, we determined the expression of MUC1 gene
products in epithelial tumors using not only a panel of antibodies
directed against the 20 amino acid tandem repeat motif but also
antibodies which specifically recognize the MUC1/Y cytoplasmic
and extracellular domains. Reverse transcriptase PCR (RT-PCR)
was used to assess the expression of MUC1 isoforms devoid of the
tandem repeat array. Our studies demonstrate that the MUC1
isoforms lacking the tandem repeat array not only are extensively
expressed in epithelial tumors but in certain cases they are almost
the only MUC1 protein products observed.
Significantly, the tyrosine and, to a lesser extent, the serine
residues of the cytoplasmic domains of the MUC1 proteins undergo
phosphorylation (Zrihan-Licht et al., 1994a). One study confirmed
the phosphorylation of the MUC1 tyrosine residues (Pandey et al.,
1995) and demonstrated in vivo interaction between the MUC1
cytoplasmic domain and the GRB-2 protein. As membrane receptor
proteins participating in signal transduction processes that involve
GRB-2, and thereby ras, are often important for tumorigenesis, we
analyzed whether MUC1 expression can affect the tumorigenic
potential of MUC1 isoform transfectants. We show that the novel
MUC1/Y protein, in contrast to MUC1/REP, can increase the transforming potential of DA3 mouse mammary tumor cell transfectants.
Our findings suggest that MUC1 isoforms lacking the tandem
repeats may act as novel marker proteins of malignant epithelial
cells and play an active role in the oncogenetic process. It is thus
likely that the novel tandem repeat array lacking MUC1 isoforms
described here perform functions distinct from those of the
MUC1/REP protein.
Stable transfectants expressing MUC1 isoforms were generated
by co-transfecting an expression plasmid harboring either MUC1/
REP or MUC1/Y cDNA with the neomycin plasmid (pSV2 neo)
selection marker into 3T3 ras-transformed fibroblasts (Wreschner
et al., 1990; Hareuveni et al., 1990; Zrihan-Licht et al., 1994a, b)
or DA3 mouse mammary tumor cells (Fu et al., 1990). The cell line
T47D is derived from a human mammary carcinoma and is a high
MUC1 expressor. HeLa cells are derived from a uterine cervical
adenocarcinoma and express low levels of the MUC1/REP isoform. All cell lines were grown in DMEM supplemented with 10%
FCS and 2 mM L-glutamine.
cDNA constructs
Generation of expression vectors harboring either the full-length
transmembrane MUC1/REP or the novel MUC1/Y cDNA and
driven by the HMG co-enzyme A reductase promoter (expression
Contract grant sponsors: Israel Cancer Association, Israel Cancer Research Fund, Israel Academy of Sciences, Federico Fund of Tel Aviv
University, Barbara Friedman Fund, Simko Chair for Breast Cancer
*Correspondence to: Department of Cell Research and Immunology, The
George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv
69978, Israel. Fax: 972-3-6422046.
first two authors contributed equally to these studies.
Received: 10 September 1996; accepted 6 January 1997
vector pCL642) has been described previously (Wreschner et al.,
1990; Hareuveni et al., 1990; Zrihan-Licht et al., 1994a,b).
Western blot analyses
Cell lysates were prepared by adding lysis buffer (50 mM NaCl,
20 mM Tris-HCl [pH 7.4], 100 µg/ml leupeptin and 0.5% Nonidet
P-40) either to cell pellets or to surgically resected breast tissues,
pulverized under liquid nitrogen, followed by vortex mixing and
sonication (3 3 10 sec bursts using a Branson sonicator). Cell
debris were removed by centrifugation at 12,000g for 10 min. All
procedures were performed at 4°C or on ice. Protein samples were
denatured by boiling in SDS-buffer containing mercaptoethanol
and analyzed on SDS/acrylamide gels. Gels were electrotransferred
for 3 hr at 1 A to nitrocellulose filters, which were then blocked in
PBS containing 5% skimmed milk, followed by incubation with the
primary antibody. Filters were washed in PBS and then incubated
with a secondary anti-rabbit (or anti-mouse) antibody conjugated to
horseradish peroxidase followed by ECL (Amersham, Aylesbury,
UK) detection.
Western blot analyses were performed with (i) monoclonal
antibodies (H23 MAbs) that recognize an epitope contained within
the tandem repeat domain of the MUC1/REP protein (Keydar et al.,
1989), (ii) a polyclonal antibody (a gift from Dr. S. Gendler,
London, UK) directed against the 17 C-terminal amino acids of the
MUC1 cytoplasmic domain, (iii) an affinity-purified polyclonal
antibody (designated anti-MUC1/Yex) prepared by injecting rabbits with the MUC1/Y extracellular domain synthesized as a
recombinant protein using the PET expression system according to
the protocol described below, and (iv) affinity-purified antibodies
recognizing MUC1/Y extracellular domain sequences that are
N-terminal to the site of proteolytic cleavage in the MUC1/REP
protein (described below).
