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J Biol Chem, Vol. 275, Issue 17, 12565-12571, April 28, 2000
From the Department of Pathology, Royal Victoria Infirmary,
Newcastle-upon-Tyne NE1 4LP, United Kingdom
Estrogens can stimulate the proliferation of
estrogen-responsive breast cancer cells by increasing their
proliferative response to insulin-like growth factors. The mechanism
underlying the increased proliferation could involve the induction of
components of the insulin-like growth factor signal transduction
pathway by estrogen. In this study we have examined the regulation of
the expression of insulin receptor substrate-1, a major intracellular
substrate of the type I insulin-like growth factor receptor tyrosine
kinase. Estradiol increased insulin receptor substrate-1 mRNA and
protein levels at concentrations consistent with a mechanism involving the estrogen receptor. Insulin receptor substrate-1 was not induced significantly by the antiestrogens tamoxifen and ICI 182,780, but they
inhibited the induction of insulin receptor substrate-1 by estradiol.
Analysis of tyrosine-phosphorylated insulin receptor substrate-1 showed
that the highest levels were found in cells stimulated by estradiol and
insulin-like growth factor-I, whereas low levels were found in the
absence of estradiol irrespective of whether type I insulin-like growth
factor ligands were present. Insulin receptor substrate-2, -3, and -4 were not induced by estradiol. These results suggest that estrogens and
antiestrogens may regulate cell proliferation by controlling insulin
receptor substrate-1 expression, thereby amplifying or attenuating
signaling through the insulin-like growth factor signal transduction pathway.
Estrogens are important in controlling the proliferation of breast
cancer cells, and therapeutic agents that inhibit the synthesis and
effects of estrogens are widely used in the treatment of breast cancer.
The mechanisms by which estrogens stimulate cell proliferation, however, are not clear. The proliferation of estrogen receptor-positive breast cancer cell lines is stimulated by estrogen in culture, and
these cell lines provide useful model systems for determining how
estrogens stimulate breast cancer cell proliferation. The proliferation
of breast cancer cells in culture is also responsive to insulin-like
growth factors, and components of the
IGF1 signal transduction
system are expressed by both breast tumors and cultured breast cancer
cells (1-5).
We along with others have suggested that estrogens control cell
proliferation by modulating the proliferative response of breast cancer
cells to IGFs (6-8). Estrogen-responsive breast cancer cells show a
much greater proliferative response to high concentrations of insulin
(acting through the type I IGF receptor) or physiological
concentrations of IGFs in the presence of estrogen, and conversely the
proliferative response to estradiol is much greater in the presence of
insulin or IGFs.
The proliferative effects of IGFs are mediated by the type I IGF
receptor a cell surface receptor comprising two We have suggested (7, 19, 20) that the ability of estrogens to increase
the response of breast cancer cells to IGFs derives from the
up-regulation of one or more critical components of the IGF signal
transduction system. Components of the IGF signal transduction pathway
have been characterized in breast cancer cells in culture and tumors,
and the expression of ligands (21, 22), receptors (7, 23-25), and
downstream signaling molecules (26, 27) have all been demonstrated.
We originally proposed that the increased response could result from
increased expression of the type I IGF receptor (7). However,
experiments in which this receptor was constitutively overexpressed in
estrogen-responsive breast cancer cells showed that overexpression did
not abrogate estrogen responsiveness (20).
In this study, we show that the expression of IRS-1, a major substrate
for the type I IGF receptor tyrosine kinase, is regulated by estrogens
and antiestrogens, whereas other members of the IRS family, IRS-2, -3, and -4, are not. This regulation could be responsible for the increased
proliferative response of breast cancer cells to IGFs in the presence
of estrogen.
