ft. Steroid Biochem. Molec. Biol. Vol. 59, No. 3/4, pp.

271-279, 1996
Pergamon Copyright © 1996 Elsevier Science Ltd. All rights reserved
Printed in Great Britain
PII: S0960-0760(96)00117-3 0960-0760/96 $15.00 + 0.00

T r i i o d o t h y r o n i n e M i m i c s the Effects of
E s t r o g e n in B r e a s t C a n c e r Cell Lines
C61ia R e g i n a N o g u e i r a x a n d M a r i a M i t z i B r e n t a n i 2.
IDepartamento de Quimica, Disciplina de Bioquimica, Instituto de Biocidncias, Universidade Estadual Paulista,
Botucatft, Rubi~o Juniol; S~o Paulo, 18618-000, Brazil and 2Disciplina de Oncologia, Departamento de Radiologia,
Faculdade de Medicina da Universidade de S~o Paulo, S~o Paulo, 01246-903, Brazil

M C F - 7 (estrogen receptor positive m E R +) and M D A - M B - 2 3 1 (estrogen receptor negative m ER-)

are h u m a n breast c a n c e r cell lines w h i c h express f u n c t i o n a l thyroid h o r m o n e receptors ( c - e r b A a l
and c-erb ill) as i n d i c a t e d by s t i m u l a t i o n o f m i t o c h o n d r i a l a - g l y c e r o p h o s p h a t e d e h y d r o g e n a s e . In
M C F - 7 , m i m i c k i n g E2, T3 s t i m u l a t e d gr owt h in a d o s e - d e p e n d e n t (101° M-10 -s M) m a n n e r , i n d u c e d
the expression o f p r o g e s t e r o n e receptor and growth factor TGF~ m R N A s and inhibited that o f TG F fl
m R N A ; T3 also i n c r e a s e d p r o g e s t e r o n e b i n d i n g and LDH5 i s o z y m e activities. N o n e o f these effects
were observed in (ER-) M D A - M B - 2 3 1 cells. 10-6M t a m o x i f e n (TAM) reverted grow t h s t i m u l a t i o n ,
s u p p r e s s e d p r o g e s t e r o n e receptor and T G F a m R N A i n d u c t i o n and restored TGFfl m R N A to control
levels in T 3 - t r e a t e d M C F - 7 cells. T h a t T3 is acting in M C F - 7 cells via its b i n d i n g to E R is suggested
b y th e i m m u n o p r e c i p i t a t i o n o f p r e - b o u n d 12SI-T3 f r o m M C F - 7 nuclear extracts b y an E R - s p e c i f i c
m o n o c l o n a l a n t i b o d y a n d b y t he d i s p l a c e m e n t o f 3 H - e s t r a d i o l b i n d i n g to E R by r a d i o i n e r t T3. C o p y -
r i g h t © 1996 Elsevier Sc i e nc e Ltd.