Generation of bacterial recombinant MUC1/Yex protein and
truncated MUC1/Yex protein
DNA coding for the MUC1/Y extracellular domain was generated by PCR using an up-stream oligonucleotide (ACAAGTACTGGTCATGCA AGCTCTACCCCAGGTGGAG) that introduces a
Sca1 restriction enzyme site (AGTACT) at the 58 terminus. The
first nucleotide of the sequence CTGGTCA corresponds to the
MUC1/Y nucleotide at position 128 (Zrihan-Licht et al., 1994b)
and the trinucleotide ACT codes for an N-terminal threonine in
place of the serine located at amino acid 24 in the MUC1/Y
sequence (Zrihan-Licht et al., 1994b) and represents the first
N-terminal amino acid following cleavage of the MUC1/Y signal
peptide sequence. The down-stream anti-sense oligonucleotide
used introduces a termination codon after the nucleotide sequences
coding for the amino acid sequence SAQSGAGV (amino acids
149–156; Zrihan-Licht et al., 1994b) located just up-stream to the
MUC1/Y transmembrane domain; the termination codon is then
followed by a BamH1 site. For PCR amplification, the MUC1/Y
cDNA was used as a template with the above up-stream and
down-stream primers. The PCR product obtained was cleaved with
Sca1 and BamH1 and ligated to Stu1- and BamH1-cleaved
pET11d-DHFR vector DNA (this pET 11d vector [Novagen,
Madison, WI] codes for a non-relevant bacterial dihydrofolate
reductase protein that at its carboxyl terminus contains the factor
Xa cleavage site [amino acids IEGR] followed by Stu1 and BamH1
restriction sites; the vector was obtained from Dr. M. Mevarech
[Tel Aviv University]). The recombinant plasmid pET11d-DHFRMUC1/Yex was introduced into BL21(DE3)pLysS bacteria, and
recombinant protein was induced by the addition of IPTG.
Affınity purification of anti-MUC1/Yex antibodies
The recombinant DHFR-MUC1/Yex fusion protein (see above)
was purified from inclusion bodies by preparative SDS-PAGE and
injected with Freund’s adjuvant into New Zealand white rabbits.
The anti-MUC1/Yex serum obtained was purified by passage
through an Affigel-10 (Bio-Rad, Hercules, CA) affinity column to
which N-terminal histidine tagged recombinant MUC1/Yex protein
(see below) had been covalently bound. The use of this affinity
resin ensures that only antibodies recognizing the MUC1/Y extracellular domain will be purified. Anti-MUC1/Yex antibodies were
eluted from the affinity column with 0.1 M glycine-HCl, pH 2.7,
buffer. Eluted fractions were immediately neutralized by the
addition of 1 M Tris base.
Generation of N-terminal histidine tagged recombinant
MUC1/Yex protein
The expression vector coding for N-terminal histidine tagged
recombinant MUC1/Yex protein was generated by utilizing the
pET16b (Novagen) expression system. The MUC1/Y extracellular
domain (nucleotides 127–525; Zrihan-Licht et al., 1994b) was
amplified by PCR using the full-length MUC1/Y cDNA as template
and up-stream and down-stream primers that introduced Nde1 and
BamH1 sites at the 58 and 38 termini, respectively. The purified
PCR fragment was cleaved with Nde1 and BamH1 and ligated to
Nde1/BamH1 cleaved pET16b vector DNA. Recombinant DNA
plasmid was introduced into BL21(DE3)pLysS bacteria and recombinant protein prepared as follows. An overnight culture of
recombinant bacteria was diluted 1:100 into fresh LB medium
containing 100 µg/ml ampicillin. After the culture reached an
optical density (at 600 nm) of 0.3, IPTG was added to a final
concentration of 1 mM and cells were grown for an additional 5 hr.
Cells were centrifuged at 5,000 g for 10 min, and the bacterial
pellet was washed with 0.9% NaCl, resuspended in lysis buffer (0.1
M phosphate buffer [pH 7.0], 10 mM EDTA, 0.1% Triton X-100;
30 ml/l culture) followed by vortexing and sonication (5 3 10 sec
bursts, using a Branson sonicator). Inclusion bodies were pelleted
by centrifugation at 12,000g for 10 min. The pellet was resuspended in buffer A (10 mM Tris-HCl [pH 8.0], 50 mM Na2HPO4, 6
M guanidine HCl; 5 ml/g of cell pellet wet weight) and incubated
for 30 min in the same buffer at 25°C. The suspension was
centrifuged at 10,000 g and the supernatant incubated with a 50%
Ni-agarose slurry for 30 min at 25°C. The slurry was loaded onto a
2 cm diameter column and washed with 10 column volumes of
buffer A, 20 column volumes of buffer B (10 mM Tris-HCl [pH
8.0], 50 mM Na2HPO4, 8 M urea) and buffer C (10 mM Tris-HCl
[pH 6.3], 50 mM Na2HPO4, 8 M urea) until an optical density at
280 nm of less than 0.01 was obtained. Elution was performed with
150 mM imidazole in buffer C. For protein renaturation, fractions
containing recombinant protein were loaded onto a 50 ml Sephadex
G-50 column pre-equilibrated with 0.1 M NaHCO3 (pH 8.4), and
0.5 ml elution fractions were collected.