Cell Culture--
MCF-7 (28) ZR-75-B (29), EFF-3 (30), EFM-19
(31), T47D (32), BT20 (33), and Hs578T (34) human breast cancer cell
lines were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1 µg/ml insulin. MDA
MB-231 cells (35) were cultured in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum only. Cells were incubated at
37 °C in a humidified atmosphere containing 4% CO2. In
experiments in which the effects of estrogen and/or insulin-like growth
factors were measured, cells were first cultured in medium depleted of
steroids. This comprised phenol red-free minimal essential medium
supplemented with charcoal-treated newborn calf serum and insulin (1 µg/ml). In experiments in which the acute effects of insulin or IGF-I
on IRS-1 tyrosine phosphorylation were examined, cells were incubated
with the type I IGF receptor ligand in medium comprising phenol
red-free minimal essential medium supplemented with 1% bovine serum
albumin for the time indicated.
RNA Preparation--
75-cm2 flasks of cells were
grown to 70% confluence and then withdrawn from steroids and growth
factors by culturing for 6 days in withdrawal medium, as described
previously (36). Medium was changed daily, and cells were washed twice
with phosphate-buffered saline before adding the new medium for the
first 3 days. Withdrawn cells were treated for 3 days with steroids
and/or growth factors in withdrawal medium as required, with a daily
change of medium. Total RNA was prepared from cells by homogenization
in lithium chloride/urea and extraction in phenol/chloroform.
Northern Analysis--
10 µg of total RNA was electrophoresed
on 1.2% agarose/formaldehyde gels. RNA was transferred to Hybond-N
nylon membrane by capillary transfer and fixed by baking at 80 °C
under vacuum and irradiating with UV light. The 4.1-kb human IRS-1
cDNA insert from plasmid phIRS-1-5 (obtained from C. R. Kahn
(37)) was radiolabeled with [32P]dCTP by random-priming
and was hybridized to the immobilized RNA at a concentration of
107 cpm/ml for 3 days at 42 °C. Membranes were then
extensively washed at 65 °C and exposed to preflashed x-ray film for
up to 1 week at Protein Preparation--
Cells were grown to 70% confluence in
35-mm wells, withdrawn for 6 days, and treated for 3 days with growth
factors and/or steroids as required. Cell monolayers were washed twice
with ice-cold phosphate-buffered saline and lysed in 0.5 ml of lysis
buffer (50 mM HEPES, pH 7.2, 150 mM NaCl, 1.5 mM MgCl2, 1 mM Na2EDTA, 10% glycerol, 1% w/v Triton X-100, 0.4 mM
Na3VO4, 1 µg/ml aprotinin, 1 µg/ml
leupeptin) on ice for 30 min. The lysate from each well was centrifuged
at 12,900 × g (12,000 rpm) for 15 min. The protein concentration of the supernatant was measured by BCA assay.
Immunoblotting--
Antibodies against IRS-1, -2, -3, and -4 were obtained from Upstate Biotechnology Inc. (Lake Placid, NY). The
IRS-1 antibody was raised against the 14 C-terminal amino acids of rat
IRS-1, the IRS-2 antibody was raised against amino acids 976-1094 of mouse IRS-2, the IRS-3 antibody was raised against a GST fusion protein
containing the N-terminal 120 amino acids of rat IRS-3, and the IRS-4
antibody was raised against amino acids 1240-1257 of human IRS-4. All
antibodies were raised in rabbits and were protein A-purified IgG. The
IRS-4 antibody was raised against the human sequence, and the IRS-1 and
2 antibodies are known to cross-react with the human protein. The IRS-3
antibody reacted with a protein of correct size (approximately 66 kDa)
in MCF-7 cell extracts. 7.5-µg samples of protein were mixed with an
equal volume of 2× concentrated SDS-polyacrylamide gel electrophoresis sample buffer, boiled, and then electrophoresed on 7% polyacrylamide gels containing SDS. Proteins were then transferred to nitrocellulose (Schleicher & Schuell). The nitrocellulose filters were incubated in
blocking solution (phosphate-buffered saline containing 5% nonfat milk
and 0.05% Tween-20), and then incubated in 0.2-0.5 µg/ml IRS-1 or
0.5 µg/ml IRS-2, -3, or -4 antibody in blocking solution overnight at
4 °C. The filters were washed thoroughly in water and then incubated
for 90 min at room temperature in a 1:3000 dilution of goat anti-rabbit
horse radish peroxidase-conjugated secondary antibody (DAKO). The
filters were then developed using enhanced chemiluminescence.
