Carcinogenesis vol.21 no.5 pp.881–886, 2000
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) inhibits growth factor
withdrawal-induced apoptosis in the human mammary epithelial
cell line, MCF-10A
John W.Davis II, Karla Melendez, Virginia M.Salas,
Fredine T.Lauer and Scott W.Burchiel1
Toxicology Program, The University of New Mexico College of Pharmacy,
Albuquerque, NM 87131, USA
1To
whom correspondence should be addressed
Email: [email protected]
Previous studies have demonstrated that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increases cell recovery in the
human mammary epithelial cell line MCF-10A grown
under growth factor-restricted conditions. TCDD was also
found to mimic growth factor signaling pathways by stimulating the tyrosine phosphorylation of numerous effector
molecules, and increased phosphatidylinositol 3-kinase
(PI3K) activity in the absence of exogenously added growth
factors. In the present studies, we have expanded on these
initial results to show that TCDD (3–30 nM) increases cell
recovery on days 2–6 by as much as 80% when insulin or
epidermal growth factor (EGF) was removed from the
media. The mechanism for this effect appears to be complex
as TCDD inhibited apoptosis stimulated by EGF, or EGF
and insulin, withdrawal by almost 80% as determined by
Annexin V binding. However, withdrawal of insulin alone
did not induce apoptosis even though TCDD did increase
cell number in its absence. These results were corroborated
by immunoblot analysis of poly(ADP-ribose) polymerase
cleavage. Since TCDD stimulates PI3K activity, the
phosphorylation status of Akt, a serine/threonine kinase
that mediates PI3K-dependent inhibition of apoptosis, was
examined. Immunoblot analysis revealed that TCDD causes
a transient increase in the phosphorylated form of Akt
that peaks at 6 h and disappears by 12 h. It appears that
EGF stimulates an anti-apoptotic pathway, while insulin
signals a pro-mitogenic pathway. By stimulating or mimicking one or both of these pathways TCDD may alter tightly
regulated growth pathways in the MCF-10A cell line.
Introduction
Cancer incidence has increased in the Western world during
the latter half of the twentieth century, with the most notable
increase in breast cancer. One in 10 women in the USA
develop breast cancer and over 40 000 per year are expected
to die from the disease (1–3). Breast cancer is a complex
disease and its etiology remains a mystery. It has been
postulated that environmental pollutants may be involved. The
exposure of women to ubiquitous environmental pollutants
such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other
Abbreviations: AhR, aryl hydrocarbon receptor; DMSO, dimethyl sulfoxide;
EGF, epidermal growth factor; IGF-I, insulin-like growth factor-I; IGF-IR,
IGF-I receptor; PARP, poly(ADP-ribose) polymerase; PI, propidium iodide;
PI3K, phosphatidylinsitol 3-kinase; PS, phosphatidylserine; SFH, EGF and
insulin-deficient media; SFHE, insulin-deficient media; SFIH, EGF-deficient
media; SFIHE, complete growth media; TCDD, 2,3,7,8-tetrachlorodibenzop-dioxin.
© Oxford University Press
halogenated aromatic hydrocarbons have been well documented (4,5). Moreover, these chemicals are resistant to
metabolic breakdown, extremely lipophilic in nature, and can
be stored in human breast fat and milk (5,6). However, the
link between exposure to environmental contaminants and
breast cancer has yet to be established. Exposure of women
to TCDD from an industrial accident in Seveso, Italy did not
result in increased incidence of breast cancer (4), while other
reports suggest that exposure to environmental contaminants
may increase risk of breast cancer (7).
Activation of growth factor receptors and their cognate
signaling pathways is a potential mechanism of mammary
tumor promotion and progression (reviewed in ref. 8). Previous
studies have demonstrated that TCDD mimics growth factor
stimulation and cell growth in the human mammary epithelial
cell line, MCF-10A (9). In the absence of insulin, TCDD
increased total tyrosine phosphorylation, as well as tyrosine
phosphorylation of the insulin-like growth factor-I receptor β
(IGF-IRβ), insulin receptor substrate-1 and Shc. In addition,
TCDD treatment led to an increase in phosphatidylinositol 3kinase (PI3K) activity, and TCDD’s growth stimulatory effects
upon co-treatment with the PI3K inhibitor LY29004 were
attenuated. Finally, Shc tyrosine phosphorylation is not unique
to IGF signaling. Shc can also bind to phosphotyrosine residues
on the epidermal growth factor receptor (EGFR) resulting in
Ras activation through Grb2–Sos interactions and stimulation
of the mitogen activated protein kinase pathways (10). These
results argue that TCDD could act as a mammary tumor
promoter by over-stimulating epidermal growth factor (EGF)
and insulin-like growth factor-I (IGF-I) signal transduction
pathways.
