From the Mayo Clinic Scottsdale, S.C. Johnson Research Building, Scottsdale, Arizona 85259
Received for publication, December 13, 2000, and in revised form, January 22, 2001
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ABSTRACT |
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MUC1 is a large (>400 kDa), heavily glycosylated
transmembrane protein that is aberrantly expressed on greater than 90%
of human breast carcinomas and subsequent metastases. The precise function of MUC1 overexpression in tumorigenesis is unknown, although various domains of MUC1 have been implicated in cell adhesion, cell
signaling, and immunoregulation. Stimulation of the MDA-MB-468 breast
cancer line as well as mouse mammary glands with epidermal growth
factor results in the co-immunoprecipitation of MUC1 with a
tyrosine-phosphorylated protein of ~180 kDa. We have generated transgenic lines overexpressing full-length (MMF), cytoplasmic tail
deleted ( The transmembrane mucin MUC1 (DF3, CD227, episialin, PEM) is a
heavily O-glycosylated protein expressed on most secretory epithelium, including the mammary gland as well as some hematopoetic cells. MUC1 is expressed abundantly in the lactating mammary gland in
addition to being overexpressed in greater than 90% of human breast
carcinomas and metastases (1). In the normal mammary gland, MUC1 is
expressed mainly on the apical surface of glandular epithelium and is
believed to play a role in anti-adhesion and immune protection (2-4).
In breast cancer, MUC1 is overexpressed, underglycosylated, and apical
localization is lost (2). Mice lacking Muc1 demonstrate no overt
phenotypic developmental abnormalities in the mammary gland, but when
crossed with the tumorigenic
MMTV1-mTag transgenic line
(5), mammary gland tumor growth was significantly slowed. Additionally,
these Muc1-null/MMTV-mTag transgenics demonstrated a trend toward
decreased metastasis, showing that the absence of Muc1 results in both
reduced tumor growth and spread (6).
MUC1 is transcribed as a large precursor gene product, which, upon
translation, is cleaved in the endoplasmic reticulum, yielding two
separate proteins that form a heterodimeric complex, bound together by
noncovalent interactions (7). The larger of the two components (the
"mucin-like" subunit) contains most of the extracellular domain,
including the signal sequence, tandem repeats, as well as some
degenerate repeats. The tandem repeats consist of 30 to 90 repeat
sequences of 20 amino acids, rich in serine and threonine residues.
Approximately 50-90% of the mass of MUC1 is derived from
O-glycosylation that occurs on these amino acids (8)). The
second component of the heterodimer consists of an extracellular stem
(where the two protein products are joined), the hydrophobic
transmembrane domain, and a short, 72-amino acid cytoplasmic domain.
The cytoplasmic domain contains potential docking sites for Src
homology domain 2 containing proteins, as well as a variety of
putative kinase recognition sites and is tyrosine-phosphorylated
in vitro (9, 10). There are 7 tyrosine residues in the
cytoplasmic tail, which are highly conserved across species (10).
MUC1 interacts with a number of proteins implicated in neoplasia
through both its tandem repeat and cytoplasmic domains. The tandem
repeat region can act as a ligand for intercellular adhesion molecule 1 on human umbilical vein endothelial cell monolayers, indicating a
potential role in metastatic intravasation (11, 12). Additionally, MUC1
binds One family of transmembrane tyrosine kinases, erbB receptors (including
erbB1 or epidermal growth factor receptor (EGFR), erbB2, erbB3, and
erbB 4) are expressed dynamically during mammary gland development (16)
and are commonly implicated in breast cancer initiation and progression
in both humans and rodents (17, 18). Overexpression of either the
receptors or ligands in this family generally occurs in advanced,
metastatic disease, resulting in poor overall patient outcomes (17).
Ligands of the epidermal growth factor family (including EGF-like
members and neuregulin family members) induce dimerization of
these receptors, leading to the activation of a variety of effector
proteins including Src, phosphatidylinositol 3-kinase, Shc,
phospholipase C To explore signaling roles of MUC1 in the mammary gland, we have
generated a number of transgenic animals overexpressing full-length and
deletion constructs of human MUC1 in the mouse mammary gland using the
mouse mammary tumor virus (MMTV) promoter. We have demonstrated that
treatment with exogenous betacellulin, in addition to other EGFR
ligands, can induce tyrosine phosphorylation of MUC1 in culture. Immunoprecipitation and co-localization experiments have revealed a
physical interaction between MUC1 and EGFR that occurs through the
cytoplasmic tail of MUC1. Furthermore, we demonstrate that EGF-dependent activation of ERK1/2 MAPK is strongly induced
in the presence of high levels of MUC1 in the mouse mammary gland.
Transgenic Constructs--
Muc1 knockout animals have been
described previously (6). The 42 tandem repeat human MUC1 (27), human
MUC1 lacking the cytoplasmic tail (28), or human MUC1 lacking the
tandem repeat domain ( Animals and Cell Lines--
All studies were performed on the
FVB strain of mice with wild-type Muc1, transgenic hMUC1, or Muc1-null
(6). Human MUC1 is designated MUC1 while the mouse homologue is
designated Muc1. For EGF injection, 1 µg/g body weight receptor grade
mouse EGF (Sigma and Collaborative Biosciences) was injected
intraperitoneally. After 20 min, animals were sacrificed and the
mammary glands were harvested. T47D and MDA-MB-468 cell lines were from
ATCC and cultured as suggested.
Antibodies--
Antibodies to MUC1 included HMFG-2 (kindly
provided by J. Taylor-Papadimitriou, ICRF, London, United Kingdom) and
B27.29 (kindly provided by Biomira), both mouse monoclonals which react
with the human tandem repeat domain, and CT1 (32) and CT2. CT2 is an
Armenian hamster monoclonal antibody generated to the last 17 amino
acids of the cytoplasmic domain of MUC1. Its reactivity against mouse
and human MUC1 appears similar to CT1 in immunoprecipitation, immunoblot, and immunohistochemistry (32, 33). Antibodies to erbB1,
erbB2, erbB3, and erbB4 as well as Grb2, SOS, and PCNA-HRP were all
from Santa Cruz, and ERCT was a kind gift from H. S. Earp
(University of North Carolin, Chapel Hill, NC). The phosphotyrosine antibody (RC20-HRP) was from Transduction Laboratories. HRP-conjugated secondary antibodies for Western blot analysis were from Pierce and
Jackson Laboratories and Alexa-conjugated secondary antibodies for
confocal imaging were from Molecular Probes. Dual-phosphorylated ERK
antibody is from Sigma and p42/44, phospho-p38, and phospho-SAPK/JNK are from New England Biolabs Cell Signaling.