J. Steroid Biochem. Moh'c. Biol., Vol. 59, No. 3/4, pp. 271-279, 1996

INTRODUCTION Thyroid hormone receptors (TR) have been

Breast development and function is influenced by a
variety of hormones [11. Several studies supported the
role of estrogen as an inducer of breast cell prolifer-
ation [2, 3]. The involvement of this hormone in the
growth of breast cancer has been established. About
30% of breast cancers maintain the estrogenic depen-
dence on growth, and the concentration of estrogen
receptors in malignant breast tissues is an indicator of
their hormonal dependence [4].
Thyroid hormones also seem to stimulate lobular
development and contribute to the differentiation pro-
cess in normal breast [15]. On the other hand, the re-
lationship between breast cancer and thyroid
hormone-related pathologies has been a controversial
topic for the past 30 years. Increased risk and inci-
dence has been correlated with hyperthyroidism or
hypothyroidism as well as with the use of supplements
to treat the latter, reviewed in [6].
*Correspondence to M. M. Brentani, Av. D r Arnaldo, 455 40
andar, S~o Paulo, S.P., Brazil, 01246-903. Tel: +55 (0)11 3061
4011 R470; Fax: +55 (0)11 282 6580.
Received 9 Jan. 1996; accepted 21 M a y 1996.
SBMB 5 9 / 3 ~ B
described in normal mammary cells and in cultured
breast cancer cells, suggesting that such hormones
might be key growth regulators of breast cells [6-10].
T o date there are few studies on the effects of triio-
dothyronine (T3) on the proliferation of human breast
cancer cells [11, 12]. The present work focused on
the comparison between the effects of estradiol (E2)
and T3 on two breast cancer cell lines: M C F - 7 , with
high levels of estrogen receptor (ER) and M D A - M B -
231, which is estrogen-insensitive. Taking E2 actions
as a reference, we investigated the effects of T 3 o n
proliferation, and on the induction of progesterone
receptor (PR), classically induced by E2 [13], as well
as on the modulation of transforming growth factor
mRNAs (TGF~ and TGFfl) at least in part controlled
by E2 [14, 15]. We have previously reported that
LDH5 activity (lactate dehydrogenase isoform 5) was
markedly affected in response to E2 [16], and there-
fore LDH5 activity was analysed in the same cell lines
after T3 treatment. T o characterize our model better,
the expression of c-erb A mRNAs (A~tl and Afll-
encoding thyroid hormone receptors) as well as the
activity of ~t-glycerophosphate dehydrogenase, a
271
272 C.R. Nogueira and M. M. Brentani
widely used index of thyroid hormone action, have
been determined in both cell lines [17].
MATERIALS AND METHODS
Chemicals and reagents
Estradiol 17fl (E2) and L-triiodothyronine (T3)
were obtained from Sigma (St Louis, MO, U.S.A.).
The absence of Ez contamination in L-T3 solution
was checked by radioimunoassay. Tamoxifen (TAM)
was a gift from Imperial Chemical Industries (ICI,
Macclesfield, Cheshire, U.K.) [3H] estradiol (41 Ci/
mmol), [3H] ORG 2058 (44 Ci/mol) and [3Zp] dATP
(3000 Ci/mM] were purchased from Amersham Little
Chalfont, Bucks. Two ER monoclonal antibodies
were used in our experiments, one (H 226) was a gift
from Dr J. T. Tomita (Abbott Diagnostics, North
Chicago, IL, U.S.A.). The second was the anti-
human estrogen receptor (clone 1D5) purchased from
DAKO, Carpinterie, CA, U.S.A. 125I-T 3 ( 8 0 0 -
1000 Ci/mmol) was donated by ICN (Trilab
Diagn6stica Ltda). RPMI-1640 medium, fetal calf
serum and molecular biology grade reagents were pro-
vided by Gibco/BRL Gaithersburg, MD, U.S.A. All
other chemicals were obtained from Sigma.
cDNA probes and cells lines
The c-erb A~t 1 probe (PsTI 0.5 Kb fragment from
pAEV-11 specific for c-erb A~ 1) [18] and the T G F~
probe (EcoRI 1.38 Kb fragment from pSP65 specific
for transforming growth factor ~) [19] were kindly
provided by Dr M. Waterfield (Ludwig Institute for
Cancer Research, London, U.K.). The c-erb Aft 1
(EcoRI 0.5 Kb fragment from p C D B - e r b 62) [20]
was a gift from Dr Mitchel A. Lazar (University of
Pennsylvania, Philadelphia, PA). EcoRI 1.0 Kb frag-
ment from pUC for TGFfl [21] was kindly provided
by Dr C. Heldin (Ludwig Institute for Cancer
Research, Uppsala, Sweden). PR mRNA expression
was determined using the BamHI fragment from
Bluescript M 13 + [22] and ER mRNA expression was