Generation of truncated MUC1/Y extracellular domain protein
Generation of bacterial recombinant MUC1/Y truncated extracellular domain cDNA was performed utilizing the pET11d expression system. The truncated MUC1/Yex construct (designated
MUC1/Y/Ssp1) was generated by replacing the entire MUC1/Yex
coding region of the pET11d-DHFR-MUC1/Yex vector with a
PCR fragment (nucleotides 128–328) that extends to the Ssp1 site
at nucleotide 328 and introduces a stop codon immediately
down-stream to the Ssp1 site, using full-length MUC1/Y cDNA as
template. BL21 bacteria were transformed with the pET11d-DHFRMUC1/Y/Ssp1 vector coding for the truncated protein. The MUC1/
Y/Ssp1 truncated protein was purified from inclusion bodies as
described above and the purified protein covalently bound to an
Affigel-10 resin. To isolate antibodies recognizing MUC1/Y/Ssp1,
the affinity-purified anti-MUC1/Yex antibodies (see above) were
passed through the MUC1/Y/Ssp1 affinity column and the antibodies eluted with 0.1 M glycine-HCl buffer (pH 2.7) as described
RNA isolated from HeLa or Lucy ovarian carcinoma cells was
reverse-transcribed using the anti-sense down-stream oligonucleotide primer ‘‘c’’ (complementary to nucleotides 1050–1018, nucleotide numbering for location of the primers as in Wreschner et al.,
1990), and 10% (2 µl) of the reverse transcription reaction
underwent PCR amplification in a final volume of 40 µl with the
same down-stream primer and up-stream sense oligonucleotide
primer ‘‘a’’ (nucleotides 18–42), using 35 cycles of 95°C, 30 sec;
55°C, 60 sec; and 72°C, 60 sec. PCR products were purified by
agarose gel electrophoresis and, in some cases, subjected to nested
PCR amplification using the same down-stream anti-sense primer
(c) and the up-stream primer (b) corresponding to nucleotides
60–88. All reagents used for RT-PCR were obtained from a Gene
Amp RNA PCR kit (Perkin Elmer Cetus, Norwalk, CT), and the
manufacturer’s protocols were followed.
DNA sequencing
MUC1-specific RT-PCR products were subcloned into M13 and
sequenced using the dideoxynucleotide chain-termination method.
The M13 universal primer or synthetic oligonucleotides prepared
according to known sequences were used to prime ssDNA.
To examine the levels of the various MUC1 isoforms in
epithelial tissues, we assessed MUC1 expression with antibodies
directed against various domains of the MUC1 proteins.
Characterization of MUC1/REP expression with anti-repeat array
Expression of MUC1 proteins in epithelial tumors was initially
analyzed with H23 MAbs that recognize an epitope present within
the 20 amino acid tandem repeat motif. As previously reported
(Wreschner et al., 1990; Hareuveni et al., 1990; Ligtenberg et al.,
1990; Gendler et al., 1990), these antibodies detect polymorphic
high m.w. MUC1 proteins of different sizes and with molecular
masses greater than 200 kDa (Fig. 1a). The polymorphism is due to
varying lengths of the tandem repeat array included in the MUC1
alleles. Immunoreactive polymorphic MUC1 proteins migrating
FIGURE 1 – MUC1/REP and MUC1/Y expression in tumor tissue.
Identical protein lysates prepared from primary breast tumor tissue
samples (BT), normal breast tissue (BN), paired tumor and normal
breast tissues from the same patients (BT*, BN* and BT9, BN9), T47D
breast cancer cells or HeLa adenocarcinoma cells were analyzed on (a)
6% or (b) 15% SDS/acrylamide gels. This was followed by Western
blotting and probing with H23 MAbs that recognize an epitope within
the tandem repeat array (a) or with polyclonal antibodies generated
against the cytoplasmic domain (b). Detection was with horseradish
peroxidase-conjugated secondary antibodies followed by enhanced
chemiluminescence. Numbers to the left indicate the molecular mass in
kDa. (a) Expression of repeat-array-containing MUC1 proteins detected with anti-repeat antibodies. The immunoreactive bands represent
the high m.w. polymorphic repeat-array-containing MUC1 proteins.
The mature (fully glycosylated) repeat-array-containing proteins present in the T47D breast cancer cells are indicated by the top 3 arrows to
the left; the bottom 3 arrows indicate the core or partially glycosylated
repeat-array-containing proteins in the T47D sample. (b) Expression of
MUC1/Y proteins detected with anti-cytoplasmic domain antibodies.
The mature 42–45 kDa MUC1/Y protein is indicated at the left and
right of the figure by the full arrow; the arrowhead indicates the
non-glycosylated MUC1/Y (see also c and d). Brackets to the left and
right indicate the cytoplasmic domain-containing proteolytic cleavage
products migrating with a molecular mass of 25–30 kDa. (d) The
MUC1/Y protein is N-glycosylated. The MUC1/Y protein was expressed in 35S-methionine-labeled BSC-1 monkey epithelial cells that
had been infected with recombinant vaccinia virus coding for T7 RNA
polymerase and transfected with the pTM1 vaccinia expression vector
harboring cDNA coding for the MUC1/Y protein, as described in
Zrihan-Licht et al. (1994b). The labeled MUC1/Y protein was immunoprecipitated from cell lysates and the immunoprecipitate collected with
protein A-Sepharose beads. The MUC1/Y protein was then incubated
without or with N-glycanase (control and N-glyc, lanes 1 and 2,
respectively), analyzed on SDS-polyacrylamide gel and visualized by
autoradiography. The mature N-glycosylated 42–45 kDa MUC1/Y
protein is indicated to the right by the full arrow; the arrowhead
indicates the nonglycosylated MUC1/Y.
with molecular masses ranging from 100 to slightly more than 200
kDa were also observed; pulse-chase experiments previously
showed that these represent the precursor MUC1/REP proteins
(Hilkens and Buijs, 1988).
Despite the high amount of MUC1/REP expression in the T47D
breast cancer cell line as well as in several breast tumor samples,
other breast tumor samples displayed only very low MUC1/REP
protein amounts, as did normal breast tissue samples. Furthermore,
exceptionally low to undetectable concentrations of MUC1/REP
proteins were observed in HeLa cells (Fig. 1a). Ponceau red
staining of the blotted proteins revealed that this lack of immunoreactivity was not due to variations in protein loading.