Protein Analysis--
Glands were homogenized in Triton X-100
lysis buffer (20 mM HEPES, pH 8.0, 150 mM NaCl,
1% Triton X-100, 2 mM EDTA, 2 mM sodium orthovanadate, 50 µM ammonium molybdate, 10 mM sodium fluoride, and Complete inhibitor mixture (Sigma).
BCA assays (Pierce) were performed to determine protein concentration
and samples were stored frozen at
Immunoprecipitations were performed with 0.5-4.0 mg of protein lysate,
using Protein A/G-agarose conjugate (Santa Cruz). Western blots were
performed using 200 µg of protein lysate per sample. Samples were
separated by SDS-PAGE and transferred to polyvinylidene difluoride
membrane (Immobilon) for Western blot analysis.
In Vitro Kinase Assay--
A GST fusion protein (generated in
the pGEX-2TK expression vector (Amersham Pharmacia Biotech)) containing
the 72-amino acid cytoplasmic tail of MUC1 (GST-CT) was purified using
glutathione-Sepharose and used as the substrate. The kinase reaction
contained 25 mM HEPES, pH 7.5, 120 µM
[ Immunofluoresence--
Tissues were fixed in methacarn, paraffin
embedded (Mayo Clinic Scottsdale Histology core), and either 5 µm (brightfield) or 20 µm (confocal) sections were cut.
Slides were deparaffinized in xylene, rehydrated, preincubated in
enhancing wash buffer (Immunex), blocked in normal goat serum and
incubated with primary antibodies overnight at 4 °C. Slides were
washed in enhancing wash buffer, incubated with either HRP- or
fluorescent-conjugated secondary antibodies, washed in enhancing wash
buffer, and for immunohistochemistry, developed with
3',3'-diaminobenzidine (Santa Cruz Biotechnology) and counterstained
with Meyers hematoxylin (Sigma). For confocal microscopy, slides were
coverslipped (1.5 µM) in antifade solution (Molecular
Probes) and visualized with a Zeiss laser scanning microscope 510, and
analyzed using LSM 510 software version 2.5. Negative controls included
antibody-specific peptide blocking and Muc1 knockout tissues. Dilutions
for the antibodies are as follows: B27.29-HRP, 1:100, CT2, 1:200, EGFR,
1:250, dpERK, 1:400, PCNA, 1:100.
MMTV-MUC1 Transgenics--
To investigate the effects of MUC1
overexpression and the contribution of the various MUC1 subdomains to
signaling, transgenic animals were created. Transgenic constructs were
derived by inserting the MUC1 cDNA (see below) into the construct
designed by Guy et al. (5) which uses the MMTV long
terminal repeat promoter and SV40 3'-untranslated region. Three
different lines of MUC1 transgenic mice were created, expressing either
full-length human MUC1 (MMF), cytoplasmic tail deleted human MUC1
(
The
To determine whether the transgenic proteins trafficked to
physiologically relevant sites, immunohistochemistry was performed. Pregnant and lactating glands displayed predominantly apical staining for all constructs, although cytoplasmic staining was also observed (Fig. 2). Note that similar to wild-type Muc1, MMF (Fig. 2A)
and MUC1 Co-immunoprecipitates a pp180 in Response to EGF Family Ligand
Treatment--
The cytoplasmic domain of MUC1 is
tyrosine-phosphorylated both in vitro (Fig.
3B, lower arrow) and in
vivo (Fig. 4A). To
determine the mechanism of this phosphorylation, a panel of potential
kinases were analyzed for activity with MUC1, and phosphorylation was observed with EGFR kinase, among others (Fig. 3A). To
examine phosphorylation of MUC1 by the EGFR kinase in culture, multiple EGF family ligands were used to treat MDA-MB-468 and T47D mammary carcinoma cells. Phosphorylation of MUC1 could be induced in a dose-dependent manner with betacellulin in MDA-MB-468 but
not T47D cells (Fig. 3B and data not shown). Additionally,
phosphorylation was induced with EGF, amphiregulin, and transforming
growth factor- EGFR Physically Associates with MUC1--
Members of the erbB
receptor tyrosine kinase family range in size from 170 to 190 kDa. To
determine whether the co-immunoprecipitating pp180 was one or more of
the erbB receptors, mammary gland lysates were immunoprecipitated with
an antibody to the erbB proteins and blotted with MUC1 antibodies. EGFR
and MUC1 complexes were observed in lysates from both wild-type animals
and MMF transgenics using antibodies to both the tandem repeat region
and the cytoplasmic domain (Fig.
5A and data not shown). While
co-immunoprecipitation experiments demonstrate an interaction between
full-length MUC1 and EGFR, this interaction is markedly reduced in the
MUC1 and EGFR Co-localize to the Apical Membrane--
To give
insight to the localization of this complex formation in the gland, we
used confocal microscopy to analyze MUC1/EGFR co-localization. We have
localized MUC1 to the apical membrane during pregnancy and lactation,
and observed it also in the alveolar lumen during lactation (Figs. 2
and 6B). Using antibodies to
EGFR, we detected protein throughout the alveolar epithelium during both pregnancy and lactation (Fig. 6A), as has been
previously reported (16). Dual staining for EGFR and MUC1 revealed that they are co-localized mainly in the apical membrane proximal region (Fig. 6C). Furthermore, by removing all but the most
intensely dual-staining colors through computer enhancement, we
determined that the co-localization appears to be concentrated at
points of cell-cell contact (Fig. 6D).