determined using the EcoRI 1.8 Kb fragment from
pS65 HEO [23] both provided by Dr P. Chambon
(Institute Chimie Biologique, Facult6 M6dicine,
Strasbourg, France). The cDNa probe (SaII/EcoRI
1.9 Kb fragment from pBR322 specific for the 18S
ribosomal RNA (rRNA) gene) [24] was used as a
control for RNA quantitation and was provided by Dr
Arnhein (Department of Biochemistry, University of
New York, NY, U.S.A.). M C F - 7 and M D A - M B -
231 cells were also supplied by Dr M. Waterfield.
Cell culture and growth experiments
Cells were grown 14 days before harvesting in
RPMI 1640 supplemented with L glutamine
(2.8 mM), insulin (8 mIU/ml), penicillin-streptomycin
100 U/ml, and 5% charcoal-stripped calf serum
(FCS) and kept at 37°C in humidified 5% CO2 and
air. The medium was changed every two days. Before
starting hormone treatments the medium was
replaced with phenol red-free RPMI 1640 to elimin-
ate all known sources of estrogen [25]. After 24 h
(day 0), the medium was changed and hormones
added dissolved in absolute ethanol, with daily med-
ium changes. Cells were harvested in triplicate at the
times indicated and cell numbers were counted. Cell
numbers were plotted as a logarithmic function
against time and cell population doubling (DT) was
estimated at the exponential phase.
Steroid-hormone receptor assay
M C F - 7 and M D A - M B - 2 3 1 cells were cultured as
described above and the confluent monolayer tripsi-
nized, centrifuged for 2 min at 800 g and resuspended
in phenol red-free RPMI containing 5% FCS
depleted of steroids. Cells were incubated for 2 h at
37°C before receptor assay. ER and PR concen-
trations were determined by a whole-cell radioligand
binding technique. Aliquots of 150pl containing
3 x l0 s cells were incubated with (3H) estradiol or
(3H) ORG 2058 at concentrations ranging from 0.5-
5 x 10 -9 M and 1.0-10 x 10 -9 M, respectively, in the
presence or absence of a 200-fold excess of the corre-
sponding unlabelled steroid. After 45 rain at 37°C,
medium was removed by centrifugation and the cells
were washed three times with ice-cold saline phos-
phate (NaC1 8g, KC1 0.2g, Na2HPO4 2. 8g and
KH2PO4 0.2 g/l, pH 7.4) containing 0.2% of albumin
(PBSA). Steroids were extracted with 200 #1 of 0.5%
Triton X-100 in PBSA and aliquots quantified for
radioactivity. All measurements were performed in tri-
plicate and specific receptor numbers were calculated
from Scatchard plots and expressed as number of
sites per cell [16]. For competition assays, cells were
cultured as above and the whole cell binding assay
performed with a fixed concentration of labelled estra-
diol (5 nM) in the presence or absence of increasing
amounts (10-1°M-10 -5 M) of radioinert Ez or T3.
The amount of residual (3H) estradiol binding was
plotted in the ordinate against the log of unlabelled
hormone concentration in the abscissa, to obtain dis-
placement curves.
RNA extraction
Total RNA was extracted from cells by the guanidi-
nium thyocianate method and analysed by electro-
phoresis using 1% agarose gel containing 10%
formaldehyde, transferred to a Hybond nylon mem-
brane (Amersham) by blotting and hybridized to 32p
labelled probes. Conditions for prehybridization, hy-
bridization, and washing were carried out according
to Maniatis [26]. The membranes were exposed to
Kodak X-AR-K films with intensifying screens for 4 -
7 days. Hybridization to cDNA probes was quantified
using scanning laser densitometry. The amount of
 T3 Effects in Breast Cancer Cells
total R N A on each lane', was evaluated relative to 18S
rRNA content.
Lactate dehydrogenase activity (LDH)
Total L D H activity and isoenzyme patterns were
determined in cell cytosols. T o prepare cytosols, cells
were cultured as described above, cell suspensions
were spun down, the supernatant discarded, the cell
pellet homogenized in 10 m M Tris p H 7.4, containing
1.5 m M E D T A , 2 m M ditiothreitol and 10% glycerol,
with a Polytron tissue., disintegrator, and the hom-
ogenate centrifuged at 105000 g for 60 min. L D H ac-
tivity and the relative proportion of L D H isoenzymes
were determined as previously described. L D H ac-
tivity was expressed as micromoles of co-factor altered
per min per mg of cytosolic protein [16]. Protein was