Characterization of MUC1 isoform expression with
anti-cytoplasmic domain antibodies shows preferential
MUC1 isoform expression
To test for differential expression of the MUC1/REP and
MUC1/Y isoforms in the various epithelial tumor tissues, the
samples analyzed above were resubjected to Western blot analysis
using antibodies that specifically bind to the C-terminal 17 amino
acids of the MUC1 cytoplasmic domain and that do not recognize
epitopes contained within the tandem repeat array. It has been
shown that early after translation, the transmembrane form of the
MUC1/REP protein undergoes proteolytic cleavage in a region that
is 45–60 amino acids N-terminal to the transmembrane domain
(Ligtenberg et al., 1992a), thereby generating a heterodimer
MUC1/REP protein. This consists of the high molecular mass
tandem-repeat-array-containing extracellular domain that is bound
non-covalently in an SDS-sensitive linkage to the remaining
C-terminal cleavage products containing the 45–60 extracellular
MUC1 amino acids, the transmembrane domain and the MUC1
cytoplasmic tail, which migrate as diffuse immunoreactive species
in the region of 25–30 kDa. Accordingly, Western blot analysis
with anti-cytoplasmic domain antibodies of proteins resolved by
SDS/15% acrylamide gels clearly demonstrated that, in the high
MUC1/REP-expressing samples, immunoreactive species migrated as a diffuse band in the region of 25–30 kDa (Fig. 1b, lanes
2, 3, 6 and 7). In line with the MUC1/REP expression analyses
described above, these immunoreactive proteins were absent from
the HeLa epithelial tumor cell sample (Fig. 1b, c, lanes 1) as well as
from those breast tissue samples which expressed very low
amounts of the MUC1/REP protein (compare Fig. 1a with b).
In contrast, prominent anti-cytoplasmic domain-reactive species
migrating in the region of 42–45 kDa were observed not only in the
high MUC1/REP-expressing samples but also conspicuously in the
MUC1/REP-negative HeLa epithelial cell sample and in the low
MUC1/REP-expressing breast tumor tissue samples (Fig. 1b). In
several samples, there were 2 closely spaced immunoreactive
bands in the 42–45 kDa region (e.g., Fig. 1b, lane 9), which may
represent variations in the glycosylation status of the MUC1
proteins (see also ‘‘Discussion’’). The 42–45 kDa MUC1 isoforms
were not observed in any of the normal breast tissue samples (Fig.
1b). By analyzing MUC1/Y-expressing transfectants with both
anti-cytoplasmic domain antibodies and anti-MUC1/Y extracellu-
lar domain antibodies (Fig. 2a), the 42–45 kDa immunoreactive
species was identified as the MUC1/Y protein.
A comparison of the expression of repeat array-containing
MUC1 proteins (Fig. 1a) and MUC1/Y proteins (Fig. 1b) demonstrates that some breast tumor samples preferentially express the
MUC1/Y protein and have only low expression levels of the repeat
array containing MUC1 proteins. This is particularly striking in the
breast tumor samples analyzed in lanes 5 and 9 of Figure 1. A
different epithelial adenocarcinoma, represented by the HeLa
cervical uterine adenocarcinoma cells, also exhibits this pattern of
MUC1 expression (compare Fig. 1a with b, lanes 1, HeLa; this is
more evident in Fig. 1c).
A faint 33 kDa band that was immunoreactive with the anticytoplasmic domain antibodies was also detected in several
samples expressing the MUC1/Y protein (Fig. 1b, c). This band
represents a precursor MUC1/Y protein, as demonstrated by
digesting immunoprecipitated radioactively labeled MUC1/Y protein with N-glycanase (Fig. 1d). The treatment reduced the mass of
the 42–45 kDa proteins to about 33 kDa, thus indicating that the
mature MUC1/Y protein is N-glycosylated. Whereas the 33 kDa
precursor MUC1/Y protein was easily observed in stable MUC1/Yexpressing transfectants (Fig. 2a, lane 3), it was present in only
very small amounts in HeLa and T47D cells (Fig. 1c) and barely
detectable in the primary breast tumor tissue samples (Fig. 1b).
MUC1/Y expression detected with antibodies directed
against the MUC1/Y extracellular domain
To obtain a reagent directed against the MUC1/Y protein,
polyclonal antibodies (designated anti-MUC1/Yex) were generated
against the bacterial recombinant MUC1/Y extracellular domain
protein. Western blotting of the MUC1/Y-expressing transfectant
with these antibodies demonstrated the presence of mature 42–45
kDa and 33 kDa precursor MUC1/Y proteins (Fig. 2a, lane 3).
When anti-MUC1/Yex antibody was used to probe lysates prepared
from either T47D breast tumor cells (Fig. 2a, lane 1) or primary
breast tumor tissues (Fig. 2a, b, lanes indicated by BT), the
FIGURE 2 – MUC1/Y expression in tumor tissue detected by anti-MUC1/Y antibodies. (a,b) Protein lysates prepared from primary breast tumor
tissue samples (BT), normal breast tissue (BN), paired tumor and normal breast tissues from the same patient (BT*, BN*), T47D breast cancer
cells (T47D), HeLa adenocarcinoma cells (HeLa), control 3T3 neomycin-resistant transfectants (control), 3T3 MUC1/REP-expressing
transfectants (M.1/REP) and 3T3 MUC1/Y-expressing transfectants (M.1/Y) were resolved on SDS-polyacrylamide (12%) gels, Western blotted
and probed with affinity-purified antibodies that had been generated against the recombinant MUC1/Y extracellular domain. The mature MUC1/Y
protein and the cytoplasmic domain containing proteolytic cleavage products are indicated by the full arrow and bracket to the right. Panel (b)
contains samples different from those presented in (a) and was analyzed with anti-MUC1/Yex antibodies. Note the expression of the MUC1/Y
protein in the breast tumor samples (BT), breast cancer cells (T47D) and the MUC1/Y transfectant (M.1/Y). Expression was not detected in the
normal breast tissue samples (BN), the MUC1/REP transfectant or the control neomycin 3T3 transfectant (control). (c) Competition of antibody
binding. Protein lysate from a breast tumor tissue sample was analyzed by SDS-acrylamide gel, and the Western blot was probed with
affinity-purified antibodies that had been generated against the recombinant MUC1/Y extracellular domain, as described above for (a) and (b).