MUC1 Effects EGF-dependent Signaling--
We next
investigated the potential effects of MUC1 overexpression on EGFR
signaling. To determine whether the presence or absence of MUC1
effected the ability of EGFR to autophosphorylate, transgenic and
Muc1-null animals were injected intraperitoneally with receptor-grade
EGF, and mammary gland lysates prepared. We detected similar levels of
phosphorylation of the EGFR in both transgenic and knockout animals in
response to EGF treatment (Fig. 7).
Multiple kinase pathways lie downstream of EGFR activation, and we next
explored whether MUC1 overexpression promotes signaling through these
molecules in the mammary gland. Using antibodies directed against the
phosphorylated forms of p38, p42/44 ERK1/2 (dpERK), and p46/54
SAPK/JNK, we observed a striking pattern of activation. MMF, Muc1
knockout, and wild-type animals were injected intraperitoneally with
receptor-grade EGF to stimulate signaling in the mammary gland. Upon
stimulation, phosphorylated ERK1/2 was strongly induced in MMF
lactating mammary gland, while it was detectable in comparably low
amounts in the wild-type and Muc1
We also examined potential EGFR effector proteins for involvement in
MUC1 signal transduction to ERK1/2 activation. It has been previously
reported that Grb2/SOS associates with MUC1 in breast carcinoma cell
lines (15). We detected MUC1 co-immunoprecipitating with both Grb2 and
SOS in wild-type, MMF, and
To examine potential effects of activated MAPK, we have investigated
the possibility of increased mitogenesis by comparing nuclear staining
(data not shown) and immunoblot detection of PCNA (proliferating cell
nuclear antigen). We compared levels of PCNA in wild-type, knockout,
and transgenic animals, and observed no significant difference between
the groups over multiple samples (Fig. 8D).
In an effort to recapitulate the overexpression of MUC1 observed
in human breast cancer, we have generated transgenic animals that
overexpress both full-length and domain-deleted human MUC1 in the mouse
mammary gland. The mammary glands of these transgenics appear
developmentally and functionally normal, and transgene expression is
localized to the apical border of both ducts and alveoli of the mammary
gland. We have demonstrated that MUC1 co-localizes with and physically
interacts with members of the erbB receptor kinase family. Finally, we
have demonstrated a strong activation of dual-phosphorylated p42/44 ERK
in the presence of transgenic, full-length MUC1 in the lactating
mammary gland.
The interaction between transmembrane mucins and members of the erbB
family has been demonstrated previously. Caraway et al. (34)
demonstrated a co-immunoprecipitation between erbB2 and MUC4 (ASGP1 and
-2) in both the metastatic ascites 13762 rat mammary carcinoma cell
line as well the pregnant rat. Unlike MUC1 and EGFR, this interaction
occurs in the extracellular domain of the proteins. Furthermore,
increased proliferation rates and a potentiation of NRG
signaling correlates with MUC4 expression. Interestingly, they have
recently shown that regulation of MUC4 expression is dependent upon
activation of the ERK pathway in 13762 cells (35). MUC4 is also a
transmembrane member of the mucin family, with a processed protein core
that is heavily O-glycosylated, similar to MUC1 (36). Unlike
MUC1, MUC4 contains EGF-like repeats in the extracellular portion of
its membrane-spanning domain, which appear to be responsible for its
interactions with erbB2. While MUC4 appears to interact only with
erbB2, MUC1-erbB associations appear to be much more permissive,
although modulation of Grb2/SOS interactions with the erbBs is
restricted only to EGFR (see below). Interestingly, ligand-independent
activation of EGFR and subsequent downstream MAPK activation has been
recently described by Pece and Gutkind (37) through interactions with
E-cadherin. This is further evidence that the activity of erbB family
of transmembrane receptors can be modulated by unique mechanisms in
addition to activation by cognate ligands.
It is important to note that while MUC1 could be detected
in erbB immunoprecipitations by a variety of MUC1 antibodies, the erbB
receptors could not be identified in MUC1 immunoprecipitations. This
may be due to the extremely high levels of MUC1 being expressed and
released into the alveolar lumen compared with the relatively modest
levels of the erbB receptors present in the apical epithelium. Proportionately, only a very small fraction of the total MUC1 being
expressed may be complexing with the erbB receptors while the opposite
may be true for the erbBs at that cellular location. Additionally, as
the pp180 that is identified in immunoprecipitates appears to be
multiple bands of the approximate same size (Fig. 2D), it is
possible that the phosphotyrosine immunoblot is in fact detecting all
four erbB receptors complexing with MUC1. If this is the case, the
detection of a single erbB from the complex becomes increasingly difficult.
Pandey et al. (15) report interactions between MUC1 (DF3)
and Grb2/SOS in MCF7 breast carcinoma cell lines through the Src homology domain 2 domain of Grb2, although no downstream signaling was
reported. We were able to observe MUC1 directly interacting with Grb2
and SOS in mammary glands from both the full-length and tandem
repeat-deleted MUC1 transgenic. As MUC1 has no intrinsic kinase domain,
a possible interpretation of these data is that EGFR phosphorylation of
MUC1 allows recruitment of Grb2 into a complex that includes both EGFR
and MUC1. Preliminary experiments have shown a significant increase in
EGFR binding to SOS in the presence of full-length MUC1, but not the
cytoplasmic tail-deleted transgenic mammary glands. Interestingly, when
the remaining erbB receptors were examined for changes in SOS-binding,
no modulation was observed in relation to the presence or absence of
MUC1, indicating a specific relation between EGFR and MUC1. The
presence of MUC1 in immunoprecipitations of erbB2, erbB3, and erbB4 is
likely due to heterodimerization complexes between the four erbB
receptors and MUC1. It has been previously demonstrated that all erbB
receptors are capable of being transphosphorylated by EGF in the
lactating mammary gland, indicating that these complexes do form during this stage of development (16).