The inhibition of apoptosis is widely accepted as one
possible mechanism of tumor promotion/progression (11 and
references therein) and PI3K activation has been demonstrated
to provide a powerful anti-apoptotic stimuli (12). PI3K in turn
activates another serine/threonine kinase, Akt (13,14). Akt,
also known as protein kinase B, is activated by many growth
factors, including EGF and IGF-I (15), and has been observed
to be up-regulated in ovarian and breast carcinomas (16).
Activated Akt protects against apoptosis by phosphorylating
Bad, a member of the Bcl-2 family of proteins (17), which in
turn prevents Bad from heterodimerizing with Bcl-2 or BclXL (18).
The present studies were initiated to characterize the ability
of TCDD to increase cell number of the human mammary
epithelial cell line, MCF-10A, in the absence of exogenously
added growth factors. Previous studies examining the effects
of TCDD on mammary epithelial cell growth have used tumorderived cell lines that over-express the estrogen receptor, a
complicating factor given TCDD’s anti-estrogenic activity
(19). MCF-10A cells are an estrogen receptor-negative cell
line, and make an attractive model for studying growth factor
pathways of human mammary epithelial cells because they
exhibit a near normal phenotype. Similar to primary cultures
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J.W.Davis II et al.
of normal human mammary epithelial cells (HMECs), the
MCF-10A cell line does not grow tumors when injected in
nude mice, and has a strict requirement for EGF and IGF-I
for growth in serum-free media (20). We hypothesized that
removal of growth factors would induce apoptosis in MCF10A cells and that TCDD would act to mimic growth factor
signaling and inhibit apoptosis. Treatment with 30 nM TCDD
was able to increase cell recovery in the absence of EGF and/
or insulin for up to 6 days. Removal of EGF induced apoptosis
and TCDD was able to attenuate the induction of apoptosis.
Interestingly, withdrawal of insulin did not induce apoptosis.
In addition, TCDD treatment resulted in a transient increase
in phosphorylated Akt under conditions that lead to decreased
apoptosis. These data suggest that TCDD is able to mimic
multiple growth factor pathways in the MCF-10A cell line.
EGF appears to inhibit apoptosis while IGF signaling produces
a mitogenic response in MCF-10A cells. Therefore, it appears
as though TCDD possesses weak mitogenic activity and is
also able to protect cells from death. These pleiotropic effects
of TCDD on MCF-10A cells under growth factor-defined
conditions may have consequences in human mammary tumor
promotion and progression.
Materials and methods
Chemicals and reagents
All chemicals were purchased from Sigma (St Louis, MO), unless otherwise
indicated. TCDD was obtained from Cambridge Isotopes Laboratories
(Andover, MD) at ⬎99% purity. TCDD was maintained as a stock solution
(300 µM) in anhydrous tissue culture grade dimethyl sulfoxide (DMSO). The
final concentration of DMSO in all experiments was 0.1%.
MCF-10A cell culture
MCF-10A cells, a non-transforming, estrogen receptor-negative human
mammary epithelial cell line that exhibits anchorage- and growth factordependent growth were a gift from Dr Stephen P.Ethier (University of
Michigan, Ann Arbor, MI). Cells were grown on Vitrogen-coated (Collagen
Corp., Palo Alto, CA) 100⫻20 mm dishes (Corning Glass, Corning, NY) in
a 10% CO2 incubator and passed every 4–5 days (~75% confluent). Cells were
maintained in a serum-free media developed by Ethier et al. (21,22). Complete
media consisted of Ham’s F-12 (JRH Biosciences, Lenexa, KS) supplemented
with 1 mg/ml bovine serum albumin (JRH Biosciences), 1 µg/ml hydrocortisone, 10 ng/ml epidermal growth factor, 5 µg/ml insulin, 5 µg/ml
gentamycin (Gibco BRL, Grand Island, NY), 5 µg/ml transferrin, 50 µM
sodium selenite, 10 µM 3,3⬘,5-triiodo-L-thryonine, 5 mM ethanolamine, 10
mM HEPES (Gibco BRL) and 5 µg/ml fungizone (Gibco BRL).