Tyrosine-phosphorylated IRS-1 was measured by immunoprecipitation of
IRS-1 following the manufacturer's protocol and Western transfer
analysis using the antiphosphotyrosine antibody PY20, again following
the manufacturer's protocol.
Expression of IRS-1 in Breast Cancer Cell Lines--
IRS-1
mRNA expression was measured in three estrogen receptor-negative
and five estrogen receptor-positive human breast cancer cell lines. The
cDNA probe used for the hybridization to total RNA extracted from
the cells encompassed the entire coding region of human IRS-1 and
detected four major mRNAs of 8.5, 6.9, 6.1, and 4.4 kb (Fig.
1). IRS-1 mRNA was detected in all
the cell lines apart from BT20, but the levels of expression were
extremely variable. For example, MCF-7 cells showed the highest level
of expression of all four mRNA, and the level of the 8.5-kb
mRNA was more than 10-fold higher than in EFF-3 and MDA-MB 231 cells. Each cell line showed a different pattern of expression of the
four transcripts. In MCF-7 and ZR-75 cells, the level of expression of
the 8.5-kb transcript was highest, in T47D cells the level of the
6.9-kb transcript was highest, whereas EFM-19 had highest levels of the 6.1-kb transcript. The 4.4-kb mRNA was a minor mRNA in all cell lines in which it was expressed. There was no clear relationship between IRS-1 expression and the estrogen receptor status of the cell
lines. However, of the four cell lines that expressed the highest
levels of IRS-1 mRNA, three were estrogen receptor-positive.
Regulation of IRS-1 mRNA and Protein by Estrogen in the MCF-7
Breast Cancer Cell Line--
To determine whether the expression of
IRS-1 is regulated by estrogen in estrogen-responsive breast cancer
cells, IRS-1 mRNA and protein were first measured in MCF-7 cells
that had been withdrawn from the effects of estrogen in the routine
culture medium and then stimulated with estradiol. A large decrease in
IRS-1 mRNA expression was observed in MCF-7 cells after 3 days of
culture in steroid-free medium (data not shown). To investigate whether this decrease was attributable to the withdrawal of the cells from the
estrogen present in the routine culture medium, MCF-7 cells were
withdrawn by culturing for 3 days in steroid-free medium and then
treated for times varying from 4 h to 6 days with
10
To determine whether estradiol induced a similar increase in IRS-1
protein expression, the above experiment was repeated, the cells were
lysed, and the IRS-1 protein in the lysates was measured. As shown in
Fig. 2C, IRS-1 protein was undetectable in withdrawn cells,
was first detected after 16 h of estrogen treatment, and then
continued to increase during 4-5 days of treatment. Compared with the
mRNA induction by estradiol, there appeared to be a lag before
IRS-1 protein was detected. It was not possible to quantitate the
absolute increase in protein levels because of the undetectable amounts
of IRS-1 in withdrawn cells, but the increase was more dramatic than
the increase in mRNA levels (Fig. 2D). Three forms of
IRS-1 were identified. The highest molecular mass form had an apparent
molecular mass of 185 kDa as described by others. The two lower
molecular mass forms that were most prominent in cells cultured for
5-6 days in estradiol are presumed to represent degradation products
of the 185-kDa form.
Cells that had been withdrawn from the effects of estradiol in the
routine cell culture medium were then stimulated with various concentrations of estradiol for 3 days to determine the concentration required to induce IRS-1 mRNA and protein (Fig.