EGFR and MUC1 have previously been localized in the mammary
epithelium in the pregnant and lactating mammary gland, MUC1 to the
apical side and EGFR throughout the cell. We have co-localized EGFR and
MUC1 mainly to apical-lateral regions between cells, leading us to ask
if these proteins may be interacting at tight junctions. To investigate
this observation, we stained sections for MUC1 and ZO-1 (zona occludens
1), a cytoplasmic protein found in the tight junctions of polarized
epithelial cells (38). In preliminary experiments, we observed
co-localization of these two proteins at the apical-lateral region of
the epithelium of lactating epithelium through confocal microscopy,
indicating that MUC1 is indeed found at the tight junctions, and is not
merely an apically localized protein. Interestingly, Chen et
al. (39) recently demonstrated that increased MAPK activity
negatively regulates tight junction formation. As MUC1 overexpression
(as is seen in the neoplastic state as well as these transgenics) increases MAPK activity and decreases cellular adhesion in
vitro (2, 3), we might speculate a role for normal (wild-type expression levels) MUC1/EGFR signaling in the preservation of the tight
junction complex.
There are numerous potential consequences for activation of the p42/p44
MAP kinase proteins including induction of proliferation, quiescence,
apoptosis, and differentiation (40-42). We have investigated levels of
PCNA in transgenic animals, and found no increase that would correlate
to increased mitogenesis. Alternately, we have examined the possibility
of driving the cells into a state of Go arrest by
activating p21. This would potentially provide a population of cells
that do not apoptose in response to the postlactational stimuli, and
remain in the gland as potential targets of transformation (26,
43-45). While we have been unable to detect increased levels of p21 in
preliminary experiments, we have observed a trend toward delayed
regression in the postapoptotic glands of some MMF transgenic animals
(data not shown). These possibilities are being explored in subsequent experiments.
It is tempting to speculate that the modulation of EGFR signaling and
MAP kinase activation are a component of the mechanism of
MUC1-associated tumorigenesis. While aberrant MUC1 expression has been
linked with a high percentage of breast carcinomas, the role of this
overexpression is undefined. This novel data points to a potential
mechanism, that of potentiating the signaling of the tyrosine kinase
receptor EGFR, a protein whose increased expression is correlated to
aggressive breast cancer (46, 47).
CT), or tandem repeat deleted (
TR)-human MUC1 under the
control of the mouse mammary tumor virus promoter to further
examine the role of MUC1 in signaling and tumorigenesis. Immunoprecipitation experiments revealed that full-length transgenic MUC1 physically associates with all four erbB receptors, and
co-localizes with erbB1 in the lactating gland. Furthermore, we
detected a sharp increase in ERK1/2 activation in MUC1 transgenic
mammary glands compared with Muc1 null and wild-type animals. These
results point to a novel function of increased MUC1 expression,
potentiation of erbB signaling through the activation of mitogenic MAP
kinase pathways.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-catenin and GSK3
, through motifs in the cytoplasmic tail
similar to those found in the adenomatous polyposis coli protein,
leading to a reduction in the binding of
-catenin to E-cadherin in
ZR-75-1 breast carcinoma cells (13, 14). This could potentially subvert
E-cadherin-mediated cell adhesion in epithelial cells, promoting cell
migration (13). Additionally, studies in MCF-7 breast carcinoma cells
demonstrated that upon phosphorylation, MUC1 can bind Grb2/SOS (15),
signaling mediators of a number of receptor tyrosine kinases.
, STAT, Grb2/SOS, and others (19-22). The activation
of many of these proteins results in the phosphorylation of nuclear
translocating kinases, including SAPK/JNK and the MAP kinases, p38 and
ERK1/2 (23-25). One mechanism of MAP kinase activation is through the
recruitment of the Grb2·SOS complex to the phosphorylated
receptor, resulting in Ras activation and phosphorylation of Raf, MEK,
and ERK1/2. Upon activation, ERK1/2 can translocate to the nucleus and
induce transcription of a variety of genes involved in mitogenesis,
differentiation, apoptosis, and quiescence (17, 19, 26).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
TR) were cloned behind the MMTV LTR promoter
(5) via HindIII and EcoRI sites. The FLAG epitope
tag was engineered into all constructs, with the tag in the full-length
and
TR construct inserted in the BsmI site (a gift from
M. A. Hollingsworth (27)). The FLAG epitope in the cytoplasmic
tail deleted construct (
CT) was generated by polymerase chain
reaction using the following primer pairs and used to replace the
AatII (forward primer A, beginning at base pairs 1143 (8))
to EcoRI cassette (reverse primer B) in the CT3 MUC1 clone
(28): Primer A, 5'-TCAGACGTCAGCGTGAGTGATGTCCCA-3' (cloning
site bolded); Primer B,
5'-GCCCCTTTCGAATTCGTCGTCGTCATCCTTGTAATCGGCGGCACT-3' (cloning site bolded, FLAG epitope tag italicized). Constructs were
excised using SalI and SpeI (New England
Biolabs), purified using QiaQuick (Qiagen), and injected into
FVB-fertilized oocytes (Mayo Clinic Scottsdale Transgenic Core
Facility). Potential founders were screened by Southern blot using a
probe generated using BamHI/SpeI that hybridizes
to a segment of the MUC1 construct (the size varies with the construct)
and the SV40 3'-untranslated region of the cDNA. Expression of the
transgene was confirmed in the founder lines by Western blot analysis
using antibodies to the FLAG epitope (M2, Sigma), and MUC1 (B27.29,
Biomira, (29, 30) HMFG-2 (29, 31), and CT2 (see below).
80 °C.
-32P]ATP (3000-5000 cpm/pmol), 50 µM
sodium vanadate, 2.2 µM GST-CT, and 0.5 µg/ml EGFR
kinase domain (Stratagene). Reactions were incubated at 22 °C for 10 min, the proteins resolved by SDS-PAGE, and exposed to film. Negative
control was 3.1 µM pGEX-2TK protein (GST).
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
CT), both on the wild-type background, or tandem-repeat
domain-deleted human MUC1 (
TR), on the Muc1 null
(Muc1
/
) background (Fig.
1A). MMF was created using the
FLAG epitope-tagged 42-tandem repeat human MUC1 described by Burdick
et al. (27). Expression was detected in the virgin (data not
shown), pregnant (Fig. 1, B-D), lactating (Fig.
1C), and post-lactational (data not shown) mammary gland by
Western blot and immunohistochemical analysis (Fig.