Cell counting assay
MCF-10A cells were plated on Vitrogen-coated 6-well plates (Corning Glass)
at 1⫻105 cells/well in complete media plus 2% fetal bovine serum to allow
for attachment. After 1 day, plating media was removed and cells were
allowed to equilibrate for 1 day in a serum-free (SF) media supplemented
with insulin (I), hydrocortisone (H) and EGF (E) for complete media (SFIHE).
Twenty-four hours later, cells were switched to one of four growth factordefined conditions: SFIHE, growth factor-free (SFH), insulin-deficient (SFHE)
and EGF-deficient (SFIH) media. The next day media was removed and cells
were treated with TCDD in the specified media. Cells were grown for 6 days
with media and treatment changed every 48 h, harvested and total nuclei
determined using a Coulter Counter (23).
Detection of apoptosis
MCF-10A cells were plated and treated as detailed above. Apoptosis was
determined by flow cytometry using a kit that employs Annexin V conjugated
to FITC (PharMingen, San Diego, CA). One of the early changes occurring
during apoptosis is a change in the plasma membrane whereby phosphatidylserine (PS) is transposed from the inner to the outer surface of the plasma
membrane (24,25). Annexin V is a 35–36 kDa Ca2⫹-dependent phospholipidbinding protein that has a high affinity for PS. To distinguish between
apoptosis and necrosis, cells that stained for propidium iodide (PI) or PI and
Annexin V were determined to be necrotic and not counted as apoptotic.
Detection of poly(ADP-ribose) polymerase (PARP) cleavage
Cells were treated under growth factor-defined conditions with 30 nM TCDD
(or 0.1% DMSO) for 18 h. Following treatment, cells were rinsed twice with
882
cold PBS and lysed on ice for 10 min in RIPA⫹⫹ buffer [50 mM Tris pH
8.0; 150 mM NaCl; 1% Triton X-100; 0.5% sodium deoxycholate; 0.1% SDS;
200 µM phenylmethylsulfonyl fluoride; protease inhibitor cocktail purchased
from Boehringer Mannheim (Indianapolis, IN) and 200 µM sodium orthovanadate]. Cell debris was pelleted for 10 min at 12 000 g in a refrigerated
microcentrifuge, and protein content determined by micro BCA assay (Pierce,
Rockford, IL). SDS–PAGE was performed according to Laemmli (26). Cell
lysates (30 µg) were diluted with 5⫻ sample buffer, boiled, separated on 12%
Tris–glycine gels and transferred overnight to PolyScreen® PVDF membrane
(NEN Life Sciences, Boston, MA). Intact and cleaved PARP were detected
with a monoclonal antibody that recognizes both forms (PharmMingen) and
visualized using NEN⬘s Renaissance® western blot chemiluminescence
reagent.
Detection of serine phosphorylated Akt
Cells were treated and harvested as described above for PARP determination.
Cell lysates (100 µg) were diluted with 5⫻ sample buffer, boiled, separated
on 12% Tris–glycine gels and transferred overnight to PolyScreen® PVDF
membrane (NEN Life Sciences). Total and Akt phosphorylated at serine 473
were detected using New England BioLabs’ (Beverly, MA) PhosphoPlus®
Akt (Ser473) antibody kit as per supplied directions and visualized using
chemiluminescence. To quantitate the extent of phosphorylation, chemiluminescent films were scanned and band densities determined using Kodak Digital
Science Image System 440 scanner and software (Rochester, NY). PhosphoAkt band densities were normalized to the density of the total Akt band.
Statistical analysis
Data were analyzed for statistical difference (P ⬍ 0.05) between control and
treated groups using SigmaStat statistical software (Jandel Scientific, San
Rafael, CA). ANOVA followed by Dunnett’s t-tests were performed on
sample means.