Competing recombinant MUC1/Y extracellular domain at a final concentration of 100 µg/ml was either added to (1 comp., lane 2) or deleted from
(2comp., lane 1) the antibody probing solution.
MUC1/Y protein was clearly expressed. The MUC1/Y extracellular domain contains sequences common to the cytoplasmic domaincontaining proteolytic cleavage products (Fig. 3d). In some samples,
these 25–30 kDa proteins also were detected by the anti-MUC1/
Yex antibodies (Fig. 2a, lanes 1 and 7). MUC1/Y expression was
not detected in the normal breast tissue samples (Fig. 2a, b, lanes
indicated by BN). Western blotting of the HeLa cell lysates with the
anti-MUC1/Yex antibodies revealed the presence of only the 42–45
kDa MUC1/Y protein (Fig. 2b, lane 5), commensurate with the
results obtained with the anti-cytoplasmic domain antibodies (Fig.
Western blot analyses thus demonstrated that tumor tissue
samples differentially express the MUC1 isoforms. To analyze
whether specific cells in the same tumor tissue are exclusively
expressing one or the other of the MUC1 isoforms, adjacent breast
tumor tissue sections were individually immunohistochemically
stained with MUC1 isoform-specific antibodies (anti-MUC1/Yex
antibodies and anti-repeat domain antibodies). These analyses
revealed that the tumor cells can simultaneously express both
MUC1 isoforms, no exclusivity in MUC1 isoform expression in
specific tumor cells being observed (data not shown).
Repeated use of the anti-cytoplasmic domain antibodies markedly reduced their immunoreactivity against the MUC1/Y isoform,
indicating lability of the antibody population able to recognize the
MUC1/Y isoform. Conversely, the affinity-purified anti-MUC1/
Yex antibodies consistently detected, even following their repeated
usage, the 42–45 kDa MUC1/Y isoform (Fig. 3). A comparison of
the immunoreactivity of the 2 polyclonal antibodies against the
MUC1/Y isoform and the cytoplasmic domain-containing proteolytic cleavage products present in primary tumor tissue (Fig. 3a, b)
clearly shows that the anti-MUC1/Yex antibodies are far more
sensitive than the anti-cytoplasmic domain antibodies in detecting
MUC1/Y expression. Indeed, in some samples, the MUC1/Y
protein was detectable only with the MUC1/Yex antibodies (compare Fig. 3a with b, lanes 3). The MUC1/Y protein is extensively
phosphorylated within its cytoplasmic domain (Zrihan-Licht et al.,
1994a). It may well be that the MUC1/Y isoform immunoreactivity
of the anti-cytoplasmic domain antibodies, generated against an
FIGURE 3 – Detection of MUC1/Y expression with antibodies recognizing different regions of the MUC1/Y extracellular domain. The scheme
presented in (d) illustrates the repeat-array-containing MUC1/REP (MUC1, upper molecule) and MUC1/Y proteins. The sites in MUC1/REP
corresponding to the splice donor and splice acceptor sites that generate the MUC1/Y protein by deleting the tandem repeat array (block of closely
spaced vertical lines) are indicated by black arrows facing downward. The region comprising the proteolytic cleavage site of the repeat array
containing MUC1 protein is indicated by the 2 white arrows facing upward. Proteins present in tumor (BT) or normal (BN) breast tissue lysates
were resolved by SDS/PAGE, transferred and immunoblotted with (a) affinity-purified anti-MUC1/Yex polyclonal antibody, (b) anti-cytoplasmic
domain polyclonal antibody and (c) antibodies directed against the extracellular domain of MUC1/Yex and affinity-purified by passage through an
Affigel-10 affinity column to which recombinant truncated MUC1/Yex protein (designated MUC1/Y/Ssp1) terminating at the Ssp1 site just
up-stream to the proteolytic cleavage site had been bound. The regions recognized by the antibodies used in (a), (b) and (c) are indicated in panel
(d) by (a), (b) and (c), respectively. Detection was performed with horseradish peroxidase-conjugated secondary antibodies followed by enhanced
chemiluminescence. Molecular mass markers are indicated in kDa on the left. The MUC1/Y protein is indicated in (a), (b) and (c) by the horizontal
arrow and the cytoplasmic domain containing proteolytic cleavage products is indicated in (a) and (b) by the bracket.
unmodified peptide sequence, is compromised by post-translational
modifications (such as phosphorylation) occurring on amino acid
residues located within the cytoplasmic domain.
Characterization of MUC1/Y isoform expression with antibodies
directed toward the N-terminal sequences of MUC1/Y
To obtain antibodies which exclusively recognize MUC1/Y
sequences that are N-terminal to the site at which proteolytic
cleavage occurs in the MUC1/REP protein, we adopted the
following approach. As cleavage occurs in the MUC1/REP protein
within a region immediately down-stream to an Ssp1 site (at
nucleotide number 328 in the MUC1/Y sequence [Zrihan-Licht et
al., 1994b]), we generated a truncated bacterial recombinant
MUC1/Yex protein (designated MUC1/Y/Ssp1) terminating at the
Ssp1 site. The purified MUC1/Y/Ssp1 protein was covalently
bound to an Affigel-10 (Bio-Rad) resin, which was then used to
affinity-purify anti-MUC1/Yex antibodies (see ‘‘Material and Methods’’). The affinity-purified antibodies thus obtained will recognize
only MUC1/Yex sequences up-stream to the proteolytic cleavage
site. Western blot analysis of proteins derived from breast cancer
cells grown in vitro (Fig. 3c, lane 1) or of breast tumor tissue (Fig.