2). The relative expression of transgenic
MUC1 compared with wild-type Muc1 in the pregnant gland is shown in Fig. 1D. Note that
TR is on the Muc1
/
background and displays levels comparable to the wild-type. This is
in contrast to MMF (wild-type background) where expression levels are
substantially higher than in the nontransgenic counterpart. Analyses
using B27.29 and HMFG-2 antibodies to the tandem repeat domain, CT2
antibody to the cytoplasmic domain or antibodies to the FLAG epitope
demonstrated a variety of glycosylation forms. CT2 detects a doublet
that represents the cytoplasmic tail and transmembrane domain, as well
as 58 amino acids of the extracellular region, up to the cleavage site
at Ser-Val-Val-Val.2 These 58 extracellular amino acids contain 13 potential glycosylation sites (12 Ser/Thr and 1 Asn site), resulting in a 20-25-kDa cytoplasmic tail-containing species at all stages of development (Fig.
1D and data not shown). An ~200-kDa form was detected
during pregnancy and lactation with HMFG-2 (Fig. 1C),
whereas a >300-kDa form was apparent most often during late pregnancy
and lactation with B27.29 (Fig. 1C). Note that B27.29 and
HMFG-2 detect only human MUC1 whereas CT2 detects both mouse and human
Muc1. HMFG-2 reacts most strongly with the ~200-kDa species, while
occasionally reacting with the >300-kDa form. B27.29, on the other
hand, reacts most strongly with the largest form (while also
recognizing the ~200-kDa form), and has been previously characterized
as binding better to highly glycosylated MUC1 (30). Importantly, we
found that the FLAG epitope was seemingly masked by glycosylation in
the mammary gland, as we were unable to detect the >300-kDa form with anti-FLAG reagents (Fig. 1B).
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Fig. 1.
Transgenic constructs and protein expression
patterns in the mouse mammary gland. A, diagram showing
the MMTV-long terminal repeat (LTR) promoter driving
expression of the various MUC1 cDNAs, including the 42 tandem
repeat containing full-length cDNA (MMF), the cytoplasmic
tail-deleted cDNA ( CT), and tandem repeat domain-deleted
cDNA (
TR) constructs (not to scale). Note the placement of the
FLAG epitope tag is in the extracellular region in both the MMF and
TR, and in the intracellular region of the
CT transgenic.
B, immunoblot detection of transgenic proteins from day 10 pregnant (mP, MMF) or lactating (
TR) glands.
Lysates (600 µg) were immunoprecipitated (IP) and
immunoblotted (IB) using antibodies against the FLAG epitope
tag (M2). The results from two separate founders (numbers 9 and 15) are shown for MMF. C, immunoblot
detection of MMF and
CT transgenic proteins from pregnant or
lactating glands (200 µg) using monoclonal antibodies against the
tandem repeat region (B27.29 and HMFG-2). D, immunoblot of
pregnant mammary gland lysates (200 µg) from wild-type, MMF, and
TR animals using an antibody to the cytoplasmic tail domain of MUC1
(CT2). P, pregnant; mP, midpregnant,
L, lactating; V, 3-5-month-old virgin
animals.
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Fig. 2.
Transgenic MUC1 is expressed on the apical
side of ductal and alveolar epithelium. Immunohistochemical
detection of lactating mammary gland using B27.29-HRP for MMF and CT
(A and B, respectively), ×200 magnification, and
using CT2 for
TR/Muc1
/
(2C), ×400 magnification.
Also note the staining of the luminal contents in both MMF and
CT
(arrows).
CT transgenic was derived from the cytoplasmic tail deleted
clone generated by Pemberton et al. (28). This clone
contains the putative stop transfer sequence, Arg-Arg-Lys, of the
cytoplasmic domain, followed by the FLAG epitope tag on its C-terminal
end. Consistently high expression was detected with both B27.29 and HMFG-2 antibodies in the pregnant and lactational mammary glands of the
CT transgenic animals (Fig. 1C). The
TR transgenic was generated on the Muc1
/
background and contains 3 N-glycosylation sites (1 on the CT domain region and 2 on
the extracellular domain). Additionally 30% of the amino acids
contained in the extracellular domain are potential sites for
O-glycosylation. As the tandem repeat domain is missing,
TR is detected using the FLAG or CT2 antibodies (Fig. 1,
B and D). The apparent molecular mass of
TR extracellular domain is ~50 kDa, indicating that the transgenic
protein is N- and
O-glycosylated.3
Also, relative expression of the transgene in this founder line is
lower than that observed for MMF or
CT (Figs. 1D and 2).
CT (Fig. 2B) are detected in the lumen of the
lactating alveoli, presumably either shed or present on the plasma
membrane during the release of milk proteins and fat into the lumen.
Although not quantitative, the lower level of expression in the
TR
transgenic is also apparent here (Fig. 2C).
, but not NRG
in MDA-MB-468 cells (data not
shown). Treatment with any of these ligands (except NRG
)
resulted in the co-immunoprecipitation of a tyrosine-phosphorylated
protein of ~180 kDa (pp180) with MUC1 in MDA-MB-468 cells (Fig.
3B, top arrow, and data not shown). To determine whether
this interaction was physiologically relevant to the intact mammary
gland, pregnant and lactating glands from both wild-type and transgenic
mice were injected intraperitoneally with receptor grade EGF, and
mammary gland lysates prepared. The pp180 could also be readily
identified in vivo as co-immunoprecipitating with MUC1 using
antibodies to both the tandem repeat region or the cytoplasmic tail
(Fig. 4B). Additionally, a pp ~120 kDa and pp ~250 kDa
also co-immunoprecipitated in the MMF samples, but not in the
wild-type. On lighter exposure, the designated pp180 band is not a
single protein species in the mammary gland, indicating it either
represents multiple forms of one protein or multiple proteins of the
same apparent size. As MUC1 is commonly overexpressed in breast cancer,
we next examined if the interaction with the pp180 was detectable in a
mouse tumor model. We observed the co-immunoprecipitation of a pp180
with MUC1 using tumor protein lysates derived from MMTV-mTag transgenic
mice (5) treated with exogenous EGF (Fig. 4C). This
interaction in mammary tumors indicates that this Muc1-pp180 association is not unique to the normal mammary gland, as is indicated by the data from the MDA-MD-468 cell line (Fig. 3).
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Fig. 3.