Results
TCDD increases MCF-10A cell number
Previously published results in our laboratory have demonstrated that TCDD increases MCF-10A cell number in the
absence of exogenously added growth factors (9). The present
studies were undertaken to further characterize these initial
results. MCF-10A cells were plated at 105 cells/well on
Vitrogen-coated 6-well plates in a serum-free (SF) media
developed by Ethier et al. (21,22) supplemented with insulin
(I), hydrocortisone (H) and EGF (E). Cells were treated with
30 nM TCDD (or 0.1% DMSO) for 6 days under one of four
growth factor-defined conditions: SFIHE, SFH, SFIH or SFHE,
harvested and counted on days 2, 4 and 6 as described in
Materials and methods. Treatment of MCF-10A cells grown
in SFIHE with TCDD had no affect on cell number (Figure
1, upper left panel). Removal of insulin and EGF from the
growth media resulted in an initial suppression of cell growth
followed by accelerated cell death. TCDD appeared to decrease
the rate of cell death at 2 and 4 days (Figure 1, upper right
panel). Removal of EGF or insulin from the media gave similar
results. However, in both cases TCDD appeared to stimulate
cell growth at later time points (Figure 1, lower panels).
Concentration–response analyses yielded similar observations
(Figure 2). While TCDD had no affect when cells were grown
for 6 days in SFIHE or SFH media, concentrations as low as
3 nM TCDD increased cell recovery in the absence of EGF
or insulin. The results suggest that insulin and EGF are required
for MCF-10A growth and/or suppression of cell death. TCDD
appears to mimic the effects of these growth factors. More
importantly, TCDD was able to alter cell growth at 3 nM,
which is in the range of aryl hydrocarbon receptor (AhR)
saturation reported for other tissues (27), suggesting a role for
AhR for mediating the effects of TCDD on growth regulation
in MCF-10A cells.
TCDD inhibits EGF withdrawal-induced apoptosis
We have shown that TCDD is able to partially reverse MCF10A cell loss due to growth factor withdrawal (Figures 1 and
TCDD inhibits apoptosis in human mammary epithelium
Fig. 1. TCDD increases MCF-10A cell number. MCF-10A cells were plated
at 1⫻105 cells/well in collagen-coated 6-well plates. One day after plating
media was removed and cells were allowed to equilibrate for 1 day in a
serum-free media supplemented with insulin, hydrocortisone and EGF for
complete media (SFIHE). Twenty-four hours later, cells were switched to
one of four growth factor-defined conditions: SFIHE, growth factor-free
(SFH), insulin-deficient (SFHE) and EGF-deficient (SFIH) media. The next
day media was removed and cells were treated with 30 nM TCDD (or 0.1%
DMSO) in the specified media (Day 0). Cells were treated for 6 days, re-fed
with fresh treatment every 2 days and total nuclei determined at 48 h
intervals using a Coulter Counter. Results shown are the means ⫾ SE for
cell counts obtained in triplicate cultures from a representative experiment
in which significant differences were observed in at least three different
experiments. #Significantly different from DMSO control (P ⬍ 0.05).
Fig. 2. Concentration–response analysis of TCDD-mediated MCF-10A cell
recovery. MCF-10A cells were plated and cultured as indicated in Figure 1.
Cells were treated with TCDD (0–300 nM) in DMSO for 6 days, re-fed
with fresh treatment every 2 days and total nuclei determined by Coulter
Counting. Results shown are the means ⫾ SE for cell counts obtained in
triplicate cultures from a representative experiment in which significant
differences were observed in at least three different experiments.
#Significantly different from DMSO control (P ⬍ 0.05).
2). In addition, when MCF-10A cells were examined under a
light microscope after 48 h without EGF or insulin, there was
a substantial amount of cell loss and irregular shaped cells,
Fig. 3. TCDD inhibits epidermal growth factor withdrawal-induced
apoptosis in MCF-10A cells. MCF-10A cells were plated, cultured and
treated with 30 nM TCDD for 4 days as indicated in Figure 1. Induction of
apoptosis was determined on days 2, 3 and 4 using flow cytometry to detect
FITC-conjugated Annexin V binding to cells. Cells stained with PI were
gated out so that only viable cells were analyzed. Results shown are the
means ⫾ SE for cell stained for Annexin V but not PI. Data were obtained
in triplicate cultures from a representative experiment repeated on at least
three occasions. #Significantly different from DMSO control (P ⬍ 0.05).
whereas 30 nM TCDD appeared to protect in the absence of
EGF and/or insulin (data not shown). To confirm our previous
observation that TCDD inhibits growth factor withdrawalinduced apoptosis, MCF-10A cells were cultured and grown
as indicated in Figure 1. At the indicated times cells were
analyzed for apoptosis, as determined by Annexin V binding
using flow cytometry. One of the early changes occurring
during apoptosis is a change in the plasma membrane whereby
PS is transposed from the inner to the outer side of the plasma
membrane (24,25). Annexin V is a 35–36 kDa Ca2⫹-dependent
phospholipid-binding protein that has a high affinity for PS.