3). IRS-1 mRNAs were induced
coordinately and were induced by the lowest concentration of estradiol
tested (Fig. 3A). Maximal induction was observed with
concentrations of 10 Regulation of IRS-1 mRNA and Protein by Antiestrogens in MCF-7
Cells--
The ability of the antiestrogens to induce the expression
of IRS-1 mRNA and to inhibit the induction by estrogen in MCF-7 cells was then examined. Tamoxifen is widely used in the treatment of
breast cancer, and although it inhibits the effects of estradiol, it
has partial estrogen agonist activity for most estrogen responses measured in breast cancer cells. Cells were withdrawn and then treated
for 3 days with various concentrations of tamoxifen alone or in the
presence of 2 × 10
ICI 182,780 is currently being evaluated for the treatment of breast
cancer (38) and has been reported to have little or no estrogen agonist
activity. Cells were withdrawn and treated for 3 days with various
concentrations of ICI 182,780 tamoxifen alone or in the presence of
2 × 10 Effects of Insulin and IGF-I on IRS-1 Tyrosine Phosphorylation in
Cells Cultured the Presence and Absence of Estradiol--
If the
enormously increased amounts of IRS-1 protein present in
estradiol-stimulated cells are activated in the presence of the ligands
that use this docking protein to transduce their signal, then the high
levels of IRS-1 protein may contribute to the increased proliferation
induced by ligands in the presence of estradiol.
The amount of tyrosine-phosphorylated IRS-1 was therefore measured in
cells grown under the conditions used in cell proliferation assays.
Cells were cultured in withdrawal medium for 6 days and then in
withdrawal medium alone or in the presence of estradiol or insulin
alone and cells grown in the presence of estradiol and insulin together
for 3 days. Cell lysate was prepared from the cells, IRS-1 was
immunoprecipitated, and its tyrosine phosphorylation was assessed by
Western transfer analysis (Fig.
5A). Cells that had been withdrawn from the effects of
estrogen in the routine culture medium had no detectable
tyrosine-phosphorylated IRS-1 either in the absence or presence of
insulin (Fig. 5A). In contrast, large amounts of
tyrosine-phosphorylated IRS-1 were detected in cells that had been
grown in medium containing estradiol and insulin. Importantly, cells
grown in medium containing estradiol but lacking insulin showed similar
low levels of tyrosine-phosphorylated IRS-1 to cells that had been
grown in medium lacking both estradiol and insulin.
To demonstrate that insulin or IGF-I is directly responsible for the
increased tyrosine phosphorylation of IRS-1 in the experiment shown in
Fig. 5A, MCF-7 cells were grown in the presence of estradiol and then treated for 5 min with insulin or IGF-I. No
tyrosine-phosphorylated IRS-1 was detected in unstimulated cells, but
both insulin and IGF-I caused a similar large induction of IRS-1
phosphorylation in these estrogen-stimulated cells. The concentrations
of the two ligands used in this experiment are consistent with this
effect being mediated by the type I IGF receptor (Fig.
5B).
Effects of Estrogen on IRS-2, IRS-3, and IRS-4 Expression in MCF-7
Breast Cancer Cells--
IRS-1 belongs to family of related molecules
including IRS-2 (39), IRS-3 (40), and IRS-4 (41) that contain a
pleckstrin homology domain and a phosphotyrosine-binding domain toward
the N terminus of the protein. The effects of estrogen on IRS-2, -3, and -4 levels in MCF-7 cells were therefore investigated to determine whether the effects of estrogen were specific for IRS-1 or involved other members of this family of proteins. Fig.
6 shows a representative Western transfer
of control and estrogen-stimulated cells reacted with IRS-1, -2, -3, and -4 antibodies together with a histogram showing the results for
three separate experiments. The IRS-2 antibody reacted with a protein
of approximately 180 kDa, and the IRS-3 antibody reacted with a protein
of 66 kDa. These sizes are consistent with those reported in the
literature for these proteins. The IRS-4 antibody reacted predominantly
with proteins of 130 and 97 kDa. Two minor proteins of 93 and 100 kDa
were also identified, and longer exposures of the filter shown in Fig.