EGFR kinase phosphorylates MUC1 and MUC1
associates with a pp180 in cell culture. A, in
vitro phosphorylation reactions on the fusion protein of the MUC1
cytoplasmic tail with glutathione S-transferase
(GST-CT) and GST alone. EGFR KD (auto)
represents autophosphorylation that occurs in the absence of excess
cold ATP. B, MDA-MB-468 cells treated with increasing
concentrations of betacellulin (BTC) immunoprecipitated
(IP) with anti-MUC1 (CT1) and immunoblotted with
anti-phosphotyrosine (RC20-HRP). Arrows indicate the
tyrosine-phosphorylated protein ~180 kDa (top) and the
cytoplasmic tail of MUC1 (bottom). IB,
immunoblotted.
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Fig. 4.
MUC1 cytoplasmic tail is tyrosine
phosphorylated and associates with a pp180. A, in each
lane, 2 mg of lysate were immunoprecipitated with anti-MUC1
(CT2) and immunoblotted with anti-phosphotyrosine
(RC20-HRP). Lanes identified as (+) represent animals injected with 1 µg/g body weight EGF, while ( ) represents endogenous EGF.
B, mammary gland lysates (4 mg) were immunoprecipitated with
anti-MUC1 antibodies (CT2, B27.29, or
HMFG-2) and immunoblotted with antiphosphotyrosine
(RC20-HRP). The lysate only lane represents 200 µg of
protein, and the pp180 in the no EGF lane is due to endogenous
phosphorylation. C, MUC1 in mammary gland tumors from
MMTV-mTag transgenic animals also co-immunoprecipitates pp180. Lanes
represent individual animals, either treated or untreated with EGF
before sacrifice, and 2 mg of lysate were immunoprecipitated with
anti-MUC1 (CT1) and immunoblotted with anti-phosphotyrosine
(RC20-HRP).
CT transgenic (Fig. 5A). Complexes between MUC1 and the
remaining 3 erbB receptors could also be identified in pregnant and
lactating mammary glands (Fig. 5C). Again, little to no
CT MUC1 protein could be found precipitating with erbB2, erbB3, or
erbB4 antibodies. Importantly, equal if not more of the
CT MUC1
protein is present in the mammary glands where little to no
co-immunoprecipitation was observed (Fig. 5C, bottom panel).
Therefore, as MMF and
CT transgenic MUC1 proteins are both present
in the same cellular location at high levels (Fig. 2, A and
B), these results suggest a requirement for the MUC1
cytoplasmic tail in this interaction with the erbB receptors.
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Fig. 5.
MUC1 associates with EGFR, erbB2, erbB3, and
erbB4 in the mouse mammary gland. A, mammary gland
lysate from day 2/3 lactating females (2 mg) was immunoprecipitated
with anti-EGFR (SC 1005) and blotted with anti-MUC1 (HMFG-2). Note that
HMFG-2 does not react with mouse Muc1. B, lysates (200 µg)
from the same mammary gland samples shown in the top panel
were blotted with anti-EGFR (ERCT) or anti-MUC1 (HMFG-2). C,
mammary gland lysates (2 mg) were immunoprecipitated with anti-erbB2,
anti-erbB3, or anti-erbB4 and blotted with anti-MUC1 (B27.29-HRP).
Membranes were then reprobed with the same antibody used to perform the
immunoprecipitation. The bottom panel shows levels of MUC1
(200 µg) in the same mammary gland samples used in the erbB2, -3, and
-4 immunoprecipitations. MUC1 immunoblotting was performed using both
HMFG-2 and B27.29 antibodies.
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Fig. 6.
MUC1 and EGFR colocalize in the lactating
mammary gland. Parrafin sections (20 µm) were probed with
anti-EGFR (SC, 1005) and anti-MUC1 (CT2) primary antibodies and Alexa
594 streptavidin/biotin-anti-rabbit and fluorescein isothiocyanate
anti-Armenian hamster secondary antibodies. These were examined at
×400 magnification using a 510 laser scanning microscope.
Arrows (D) indicate areas of intense
co-localization.
/
lactating mammary
gland (Fig. 8A). ERK1/2 is
activated in the wild-type pregnant gland, making phosphorylation
increases in the transgenic lysates difficult to detect. Given this, we
do observe an increase in phospho-ERK1/2 in some MMF pregnant lysates compared with the pregnant gland of wild-type mice (Fig.
8B). We observed that this activation of ERK is limited to
early lactation (day 2/3), as by day 10 lactation, dpERK levels in
transgenic glands resembled that of the wild-type (Fig. 8B).
The overall levels of ERK1/2 are similar in both wild-type and
transgenic mammary glands (Fig. 8B, bottom panel). Note that
lysates from some MMF transgenic mammary glands do not show ERK1/2
activation. This may be due to reduced amounts of EGF reaching the
gland in that experiment, different physiological makeup of that
particular gland, or simply missing the kinetic window of kinase
activation with that animal. Importantly, activation of ERK1/2 was
consistently and repeatedly demonstrated in mammary gland lysates from
EGF-injected MMF transgenics. Phospho-p46 appeared similar in all
genotypes and conditions examined (Fig. 8C), and while p54
shows a modest increase of phosphorylation in some samples (Fig.
8C), this increase was not duplicated in subsequent
experiments. Phosphorylated p38 was undetectable by these methods.
These results indicate that only one of the kinase pathways analyzed,
ERK1/2, is selectively activated in response to heightened levels of
full-length MUC1 in the lactating mammary gland.
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Fig. 7.
EGFR kinase activity is largely unaffected by
MUC1 expression. Mammary gland lysates (2 mg) were
immunoprecipitated with anti-EGFR antibody (SC, 1005) and immunoblotted
with an anti-phosphotyrosine antibody (RC20-HRP). Lysates (200 µg)
were then immunoblotted with anti-EGFR antibody (ERCT) (bottom
panel). EGF represents either endogenous EGF ( ) or
exogenous treatment with 1 µg/g body weight EGF (+).
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Fig. 8.