To distinguish between apoptosis and necrosis, cells that
stained for PI, or PI and Annexin V, were determined to be
necrotic and not counted as apoptotic. Withdrawal of EGF
(SFH and SFIH media) resulted in the induction of apoptosis
in ~20% of the cells, and treatment with 30 nM TCDD
inhibited apoptosis by as much as 66% when compared with
DMSO controls (Figure 3, upper right and lower left panel).
Interestingly, withdrawal of insulin did not induce apoptosis
(Figure 3, lower right panel) even though it did decrease cell
number (Figure 1). These results correlated with the observation
that TCDD increased cell viability in the absence of EGF (as
determined by those cells that did not stain positive for
Annexin V or PI, data not shown). The results of a concentration–response analysis for the inhibition of apoptosis yielded
results similar to those observed for cell recovery. TCDD
suppresses apoptosis induced by EGF at concentrations as low
as 3 nM (Figure 4). These results demonstrate that EGF
signaling produces an anti-apoptotic signal in MCF-10A cells,
and that TCDD is able to mimic EGF and protect against
EGF withdrawal-induced apoptosis. Furthermore, it appears as
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J.W.Davis II et al.
Fig. 4. Concentration–response analysis of TCDD-mediated inhibition of
apoptosis in MCF-10A cells. MCF-10A cells were plated and cultured as
indicated in Figure 1. Cells were treated with TCDD (0–300 nM) in DMSO
for 3 days and apoptosis determined as in Figure 3. Results shown are the
means ⫾ SE for cell stained for Annexin V but not PI. Data were obtained
in triplicate cultures from a representative experiment repeated on at least
three occasions. #Significantly different from DMSO control (P ⬍ 0.05).
Fig. 5. TCDD inhibits EGF withdrawal-induced PARP cleavage in MCF10A cells. Sub-confluent cultures were grown for 24 h in SFIHE, SFIH or
SFHE (as indicated) and then treated for 18 h with 30 nM TCDD (or 0.1%
DMSO) as described in Materials and methods. Total cell lysate proteins
(30 µg) were resolved on a 12% polyacrylamide gel, transferred to a PVDF
membrane and probed with an anti-PARP antibody. The numbers and
arrows on the left of the gel indicate full-length (116 kDa) and cleaved
(85 kDa) PARP. The blot is representative of three separate experiments.
though insulin (used as a surrogate for IGF-I) produces a promitogenic signal as its withdrawal results in a decrease in cell
recovery, without an induction of apoptosis, and TCDD mimics
insulin’s effects.
TCDD inhibits EGF withdrawal-induced PARP cleavage
Treatment of MCF-10A cells with TCDD suppresses apoptosis
induced by EGF withdrawal (Figures 3 and 4). To further
characterize these observations cells were analyzed for PARP
cleavage by western blot as another marker of apoptosis.
PARP is a 116 kDa nuclear chromatin-associated enzyme that
catalyzes the transfer of ADP-ribose units from NAD⫹ to a
variety of nuclear proteins. During early apoptosis, caspases
become active and cleave PARP from the full-length form to
an 85 kDa fragment (28). Although the role of PARP in
apoptosis is unknown it is considered a marker for the induction
of apoptosis.