3c, lane 2) with these anti-MUC1/Y/Ssp1 antibodies revealed the
MUC1/Y protein. These antibodies were not immunoreactive
against proteins extracted from normal breast tissue (Fig. 3c, lane
3). As expected, the cytoplasmic domain-containing MUC1/REP
proteolytic cleavage products with molecular masses of 25–30 kDa
were not detected by this antibody preparation. The immunoreactivity of the MUC1/Y isoform against antibodies which recognize
epitopes N-terminal to the proteolytic cleavage site present in
MUC1/REP (designated ‘‘(c)’’ in Fig. 3) provides additional
evidence that MUC1/Y, in contrast to MUC1/REP, does not
undergo proteolytic cleavage. These results not only confirm
MUC1/Y expression in breast tumor tissue but also rule out the
possibility that the MUC1/Y protein derives from proteolytic
breakdown of the MUC1/REP isoform.
RT-PCR characterization of MUC1 isoforms lacking
the tandem repeat array
To ascertain the exact nucleotide sequence of the MUC1
isoforms expressed in malignant epithelial cells, HeLa RNA as well
as RNA isolated from the ovarian carcinoma cell line Lucy were
subjected to RT-PCR analysis, using primers located down-stream
and up-stream to the tandem repeat array. Ethidium bromide
staining of the amplified products revealed the presence of several
prominent bands (Fig. 4a) which hybridized extensively to an
internal MUC1 probe (Fig. 4b), suggesting that they were all
derived from the MUC1 gene. PCR products deriving from the
MUC1/REP mRNA were not detected. This is most likely due to
the low MUC1/REP expression levels in these samples, the large
size of MUC1/REP and/or its high G/C content. Nucleotide
sequencing using both sense and anti-sense strands of the RT-PCR
DNA products subcloned in M13 revealed, as expected, a sequence
corresponding to the MUC1/Y isoform. MUC1/Y sequences also
were observed which contained a 27 bp insert (designated MUC1/
Yalt) located down-stream to the 58 terminus, which is generated by
a previously described alternative splicing event (Wreschner et al.,
1990; Ligtenberg et al., 1990) occurring in the first intron (Fig. 4c).
Several additional M13 clones revealed the existence in both the
HeLa and Lucy samples of another novel MUC1 isoform that is
also devoid of the tandem repeat array. Nucleotide sequencing
showed that this isoform (designated MUC1/X) is generated by an
alternative splicing event which has the same splice donor site as
MUC1/Y ( gtgag) but utilizes an alternative splice acceptor (SA)
site located 54 nucleotides up-stream to that used by MUC1/Y. The
flanking sequence at this divergent SA site (58 ttctccccag-TTG 38)
is composed exclusively of pyrimidine residues immediately
up-stream to the invariant ag SA site and thus corresponds to
consensus SA sequences (Fig. 4c). RT-PCRs utilizing different
down-stream primers that were more distal to those used here
consistently demonstrated the MUC1/X sequence (data not shown).
As a consequence of this splice event, the expected MUC1/X
protein, except for an additional 18 amino acid insert, will be
identical to the MUC1/Y protein (Fig. 4e). The major DNA
products appearing in the HeLa and Lucy RT-PCRs are thus
derived from MUC1/Y, MUC1/Yalt and MUC1/X sequences.
Expression of the MUC1/Y isoform in mouse mammary epithelial
cells promotes in vivo tumorigenicity
Having demonstrated MUC1/Y isoform expression in epithelial
tumors, the next issue of obvious concern was its possible function
within the epithelial cell. Previous studies have demonstrated that
the cytoplasmic domains of the MUC1 proteins are phosphorylated
on serine as well as on tyrosine residues (Zrihan-Licht et al.,
1994b), and co-immunoprecipitation analyses revealed that the
tyrosine-phosphorylated MUC1 cytoplasmic domain interacts with
the GRB-2 protein (Pandey et al., 1995). These results indicate that
the MUC1 proteins may participate in cell growth and proliferation
and that they are thus likely to be functionally important in the
oncogenetic process.
To investigate whether expression of one or more of the MUC1
isoforms can transform cells or promote cell tumorigenicity, we
generated stable MUC1/Y- and MUC1/REP-expressing transfectants in several different cell lines. Expression of either isoform in
NIH/3T3 fibroblasts was not transforming (data not shown). To
answer the question whether MUC1 expression can promote
epithelial cell tumorigenicity, stable MUC1 isoform-expressing
transfectants were generated using DA3 mouse mammary epithelial cells transformed by the carcinogen DMBA (Fu et al., 1990).
Expression of the various MUC1 isoforms was confirmed by
probing cell lysates with the appropriate anti-MUC1 antibodies.