6 also revealed a protein of 160 kDa. Immunoprecipitation of IRS-3 and
IRS-4 from cell lysates of control or IGF-I-treated MCF-7 cells and
immunoblotting with antiphosphotyrosine antibody failed to demonstrate
that these proteins were tyrosine-phosphorylated or that tyrosine
phosphorylation could be induced by IGF-I. We therefore conclude that
although IRS-3 and -4 are expressed in MCF-7 breast cancer cells, they
are not tyrosine-phosphorylated by the type I IGF receptor in response
to IGF-I. IRS-1 was induced approximately 100-fold in this experiment,
whereas IRS-2 and -3 and none of the forms of IRS-4 were induced
significantly by estradiol. These experiments show that of the members
of the IRS family analyzed, only IRS-1 levels are significantly
regulated by estradiol.
Changes in the structure, function, and expression of
intracellular signaling molecules have been implicated in the aberrant control of cell proliferation resulting in malignant transformation. The intracellular signaling pathways involved in the control of breast
cancer cell proliferation are not known, although alterations to a
number of different signaling molecules have been reported.
Understanding the mechanisms underlying the responsiveness of breast
cancer cells to estrogen is clinically important given the incidence of
the disease and the widespread use of hormonal manipulation to control
the progression of the disease by abrogating the effects of estrogens.
A number of laboratories have focussed on the involvement of the IGF
signal transduction pathway and have shown: (i) that breast cancer
cells are responsive to the proliferative effects of IGFs, (ii) that a
number of components of the IGF signal transduction pathway can be
regulated by estrogen including IGF-II (21, 22), IGF-binding proteins
(42), and the type I IGF receptor (7), and (iii) that estrogens control the proliferative response of cells to IGFs (6-8). In this study we
report that estrogens regulate the expression of IRS-1, a major substrate of the type I IGF receptor tyrosine kinase, and suggest that
this regulation may be responsible for mediating the effects of
estrogen on breast cancer cell proliferation.
No previous studies have analyzed IRS-1 mRNA in breast cancer
cells. Four major mRNAs were identified of 8.5, 6.9, 6.1, and 4.4 kb in MCF-7 cells. mRNAs of 6 and 6.9 kb have been reported previously in a variety of human tissues (skeletal muscle, heart, and
brain) (37, 43), mRNAs of 9 and 6 kb have been found in human
pancreas and a variety of cell lines (44), and a single mRNA of 5 kb has been found in normal human liver and hepatocellular carcinoma
(45). The reasons for the differences in the sizes of the mRNAs is
not known but may result, at least in part, from multiple transcription
start points as has been observed for mouse IRS-1 mRNA (46).
The majority of published studies of IRS-1 protein in breast cancer
cells present illustrations which showing the 185-kDa form only (27,
47), and it is not possible to evaluate whether other forms were
observed. We consistently observed lower molecular mass forms that we
presume to be degradation products e.g. Fig. 2. The
degradation pathway of IRS-1 is not known, but the identification of
well defined intermediates suggests that there may be specific degradation pathways. Because the role of IRS-1 is to act as a docking
protein that assembles signaling complexes, IRS-1 degradation may be an
important way of regulating IRS-1 signaling. IRS-1 contains PEST
sequences and is a substrate for the calcium-dependent
protease calpain. Whether, the lower molecular mass forms of IRS-1 seen in this study reflect calpain digestion, however, remains to be established.
IRS-1 levels were highly regulated by estrogen in MCF-7 cells, and,
overall, the dose response curve for the induction of IRS-1 by
estradiol and the concentrations of the two antiestrogens, tamoxifen
and ICI 182,780, required to inhibit the effects of estradiol are
consistent with this effect being mediated by the estrogen receptor.
Although tamoxifen is generally considered to be a partial estrogen
agonist, we observed no estrogen agonist activity of tamoxifen on IRS-1
expression. It is recognized, however, that the estrogen agonist
activity of tamoxifen varies with the response being measured (48) and
that estrogen-responsive genes have been identified that are induced
minimally by tamoxifen (49).