MCF-10A cells were grown in the absence of EGF (SFIH)
or insulin (SFHE) for 24 h, followed by treatment with 30 nM
TCDD (or 0.1% DMSO) for 18 h. Cell lysates were then
analyzed for PARP cleavage using an antibody that recognizes
both full-length and cleaved PARP (Figure 5). As expected,
cleaved PARP was not detected in cells grown in SFIHE (lane
1). However, the 85 kDa fragment was detected in DMSO884
Fig. 6. TCDD-mediated Akt phosphorylation in MCF-10A cells. (A) Subconfluent cultures were grown for 18 h in SFIH, followed by treatment with
30 nM TCDD (T) or 0.1% DMSO (D) for the indicated times. Also
included were untreated cells (unt) and cells treated with 10 ng/ml EGF (E)
for 15 min. Total cell lysate proteins (100 µg) were resolved on a 12%
polyacrylamide gel, transferred to a PVDF membrane and probed with an
anti-phospho Ser473-Akt antibody (upper panel) or anti-total Akt antibody
(lower panel). (B) In a separate experiment, triplicate cultures were treated
with 30 nM TCDD (or 0.1% DMSO) for 6 or 8 h. Total cell lysates were
analyzed as in (A) for phospho-Ser473 Akt and total Akt. Chemiluminescent
films were scanned and band densities determined using Kodak Digital
Science Image System 440 scanner and software. Phosph-Akt band densities
were normalized to the density of the total Akt band. Results shown are the
means ⫾ SE and the P-values were obtained as indicated in Materials and
methods.
treated cells grown in the absence of EGF (SFIH) and
TCDD suppressed PARP cleavage (Figure 5, lanes 2 and 3,
respectively). Finally, removal of insulin had no effect on
PARP cleavage (Figure 5, lanes 4 and 5). These results confirm
that TCDD reverses EGF withdrawal-induced apoptosis in
MCF-10A cells.
TCDD-mediated Akt phosphorylation correlates with inhibition
of apoptosis
We have demonstrated by Annexin V staining and PARP
cleavage that removal of EGF stimulates apoptosis in MCF10A cells and that TCDD protects against apoptosis (Figures
3–5). In addition, previously published results have indicated
that TCDD stimulates PI3K under insulin-deficient conditions
(9). PI3K signaling can activate other downstream kinases
such as Akt through PDK1-dependent phosphorylation (14),
which in turn may result in an inhibition of apoptosis (29).
The ability of TCDD to increase phosphorylation of Akt was
examined in the absence of EGF or insulin using an antibody
that recognizes Akt phosphorylated at serine 473. MCF-10A
cells were grown and treated as described previously. Treatment
of cells with 30 nM TCDD transiently increased Akt phosphorylation, the maximal effect was observed at 6 h and disappeared by 12 h (Figure 6A), this is similar to EGF which
increased Akt phosphorylation after 15 min. To confirm this
observation, cells were cultured overnight in SFIH media and
then treated in triplicate with 30 nM TCDD (or 0.1% DMSO)
for 6 or 8 h. Total and phospho-Akt bands were quantitated
TCDD inhibits apoptosis in human mammary epithelium
by densitometric analysis as described in Materials and
methods. As expected by visual examination of the blot in
Figure 6A, TCDD significantly increased Akt phosphorylation
at 6 h (Figure 6B). These results suggest that TCDD inhibits
apoptosis in an EGF-like manner through the phosphorylation
of Akt.
Discussion
Recent observations indicate that TCDD increases cell recovery
and alters tightly controlled growth regulatory pathways in the
human mammary epithelial cell line, MCF-10A (9). Activation
of growth factor receptors, particularly EGFR and IGF-IR,
and their cognate signaling pathways, is a potential mechanism
of mammary tumor promotion and progression (8). The inhibition of apoptosis by growth factor signaling pathways is one
possible mechanism of tumor promotion/progression (11).
TCDD is able to act as a rodent tumor promoter by inhibiting
apoptosis in initiated livers (30,31). Therefore, we attempted
to characterize the ability of TCDD to alter cell growth in
the human mammary epithelial cell line MCF10A, with the
hypothesis that TCDD increases cell number by inhibiting
apoptosis in this cell line.
TCDD (30 nM) increased MCF-10A recovery in the absence
of EGF and/or insulin (SFIH, SFHE and SFH media) for up
to 6 days (Figure 1). The ability of TCDD to positively
regulate cell growth in human mammary epithelial cells differs
from previously published reports. TCDD was found to inhibit
IGF signaling exerting an anti-proliferative effect in MCF-7
cells a malignant human mammary epithelial cell line (32,33).
However, MCF-7 is a malignant cell line that grows independently of added growth factors in culture, and forms tumors
when injected into nude mice. The MCF-10A cell line is an
attractive model because it exhibits a normal phenotype and,
under serum-free conditions, its proliferation is dependent upon
addition of EGF and insulin (20). Furthermore, preliminary data
demonstrate that TCDD increases cell number in primary
cultures of HMECs under similar growth factor-defined conditions (S.L.Tannheimer and S.W.Burchiel, unpublished data).