The tumorigenic potential of 5 independent MUC1/Y and 3
independent MUC1/REP transfectants was investigated by inject-
FIGURE 4 – Detection by RT-PCR of MUC1 isoforms devoid of the
tandem repeat array in HeLa and Lucy ovarian carcinoma cells. (a, b)
RT-PCR products of MUC1 mRNA isolated from epithelial carcinoma
cells. Poly A1 RNA isolated from Lucy ovarian carcinoma cells and
HeLa cervical carcinoma cells was reverse-transcribed with the
down-stream oligonucleotide ‘‘c’’, followed by addition of the upstream oligonucleotide ‘‘a’’ and PCR amplification as described in
‘‘Material and Methods’’ (see c for location of primers). PCR products
were purified on agarose gel and subjected to nested PCR amplification, using the up-stream and down-stream oligonucleotides ‘‘b’’ and
‘‘c’’, respectively. The Lucy and HeLa RT-PCR products (lanes 1 and
2) were analyzed by agarose gel electrophoresis and ethidium bromide
staining (a) followed by Southern blotting and hybridization with an
internal MUC1 DNA probe (b). Sizes (in bp) of the DNA markers are
indicated to the right of (a). (c, d) Location of splice donor (S.D.) and
acceptor (S.A.) sites that generate the MUC1/Y and MUC1/X isoforms.
The MUC1-specific RT-PCR products (a) were subcloned in M13
followed by dideoxynucleotide sequencing of the single-stranded
DNA. The DNA sequence obtained was compared with that of the
repeat-array-containing MUC1/REP (Wreschner et al., 1990), allowing
identification of the splice donor (GTgag) and splice acceptor sites
(cacttctccccAG and tttcaaacctccAG) generating the MUC1/X and
MUC1/Y isoforms, respectively, shown in (c). As a consequence of
these splice events, the tandem repeat array and its flanking sequences
are deleted in the MUC1/X and MUC1/Y isoforms. Down-stream to
the splice site, both isoforms retain the same reading frame (RF) as the
repeat-array-containing MUC1 protein. The previously described
(Wreschner et al., 1990; Ligtenberg et al., 1990) alternative splicing
event occurring in the first intron is designated in (c) by ‘‘alt’’. The
abbreviations SP, TM and CYT in (d) indicate the signal peptide,
transmembrane and cytoplasmic domains, respectively. (e) The deduced amino-terminal amino acid sequences of the MUC1/Y and
MUC1/X isoforms. The signal peptide is boxed and followed by the
N-terminal amino acid sequence of the mature protein. The location of
the splice deleting the tandem repeat array is indicated by the first black
dot. This is followed by the smaller letters indicating the 18 amino
acids present in the MUC1/X isoform. The MUC1/Y amino acid
sequence continues from the second black dot; the full sequence
appears in Zrihan-Licht et al. (1994b).
ing transfected cells into the hind-leg muscle of syngeneic BALB/c
mice. Controls were parental, non-transfected DA3 cells as well as
DA3 cells transfected only with the neomycin-resistance gene.
Inoculated mice were then monitored for both tumor latency and
tumor size. Tumor latency in all 5 MUC1/Y-expressing transfectants was markedly shorter than that observed either in the
MUC1/REP-expressing transfectants or in the control neomycinresistant transfectant (Fig. 5). The difference in tumorigenic
potential of the various transfectants was subjected to a statistical
analysis using the Mantel-Cox and Breslow tests. Significant
differences ( p , 0.05) were noted between the MUC1/Y and the
MUC1/REP transfectants; no statistical differences were noted
between the MUC1/REP transfectants and the control cells (not
expressing MUC1). Furthermore, the average diameter of tumors at
100 days post-tumor cell inoculation, developing from the MUC1/Y
transfectants, was 35 mm compared to 13 mm for tumors developing from either the control neomycin transfectant or the MUC1/REPexpressing transfectants. In a separate experiment, by 30 days
following tumor cell inoculation, 100% of mice injected with the
MUC1/Y transfectant had developed visible tumors, whereas only
FIGURE 5 – Tumorigenicity of MUC1 DA3 transfectants. DA3 mouse mammary carcinoma cells were co-transfected with both G418
(neomycin)-resistant cDNA and MUC1/REP cDNA (MUC1/REP#), G418-resistant cDNA and MUC1/Y cDNA (MUC1/Y#) or with the
G418-resistant cDNA alone (neo#). Cells from stable transfectants (indicated by number) were injected into BALB/c mice (5 3 105 cells/animal).
Mice were examined at several time points post-injection for the appearance of tumor.
50% of mice injected with the MUC1/REP form showed tumor
formation. The increase in tumorigenic potential of the MUC1/Y
transfectants was observed at 2 different cell concentrations (data
not shown).
In summary, a comparison of the tumorigenicity of 5 independent MUC1/Y-expressing transfectants revealed that all were
significantly more tumorigenic than both the control neomycinresistant DA3 cells and the 3 MUC1/REP-expressing transfectants.
Notably, no significant differences in tumorigenic potential were
observed within the MUC1/Y-expressing transfectant group.
Using novel antibody reagents which recognize different regions
of the MUC1 isoforms, we have shown that MUC1 protein
isoforms are differentially expressed in malignant epithelial cells.
Indeed, in certain breast tumor tissues, as well as in HeLa cervical
adenocarcinoma cells, almost exclusive expression of the MUC1/Y
isoform was observed. These findings indicate that decisions taken
at the level of RNA splicing are likely to determine the outcome of
MUC1 protein isoform expression and that epithelial cells at a
specific stage of differentiation or ‘‘dedifferentiation’’ (i.e., malignant cells) preferentially express a specific MUC1 isoform. Malignancy-specific splicing events have been demonstrated (Matsumara and Tarin, 1992; Dall et al., 1994; Itoh et al., 1994).
Alternative CD44 mRNA transcripts in breast, colon and uterine
cervical cancers are both quantitatively and qualitatively specific to
the malignant state (Matsumara and Tarin, 1992; Dall et al., 1994).