Overexpression of full-length MUC1 activates
p42/p44 ERK. A, samples from mammary gland lysates were
immunoblotted with anti-dpERK1/2. B, lysates were
immunoblotted with anti-dpERK (top panel) or anti-ERK 1/2
(bottom panel). C, JNK/SAPK immunoblots are from
lactational samples only, during early (d 2/3) or late
(d 10) lactation. D, MMF (full-length MUC1
transgenic), wild-type (WT), Muc1 knockout (KO),
TR and
CT lysates were immunoblotted with an anti-PCNA antibody.
In A-D, 200 µg of protein lysate was separated on a 10%
SDS-PAGE for each sample and EGF represents either endogenous EGF (
)
or exogenous treatment with 1 µg/g body weight EGF (+).
TR mammary gland lysates (Fig.
9). Collectively, these results further
implicate the presence of Muc1 in a complex with EGFR in the mammary
gland.
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Fig. 9.
MUC1 associates with Grb2 and SOS.
Mammary gland protein lysates (2 mg) were immunoprecipitated with
either anti-Grb2 or anti-SOS antibodies and immunoblotted with an
anti-MUC1 antibody (CT2). MMF, full-length MUC1 transgenic;
WT, wild-type; KO, knockout; LC,
immunoglobulin light chain.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We are grateful to M. A. Hollingsworth
for the MUC1 and TR flag-tagged constructs, J. Taylor-Papadimitriou
for the HMFG-2 antibody, Biomira Inc. for the B27.29 antibody, H. S. Earp for the EGFR antibody, Mike McGuckin for helpful discussions,
and Todd D. Camenisch for critical reading of the manuscript. We also thank Suresh Savarirayan, Stephanie Munger, and animal care attendants for excellent animal care and Marvin H. Ruona for computer graphics.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants CA64389 (to S. J. G.) and CA81703 (to J. A. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom all correspondence should be addressed: Mayo Clinic, S.C.
Johnson Research Bldg., 13400 E. Shea Blvd., Scottsdale, AZ 85259. Tel.: 480-301-7062; Fax: 480-301-7017; E-mail: gendler.sandra@mayo.edu.
Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M011248200
2 A. Harris, personal communication.
3 W. Xie and S. J. Gendler, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: MMTV mouse mammary turmor virus, EGFR, epidermal growth factor receptor; TR, tamdem repeat; CT, cytoplasmic tail; PCNA, proliferating cell nuclear antigen; HRP, horseradish peroxidase; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; MAPK, mitogen-activated protein kinase; ERCT, EGFR cytoplasmic tail; MMF, MMTV-MUC1FLA6; NRG, neuregulin.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Zotter, S., Hageman, P. C., Lossnitzer, A., Mooi, W. J., and Hilgers, J. (1988) Cancer Rev. 11-12, 55-101 |
2. | Hilkens, J., Vos, H. L., Wesseling, J., Boer, M., Storm, J., van der Valk, S., Calafat, J., and Patriarca, C. (1995) Cancer Lett. 90, 27-33[CrossRef][Medline] [Order article via Infotrieve] |
3. | Wesseling, J., van der Valk, S. W., Vos, H. L., Sonnenberg, A., and Hilkens, J. (1995) J. Cell Biol. 129, 255-265[Abstract] |
4. | Wesseling, J., van der Valk, S. W., and Hilkens, J. (1996) Mol. Biol. Cell 7, 565-577[Abstract] |
5. | Guy, C. T., Cardiff, R. D., and Muller, W. J. (1992) Mol. Cell. Biol. 12, 954-961[Abstract] |
6. |
Spicer, A. P.,
Rowse, G. J.,
Lidner, T. K.,
and Gendler, S. J.
(1995)
J. Biol. Chem.
270,
30093-30101 |
7. |
Ligtenberg, M. J. L.,
Kruijshaar, L.,
Buijs, F.,
van Meijer, M.,
Litvinov, S. V.,
and Hilkens, J.
(1992)
J. Biol. Chem.
267,
6171-6177 |
8. |
Gendler, S. J.,
Lancaster, C. A.,
Taylor-Papadimitriou, J.,
Duhig, T.,
Peat, N.,
Burchell, J.,
Pemberton, L.,
Lalani, E. N.,
and Wilson, D.
(1990)
J. Biol. Chem.
265,
15286-15293 |
9. | Zrihan-Licht, S., Baruch, A., Elroy-Stein, O., Keydar, I., and Wreschner, D. H. (1994) FEBS Lett. 356, 130-136[CrossRef][Medline] [Order article via Infotrieve] |
10. | Spicer, A. P., Duhig, T., Chilton, B. S., and Gendler, S. J. (1995) Mamm. Genome 6, 885-888[Medline] [Order article via Infotrieve] |
11. | Regimbald, L. H., Pilarski, L. M., Longenecker, B. M., Reddish, M. A., Zimmermann, G., and Hugh, J. C. (1996) Cancer Res. 56, 4244-4249[Abstract] |
12. | Kam, J. L., Regimbald, L. H., Hilgers, J. H., Hoffman, P., Krantz, M. J., Longenecker, B. M., and Hugh, J. C. (1998) Cancer Res. 58, 5577-5581[Abstract] |
13. |
Li, Y.,
Bharti, A.,
Chen, D.,
Gong, J.,
and Kufe, D.
(1998)
Mol. Cell. Biol.
18,
7216-7224 |
14. |
Yamamoto, M.,
Bharti, A.,
Li, Y.,
and Kufe, D.
(1997)
J. Biol. Chem.
272,
12492-12494 |
15. | Pandey, P., Kharbanda, S., and Kufe, D. (1995) Cancer Res. 55, 4000-4003[Abstract] |
16. | Schroeder, J. A., and Lee, D. C. (1998) Cell Growth Differ. 9, 451-464[Abstract] |
17. |
Olayioye, M. A.,
Neve, R. M.,
Lane, H. A.,
and Hynes, N. E.
(2000)
EMBO J.
19,
3159-3167 |
18. | Schroeder, J. A., and Lee, D. C. (1997) J. Mamm. Gland Biol. Neoplasia 2, 119-129[Medline] [Order article via Infotrieve] |
19. | Alroy, I., and Yarden, Y. (1997) FEBS Lett. 410, 83-86[CrossRef][Medline] [Order article via Infotrieve] |
20. | Carpenter, G. (2000) Bioessays 22, 697-707[CrossRef][Medline] [Order article via Infotrieve] |
21. |
Olayioye, M. A.,
Graus-Porta, D.,
Beerli, R. R.,
Rohrer, J.,
Gay, B.,
and Hynes, N. E.