The mechanism by which TCDD exerts its effects in MCF10A cells was further investigated by concentration–response
analysis (Figure 2). After 6 days of treatment, TCDD was able
to replace EGF or insulin (SFIH and SFHE media, respectively)
at concentrations as low as 3 nM. In addition, 0.3 nM TCDD
also increased cell number, although it was not significantly
different from control. These results are interesting because
TCDD-mediated cell growth occurs at a concentration range
(0.3–3.0 nM) that would suggest AhR occupation (34,35).
Moreover, a recent report from our lab demonstrates that MCF10A cells express AhR and its heterodimerization partner,
AhR nuclear translocator (36). These observations suggest that
TCDD could exert its growth regulatory effects in MCF-10A
cells in an AhR-dependent manner. Further experiments are
underway in an effort to address this question.
In these studies, we have clearly demonstrated that TCDD
is able to mimic EGF or insulin and support MCF-10A growth.
Since TCDD is able to induce an insulin-like signaling pathway
in these cells resulting in an increase in PI3K activity (9),
we hypothesized that TCDD would inhibit the induction of
apoptosis that was due to growth factor withdrawal. As
expected, removal of EGF from the growth media resulted in
an induction of apoptosis (Figure 3, SFH and SFIH media),
while 30 nM TCDD suppressed apoptosis on days 2–4.
However, removal of insulin did not result in apoptosis (Figure
3, SFHE media), even though it is required for optimal cell
growth. EGF and IGF signaling work in concert to drive
proliferation of human mammary epithelial cells. It would
appear that EGF delivers an anti-apoptotic signal while insulin
provides pro-mitogenic stimuli in this cell line. TCDD is
seemingly able to mimic either pathway in a manner that is
similar to neu differentiation factor/heregulin (NDF/HRG),
which has been demonstrated to be a dual specificity growth
factor in human mammary epithelial cells (20). Similar to the
cell recovery data, TCDD at concentrations as low as 3 nM
was able to protect MCF-10A cells from cell death (Figure 4).
TCDD is able to increase PI3K activity in MCF-10A, which
is a potential route for delivery of an anti-apoptotic signal
(12). Since PI3K is known to phosphorylate Akt, thereby
propagating the anti-apoptotic signal (13,14) the ability of
TCDD to increase Akt phosphorylation was investigated.
TCDD treatment transiently increased Akt phosphorylation
under conditions that lead to suppression of cell death (Figure
6). More importantly, TCDD appears to deliver an EGF-like
signal in MCF-10A cells as EGF treatment also resulted in an
increase in Akt phosphorylation (Figure 6) (15). Phosphorylated Akt is thought to mediate an EGF-dependent anti-apoptotic
stimulus by phosphorylating Bad (17), preventing it from
dimerizing with Bcl-2 or Bcl-XL (18).
In summary, TCDD is able to mimic both EGF and insulin
signaling in MCF-10A cells. The end result of this is an
increase in cell number with EGF producing an anti-apoptotic
signal while insulin yields a pro-mitogenic stimulus. The
mechanism by which TCDD exerts an anti-apoptotic effect
(and possibly a pro-mitogenic stimulus) was not resolved in
these studies. TCDD’s ability to suppress apoptosis occurred
at concentrations that suggest an AhR involvement. It is
possible that TCDD is able to regulate MCF-10A growth by
altering the expression of target genes involved in these
pathways. There are numerous examples of TCDD-dependent
alteration of growth regulatory genes, although the exact role
of AhR in mediating the expression of these genes is unknown
(37–40). In MCF-10A cells, TCDD could produce an autocrine
effect by up-regulating the expression of growth factors such
as NDF/HRG, which could deliver a dual signal through both
an EGFR and an IGF-IR pathway, the end result being an
inhibition of apoptosis and a stimulation of mitogenesis.
Further studies are required to fully delineate the mechanism
by which TCDD regulates MCF-10A cell growth, and to
explore signaling pathways by which it may act as a tumor
promoter.
Acknowledgements
The authors would like to thank Dr Laurie G.Hudson for critical evaluation
of the data and her invaluable input. This work was supported by NIEHS
RO1-ES-07259. J.W.D.II was supported by NIEHS 1-F32-ES-05895-01
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Received June 17, 1999; revised January 12, 2000; accepted January 21, 2000