assayed by the m e t h o d of Lowry et aL [27].
Analysis of glycerol phosphate dehydrogenase (GPD) ac-
tivity
Cells were cultured as described above, suspended
in Tris-HC1 10 m M , p H 7.4 containing 0.32 M sac-
charose, 5 m M 2-fl:mercaptoethanol and 2 m M
E D T A and ruptured by D o u n c e homogenization fol-
lowed by centrifugat!ion at 1000 g x 10 min. T h e
supernatant was further centrifuged at
10000 g x 10 min and the pellets were suspended in
the same buffer to obtain mitochondrial suspensions
which were prepared from both controls and T3 trea-
ted cells. G P D activil~ was measured as published
elsewhere [28].
Quantitation of ER by immunoprecipitation
Cells (6 × 105) were: suspended in 1.5 volumes of
1 0 m M Hepes, p H 7 . 3 , 1 m M E D T A , 2 0 m M
sodium molybdate and ruptured in a D o u n c e hom-
ogenizer. Nuclei were recovered by centrifugation at
600 g for 15 rain. Nuclear pellets were washed with
the suspension buffer, incubated in extraction buffer
(10 m M sodium phosphate p H 7.35, 1% N P - 4 0 , 1%
sodium deoxycholate. 0.1% SDS, 2 r a M E D T A ,
0.5 m M benzamidine, 0.5 m M P M S F , 5 #g/ml leu-
peptin) for 20 min on ice and centrifuged at 105000 g
for 60 min to yield a clear nuclear extract (400/d).
Aliquots of nuclear extracts were diluted with an
equal volume of 1 0 r n M T E S , 5 0 m M NaC1, 10%
glycerol, 4 m M E D T A and 20 m M sodium molyb-
date, p H 7.6 and incubated with 100 n M (125I) T3 or
(3H) estradiol. Anti-ER-specific monoclonal antibody
or normal mouse immunoglobulin G (at the same
concentration as that of E R antibody) was added at
5% of the final volume and samples incubated on ice
for 1 2 - 1 6 h . Each mixture, after incubation was
adsorbed to protein A-sepharose which was then pel-
leted by centrifugation, the supernatant was removed
and radioactivity was determined in the pellet after
three washes, according to the protocol previously
described [29]. Radioactivity in the pellet was quanti-
273
tated in a gamma counter for 125I T3 and in a scintil-
lation counter for 3H estradiol and expressed as fi'nol
per mg of nuclear extract protein.
Statistical analysis
T h e values presented in this paper are means ___SD.
T h e paired t-test (Student) was used to test the differ-
ences between the means of experimental groups. P
values <0.05 were considered significant.
RESULTS
Estrogen (ER) and progesterone (PR) receptor determi-
nation
As a first step we evaluated the steroid-hormone
receptor binding activity levels in M C F - 7 and M D A -
M B - 2 3 1 breast cancer cells by a whole cell binding
assay using labelled estradiol and a synthetic pro-
gesterone ( O R G - 2 0 5 8 ) . E R and P R values calculated
by Scatchard analysis of binding data determined in
M C F - 7 cells are presented in Table 1. Northern
analysis of m R N A from M C F - 7 cells detected a
single E R - m R N A species around 6.4 Kb (data not
shown). M D A - M B - 2 3 1 did not express measurable
concentrations of E R or PR.
Te recepwrs
T h e presence of thyroid receptor was verified by
Northern analysis using c-erb al (5.4 and 2.5 Kb)
and fll (7.0 Kb) probes and in both breast cancer cell
lines we have identified c-erb A m R N A ~1 and fll
transcripts, although M C F - 7 cells presented lower
message levels of both receptors as compared to
M D A - M B - 2 3 1 cells (Fig. 1).
Analysis of I"3 induction of ~glycerophosphate dehydrogen-
ase (~GPD)
T h e basal ~ G P D activity was higher in M D A - M B -
231 cells than in M C F - 7 cells. T3 treatment induced
Table 1. Estrogen receptor (ER) and progesterone receptor (PR)
levels in MCF-7 and MDA-MB-231 cells
Cells
MCF-7 MDA-MB-231
ER 145504 + 9801 ND
Kd 1.48 + 0.22 n M
PR 49913 + 3046 ND
Kd 6.71 + 1.69 n M
After being maintained in harvest m e d i u m containing stripped fetal
calf s e r u m (5%) for 14 days, cells were changed to phenol red-
free m e d i u m . E R and P R were determined by the whole cell
binding assay with 3H-17fl estradiol or 3 H O R G 2 0 5 8 (PR) in
the presence or absence of unlabelled h o r m o n e s . E R and P R
levels were calculated by Scatchard analysis of specific binding
data and expressed in sites/cell. Values are m e a n + SD of three
experiments.
N D , not detected.
274 C.R. Nogueira and M. M. Brentani