(1998)
Mol. Cell. Biol.
18,
5042-5051 |
22. |
Olayioye, M. A.,
Beuvink, I.,
Horsch, K.,
Daly, J. M.,
and Hynes, N. E.
(1999)
J. Biol. Chem.
274,
17209-17218 |
23. | Pinkas-Kramarski, R., Soussan, L., Waterman, H., Levkowitz, G., Alroy, I., Klapper, L., Lavi, S., Seger, R., Ratzkin, B. J., Sela, M., and Yarden, Y. (1996) EMBO J. 15, 2452-2467[Abstract] |
24. | Amundadottir, L. T., and Leder, P. (1998) Oncogene 16, 737-746[CrossRef][Medline] [Order article via Infotrieve] |
25. | Daly, J. M., Olayioye, M. A., Wong, A. M., Neve, R., Lane, H. A., Maurer, F. G., and Hynes, N. E. (1999) Oncogene 18, 3440-3451[CrossRef][Medline] [Order article via Infotrieve] |
26. | Campbell, S. L., Khosravi-Far, R., Rossman, K. L., Clark, G. J., and Der, C. J. (1998) Oncogene 17, 1395-1413[CrossRef][Medline] [Order article via Infotrieve] |
27. |
Burdick, M. D.,
Harris, A.,
Reid, C. J.,
Iwamura, T.,
and Hollingsworth, M. A.
(1997)
J. Biol. Chem.
272,
24198-24202 |
28. |
Pemberton, L. F.,
Rughetti, A.,
Taylor-Papadimitriou, J.,
and Gendler, S. J.
(1996)
J. Biol. Chem.
271,
2332-2340 |
29. | Price, M. R., Rye, P. D., Petrakou, E., Murray, A., Brady, K., Imai, S., Haga, S., Kiyozuka, Y., Schol, D., Meulenbroek, M. F., Snijdewint, F. G., von Mensdorff-Pouilly, S., Verstraeten, R. A., Kenemans, P., Blockzjil, A., Nilsson, K., Nilsson, O., Reddish, M., Suresh, M. R., Koganty, R. R., Fortier, S., Baronic, L., Berg, A., Longenecker, M. B., and Hilgers, J. (1998) Tumour Biol. 19, 1-20[Medline] [Order article via Infotrieve] |
30. | Sikut, R., Sikut, A., Zhang, K., Baeckstrom, D., and Hansson, G. C. (1998) Tumour Biol. 19, 122-126[Medline] [Order article via Infotrieve] |
31. |
Burchell, J.,
Durbin, H.,
and Taylor-Papadimitriou, J.
(1983)
J. Immunol.
131,
508-513 |
32. | Pemberton, L., Taylor-Papadimitriou, J., and Gendler, S. J. (1992) Biochem. Biophys. Res. Commun. 185, 167-175[Medline] [Order article via Infotrieve] |
33. | Braga, V. M., Pemberton, L. F., Duhig, T., and Gendler, S. J. (1992) Development 115, 427-437[Abstract] |
34. |
Carraway, K. L., 3rd,
Rossi, E. A.,
Komatsu, M.,
Price-Schiavi, S. A.,
Huang, D.,
Guy, P. M.,
Carvajal, M. E.,
Fregien, N.,
Carraway, C. A.,
and Carraway, K. L.
(1999)
J. Biol. Chem.
274,
5263-5266 |
35. | Zhu, X., Price-Schiavi, S. A., and Carraway, K. L. (2000) Oncogene 19, 4354-4361[CrossRef][Medline] [Order article via Infotrieve] |
36. | Carraway, K. L., Price-Schiavi, S. A., Komatsu, M., Idris, N., Perez, A., Li, P., Jepson, S., Zhu, X., Carvajal, M. E., and Carraway, C. A. (2000) Front. Biosci. 5, D95-D107[Medline] [Order article via Infotrieve] |
37. |
Pece, S.,
and Gutkind, J. S.
(2000)
J. Biol. Chem.
275,
41227-41233 |
38. | Denker, B. M., and Nigam, S. K. (1998) Am. J. Physiol. 274, F1-9[Medline] [Order article via Infotrieve] |
39. |
Chen, Y.,
Lu, Q.,
Schneeberger, E. E.,
and Goodenough, D. A.
(2000)
Mol. Biol. Cell
11,
849-862 |
40. | Cowley, S., Paterson, H., Kemp, P., and Marshall, C. J. (1994) Cell 77, 841-852[Medline] [Order article via Infotrieve] |
41. | Woods, D., Parry, D., Cherwinski, H., Bosch, E., Lees, E., and McMahon, M. (1997) Mol. Cell. Biol. 17, 5598-5611[Abstract] |
42. | Ishikawa, Y., and Kitamura, M. (1999) Biochem. Biophys. Res. Commun. 264, 696-701[CrossRef][Medline] [Order article via Infotrieve] |
43. | Sandgren, E. P., Schroeder, J. A., Qui, T. H., Palmiter, R. D., Brinster, R. L., and Lee, D. C. (1995) Cancer Res. 55, 3915-3927[Abstract] |
44. | Lloyd, A. C., Obermuller, F., Staddon, S., Barth, C. F., McMahon, M., and Land, H. (1997) Genes Dev. 11, 663-677[Abstract] |
45. | Chapman, R. S., Lourenco, P., Tonner, E., Flint, D., Selbert, S., Takeda, K., Akira, S., Clarke, A. R., and Watson, C. J. (2000) Adv. Exp. Med. Biol. 480, 129-138[Medline] [Order article via Infotrieve] |
46. | Lundy, J., Schuss, A., Stanick, D., McCormack, E. S., Kramer, S., and Sorvillo, J. M. (1991) Am. J. Pathol. 138, 1527-1534[Abstract] |
47. | Fabian, C. J., Kamel, S., Zalles, C., and Kimler, B. F. (1996) J. Cell. Biochem. 25S, 112-122[CrossRef] |