These results are, at first sight, in contrast to those of Guvakova and
Surmacz (47) who reported that tamoxifen, although reducing the amount
of tyrosine-phosphorylated IRS-1, had no effect on IRS-1 protein levels
in MCF-7 breast cancer. Our results are, however, consistent with those
of Guvakova and Surmacz (47) in that we found no estrogen agonist or
antagonist effect of tamoxifen at the concentration used in their study
(10 nM). We did find, however, that higher concentrations
of tamoxifen inhibit the induction of IRS-1 by estradiol.
The main impetus for studying the effect of estradiol on IRS-1
expression was to explain how estrogens can increase the response of
estrogen-responsive breast cancer cell lines to IGFs. Although IGFs
alone stimulate the proliferation of breast cancer cells, the
stimulation is markedly increased by estrogen in hormone-responsive breast cancer cells (6-8). Binding studies and dose response experiments had suggested that the increased responsiveness was mediated via the type-I IGF receptor, and experiments in breast cancer
cells and other cell types had suggested that estrogens can increase
the proliferative response to insulin-like growth factors by increasing
type I IGF receptor levels (7).
To investigate this hypothesis, Daws et al. (20) used a
retroviral expression vector to produce MCF-7 cells that constitutively overexpressed the type-I IGF receptor. The cells overexpressing the
type-I IGF receptor showed the same magnitude of response to IGF-I as
the parental cells, suggesting that the level of the type I IGF
receptor does not control the response of breast cancer cells to IGFs.
In addition, estrogen increased the response of both the parental and
transfected cells to IGFs, confirming that the increased response of
MCF-7 cells to IGFs in the presence of estradiol does not result from
the induction of the receptor by estradiol.
The results of the present study provide an alternative explanation for
the increased response of breast cancer cells to IGFs in the presence
of estrogen. We have shown that IRS-1 is induced by estrogen and
suggest that the induction of IRS-1 by estrogen could account for the
increased response to IGFs in the presence of this steroid. Although
not examining the estrogen regulation of IRS-1 expression by estradiol,
the data of Surmacz and Burgaud (50) are consistent with this model.
Surmacz and Burgaud derived MCF-7 clones overexpressing IRS-1 and
showed a loss of estrogen dependence for growth. They also showed that
IRS-1 antisense oligonucleotides completely inhibited the growth of
normal MCF-7 cells as well as cells overexpressing IRS-1, again
emphasizing the importance of this protein in estrogen-responsive cell proliferation.
Estrogen regulation of other downstream signaling molecules apart from
IRS-1 may also be involved in mediating the effects of estrogen on the
responsiveness of cells to IGFs. Although we have not analyzed all
signaling molecules in this pathway, we investigated the effect of
estrogen on three other members of the IRS family. The lack of effect
of estrogen on IRS-2, -3, and 4 suggests that estrogen mediates its
effects by regulating a limited repertoire of signaling molecules, and
the magnitude of the induction of IRS-1 suggests that this molecule may
be very important in mediating the effects of estrogen on the
responsiveness of these cells to IGFs.
The importance of IRS-1 in mediating the effects of IGFs and estradiol
suggests that measurement of IRS-1 expression may be of clinical
importance in predicting the proliferative behavior of breast tumors.
There has been only one study to date (27) on the prognostic value of
IRS-1 expression in breast cancer, and this measured total IRS-1,
rather than tyrosine-phosphorylated IRS-1, in a series of 195 node-negative primary cancers. Consistent with the view that IRS-1
expression is regulated by estrogen, IRS-1 expression was positively
correlated with estrogen receptor levels. Importantly, IRS-1 expression
was associated with a poor prognosis, and this was most pronounced in
small tumors. Although these findings are consistent with the view that
IRS-1 is an important molecule in breast cancer cell growth, a stronger
association may have been found if phosphorylated IRS-1,
i.e. the active form of IRS-1, had been measured, because
this may more accurately reflect the activity of the estrogen/IGF pathways.
We thank Professor C. R. Kahn for the
plasmid phIRS-1-5.
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed. Tel.: 191-222-6000, Ext. 7200; Fax: 191-222-8100; E-mail: b.r.westley@ncl.ac.uk.