Another striking example is the K-sam gene, which encodes a
receptor for keratinocyte growth factor. In malignant tissues, an
alternatively spliced mRNA transcript codes for a carboxylterminal truncated receptor with high affinity for its ligand, whereas
in normal tissues the non-truncated receptor is the major isoform
and has a lower affinity for its ligand (Itoh et al., 1994). Although
mechanisms operative at the level of RNA splicing are likely to
determine which specific MUC1 isoform is expressed, the decision
whether to express the MUC1 gene is probably transcriptionally
regulated (Abe and Kufe, 1993). In this context, it is notable that
the CpG dinucleotide, present within the CCCGGG hexanucleotide
found in every 60 bp repeat unit comprising the tandem repeat
array, is fully methylated in cells that cannot express MUC1 but is
completely non-methylated in tissues that can express the MUC1
gene (Zrihan-Licht et al., 1995). The specific MUC1 isoforms to be
expressed will be determined, however, at the level of RNA
By Western blotting we have shown that different tumor tissue
samples express the MUC1 isoforms to varying extents. The
relative expression of the various MUC1 isoforms may thus
correlate with the stage of the breast cancer or with the specific type
of epithelial adenocarcinoma in question. Research is presently in
progress to resolve this issue.
As the MUC1/REP and MUC1/Y proteins are both tyrosinephosphorylated (Zrihan-Licht et al., 1994a), they are likely to be
involved in signal transduction processes. Our findings are also
consistent with data showing that the MUC1 cytoplasmic domain
can interact with the GRB-2 protein (Pandey et al., 1995). The
GRB-2 protein is known to serve as the link between growth factor
receptors and the ras signaling pathway. These data indicate that, as
with other cell surface receptors (e.g., receptor tyrosine kinases,
cytokine receptors), MUC1 may form a signaling complex which is
intimately related to the oncogenetic process. Furthermore, tumor
development is delayed in MUC1 null mice (Spicer et al., 1995),
thereby implicating MUC1 protein expression in tumor progression. Consistent with these findings, we present data showing that
expression of the MUC1/Y protein in DA3 mouse mammary
epithelial cells increases the tumorigenic potential of these cells.
MUC1/Y expression did not, however, lead to the transformation of
NIH/3T3 fibroblasts. This is likely due to the absence in the
fibroblast cell of accessory proteins that are likely required for
MUC1 signal-transducing functions. As reported previously, such
proteins could be cell surface molecules that generate heteromeric
complexes with MUC1/Y (Zrihan-Licht et al., 1994a) or secreted
proteins (ligands) that bind to and activate MUC1/Y function.
The marked difference in the tumorigenic potential of the
MUC1/Y and MUC1/REP transfectants may be due to different
immunological responses elicited by these molecules in the host
animal. Since the 2 molecules have a distinct structure, it could also
well be that the MUC1/Y and MUC1/REP proteins are involved in
different cellular processes. As previously shown, the MUC1/REP
isoform, in contrast to MUC1/Y, is involved in cell to cell and cell
to ECM repulsion via its large repeat-array-containing extracellular
domain (Ligtenberg et al., 1992b; Hartman et al., 1992) and can
inhibit integrin-mediated and E-cadherin-mediated cell adhesion.
Significantly, MUC1/Y is devoid of this tandem repeat array and is
apparently not involved in anti-cell-adhesive processes. It is thus
probable that, although the MUC1/REP and MUC1/Y proteins are
likely involved in signal transduction pathways, each isoform has
both a unique activating mechanism and function in malignant
epithelial cells.
It is noteworthy that RT-PCR revealed an additional MUC1
isoform, MUC1/X, that is also devoid of the tandem repeat array
and, except for an 18 amino acid insert in the extracellular domain,
is identical to the MUC1/Y isoform. As previously noted, by
probing immunoblots of breast cancer cell lysates with anticytoplasmic domain antibodies (Fig. 1b), a doublet band migrating
in the region of 42–45 kDa was detected in certain samples. This
heterogeneity could result from post-translational modifications,
such as glycosylation and/or phosphorylation of the MUC1/Y
protein. Another possibility is that the slower migrating band
represents the MUC1/X isoform, which we deduce as being about 2
kDa larger than the MUC1/Y isoform. To resolve this issue and to
demonstrate MUC1/X expression in malignant epithelial cells, we
are now developing antibody reagents specific for the MUC1/X
isoform. It should be noted that hundreds of isoforms for the
neurexin neuronal cell surface proteins are generated by alternative
splicing events and that a splice site-specific ligand for one
particular neurexin isoform has been documented (Ichtchenko et
al., 1995). Indeed, using a receptor gel overlay assay, we have
identified proteins that specifically recognize and bind to the
MUC1/Y extracellular domain. Variations in the extracellular
domain sequences of the MUC1/Y and MUC1/X isoforms could
thus result in different affinities for putative interacting ligands. We
are now investigating whether the ligand proteins that specifically
interact with the MUC1/Y isoform are recognized in a similar
manner by the MUC1/X protein.
Our results indicate that MUC1 isoforms lacking the tandem
repeat array may be useful tumor marker proteins and that in the
cancer cell they are likely to perform functions different from those
of the MUC1/REP protein. In particular, our results indicate that
MUC1/Y expression may be intimately related to the uncontrolled
growth and proliferation of breast cancer cells. Furthermore, the
fact that the MUC1/Y isoform is expressed in some malignant
epithelial cells displaying very low levels of the MUC1/REP
protein suggests that the novel MUC1 isoforms, rather than the
MUC1/REP proteins, are a hallmark of epithelial malignancy.
This work was supported in part by the Israel Cancer Association, the Israel Cancer Research Fund and the Israel Academy of
Sciences (D.H.W.) and by the Simko Chair for Breast Cancer
Research, the Federico Fund for Tel Aviv University and the
Barbara Friedman Fund (I.K.).
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