The abbreviations used are:
IGF, insulin-like
growth factor;
IRS-1, insulin receptor substrate-1;
kb, kilobase(s).
Insulin Receptor Substrate-1 Expression Is Regulated by Estrogen
in the MCF-7 Human Breast Cancer Cell Line*
,
ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and two 
subunits (9). Binding of the growth factor activates tyrosine kinase
activity in the C terminus of the subunit. The activated tyrosine
kinase of the receptor then phosphorylates a number of intracelluar
signaling molecules, of which insulin receptor substrate 1 (10, 11) is
a key signaling molecule involved in mediating the proliferative
response. IRS-1 acts as a multisite docking protein that links multiple
downstream signaling pathways by binding to the SH2 domains of a
variety of signaling molecules, including Syp phosphotyrosine
phosphatase (12, 13), phosphatidylinositol 3'-kinase (14), Fyn tyrosine
kinase (15), and the adapter proteins Grb-2 (16), Nck (17), and Crk
(18).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
70 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Expression of IRS-1 mRNA in breast cancer
cell lines. Northern transfer of BT20, Hs578T, MDA MB-231, MCF-7,
ZR75-B, EFF-3, EFM-19, and T47D human breast cancer cell line RNA was
hybridized with 32P-labeled IRS-1 cDNA. BT20, Hs578T,
and MDA MB-231 cells are estrogen receptor-negative, whereas the rest
are estrogen receptor-positive. The positions of 28 and 18 S ribosomal
RNA are shown on the left, and the sizes of the IRS-1
mRNAs are shown on the right.
9 M estradiol. RNA was then extracted for
the measurement IRS-1 mRNA expression. From the autoradiograph
shown in Fig. 2A, it is clear
that there is a dramatic induction in the levels of IRS-1 mRNA by
estradiol and that the effect is visible after only 4 h treatment.
The intensity of hybridization was quantified by scanning densitometry
(Fig. 2B), which showed that IRS-1 mRNA levels were
increased more than 2-fold after only 4 h of estradiol treatment
and continued to increase with time so that after 4 days IRS-1 mRNA
levels were more than 20-fold higher in estrogen treated compared with
control cells. IRS-1 mRNA levels started to plateau between 4 and 6 days.
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Fig. 2.
Time course of the induction of IRS-1
mRNA and protein by estradiol. MCF-7 cells were withdrawn from
the steroids present in routine culture medium for 6 days and then
treated for varying lengths of time with 10 9
M estradiol. RNA and proteins were extracted, and the
relative levels of IRS-1 mRNA and protein were detected by Northern
and Western transfer analysis, respectively. A, typical
Northern blot showing increased IRS-1 mRNA levels with length of
estradiol treatment. The position of 28 S rRNA is shown on the
left, and the sizes of the IRS-1 mRNAs are shown on the
right. B, graph showing the increase in IRS-1
mRNA with time of treatment. Each point represents the mean of two experiments. C,
typical Western transfer showing increased IRS-1 protein with time of
treatment with estradiol. The positions of the size markers are shown
on the left, and the approximate molecular masses of IRS-1
(185 kDa) and two smaller immunoreactive proteins are shown on the
right. D, graph showing the increase in IRS-1
protein with time of treatment. Each point represents the mean of two
experiments.
10 M estradiol and
higher. The induction of IRS-1 protein showed a similar dose response
curve to that of the mRNA with low levels of IRS-1 being detected
at 1013 and 1012 M estradiol
and maximal induction occurring at 1010 M
estradiol and higher (Fig. 3B). These dose response
experiments therefore showed that the induction of both IRS-1 mRNA
and protein was very sensitive to estradiol consistent with this effect
being mediated by the estrogen receptor. The two lower molecular mass forms of IRS-1 were less apparent in this experiment, which is consistent with the protein profile seen in time course experiment after 3 days of estradiol treatment.
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Fig. 3.
Dose response effect of estrogen on IRS-1
expression in MCF-7 cells. MCF-7 cells were withdrawn from the
steroids present in the routine culture medium for 6 days and then
treated with various concentrations of estradiol for 3 days. RNA and
proteins were extracted, and the relative levels of IRS-1 mRNA and
protein detected by Northern and Western transfer analysis,
respectively. A, representative autoradiograph showing the
induction of IRS-1 mRNA by different concentrations of estradiol.
The positions of 28 and 18 S ribosomal RNA are shown on the
left, and the sizes of the IRS-1 mRNAs are shown on the
right. B, representative Western transfer showing
increased IRS-1 protein by different concentrations of estradiol. The
positions of the size markers are shown on the left, and the
approximate molecular masses of IRS-1 (185 kDa) and a smaller
immunoreactive protein are shown on the right.
10 M estradiol, a
concentration that induces IRS-1 mRNA to maximal levels. Tamoxifen
alone had negligible estrogen agonist activity over a wide range of
concentrations (1010-105 M)
but inhibited the induction of IRS-1 mRNA by 2 × 1010 at concentrations above 107
M. The concentration of tamoxifen required for half maximal
inhibition was approximately 106 M (Fig.
4).
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Fig. 4.
Estrogen agonist and antagonist effects of
tamoxifen and ICI 182,780 on IRS-1 mRNA. Withdrawn MCF-7 cells
were treated with various concentrations of tamoxifen (A) or
ICI 182,780 (B) in the presence ( 
) or absence (
) of
2 × 1010 M estradiol for 3 days. RNA
was extracted, and the relative levels of IRS-1 mRNA were measured
by image analysis of autoradiographs resulting from hybridization to
Northern transfers. Each point represents the mean from two
experiments.
10 M estradiol. ICI 182,780 alone
also had negligible estrogen agonist activity over a wide range of
concentrations (1010-105 M)
but was somewhat more potent than tamoxifen in inhibiting the induction
of IRS-1 by estradiol. ICI 182,780 inhibited the induction of IRS-1
mRNA at concentrations above 109 M, and
the concentration required for half-maximal inhibition was
approximately 7 × 109 M. The
concentrations of the two antiestrogens required to inhibit the
induction of IRS-1 were consistent with the known antiestrogenic potencies of these two estrogen antagonists. Both tamoxifen and ICI
182,780 also inhibited the induction of IRS-1 protein levels by
estradiol (data not shown).
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Fig. 5.
Effects of estradiol, IGF-I, and insulin on
tyrosine phosphorylation of IRS-1. A, MCF-7 cells were
withdrawn from the effects of estrogen and then grown in withdrawal
medium alone (C), medium supplemented with 1 µg/ml insulin
(Ins), 10 9 M estradiol
(E2), or medium supplemented with 1 µg/ml insulin
and 109 M estradiol together (Ins + E2). B, MCF-7 cells were withdrawn from the
effects of estrogen by culturing in withdrawal medium and then
stimulated with 1 µg/ml insulin (Ins) or 50 ng/ml IGF-I
(IGF-I) for 5 min in binding buffer. Protein extracts were
prepared and tyrosine-phosphorylated IRS-1 measured as described under
"Materials and Methods."
View larger version (15K):
[in a new window]
Fig. 6.
Effects of estradiol on IRS-1, IRS-2, IRS-3,
and IRS-4 expression in MCF-7 breast cancer cells. MCF-7 cells
were withdrawn from the steroids present in the routine culture medium
for 6 days and then treated with estradiol (10 9
M) for 3 days. Cell lysates were prepared, and the relative
levels of IRS-1, IRS-2, IRS-3, and IRS-4 proteins were measured by
Western transfer analysis. The autoradiographs on the left-hand
side of the figure show the results of one representative
experiment, whereas the histograms on the right-hand side
show the mean and standard errors of three experiments. For the
estrogen-treated cells, samples from the three experiments were
analyzed together on one gel, and the levels of expression were
expressed as percentages of the experiment showing the highest level of
expression.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENT
FOOTNOTES
Supported by funds from the University of Newcastle-upon-Tyne and
the North of England Cancer Research Campaign.
ABBREVIATIONS
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INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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