Table 2. Effects of 7"3 on ~-glycerophosphate ~ desidrogenase

(a) 5.4kb - - ~ (~GPD) activity of breast cancer cells
7kb --~
2.5kb Cells
MCF-7 MDA-MB-231

18S - - ~ 18S Control 0.039 ± 0.005 ~ 0.071 + 0.012 ~

T 3 (10 -8 M) 0.086 ± 0.002 b 0.132 ± 0.020 a
(b) 0.3 T 3 (10 -s M) + T A M
(10 -6 M) 0.079 ± 0.002 0.158 ± 0.024

e G P D activities were determined in mitochondrial extracts of cells

cultured as described in Table 1 and expressed as optical den-
O3 02 sity/mg of protein/rain. Values refer to mean + SD of three ex-
O periments, (a) vs (b) P < 0.010; (c) vs (d) P < 0.025.
O
O3
._>
Progesterone receptor (PR) levels are induced by 7"3 and
E2
PR has been proposed as an indicator of respon-
siveness to estrogen in M C F - 7 cells, and therefore
I PR induction was studied and treatment with T3
(10 -s M) resulted in a progressive increase with time
in PR binding activity without changes in affinity
• MDA-MB-231 ~ J~ (Table 3). When M C F - 7 cells received 10-aM T 3
[ ] MCF-7 6

Fig. 1. N o r t h e r n analysis of c-erb A ~1 and c-erb A fl] R N A s .

(a) T o t a l R N A s were isolated from M C F - 7 and M D A - M B -
231 cells and 30 #g of each total R N A in duplicate was hybri-
d i z e d to c-erb A ~1 and c-erb A /~1 probes. Blots were
reprobed w i t h a S 2 P - r i b o s o m a l R N A ( r R N A ) in order to
quantify the a m o u n t of m R N A layered on each lane.
A u t o r a d i o g r a m s were s c a n n e d d e n s i t a m e t r i c a l l y and the d a t a
in (a) was n o r m a l i z e d to the internal reference s i g n a l (18S).
(b) Graphic representation of the m e a n relative content
( ± S D ) o f c - c r b A ~1 and/31 m R N A s m e a s u r e d in two separate
experiments.

eGPD activity by 2.2-fold in the M C F - 7 and by 1.8- CT 10 "10 10 "w 10 4

T3 Dose (M)
fold in the M D A - M B - 2 3 1 cells (Table 2) as com-
pared to control cells. (b) (e) t-

Actions of 7"3, E2 and the steroidal antagonist tamoxifen

(TAM) in cell proliferation
T3 decreased the doubling time (DT) of control
M C F - 7 (62 h) in a dose-dependent manner to 56.7, ~ g
52.3 and 33.6 h when cells were treated with 10-1°,
10 . 9 and 1 0 - aM T3, respectively. Only with the
maximal dose, however, was the decrease statistically
significant (P < 0.005) (Fig. 2a). The effect of T 3 o n ÷ + ,,~ +

proliferation of M C F - 7 was less marked than that

produced by estradiol (10 -7 M) which depressed the
D T to 24 h (P < 0.005). Although Fig. 2 reports stu-
dies using 10-TM E2 we have observed 10-gM and
10-SM E2 to be equally effective in stimulating cell
proliferation (data not shown). The simultaneous ad-
dition of TAM (10-6M) inhibited the proliferative
effect of E2 (DT increased to 57.6 h) and T3 (D T
increased to 46.6h, P < 0.05) (Fig. 2b). T he growth
of M D A - M B - 2 3 1 cells was independent from the ad-
dition of E2, T 3 and T A M (Fig. 2c).
Fig. 2. Growth e x p e r i m e n t s w e r e c o n d u c t e d in M C F - 7 (a and
b) and M D A - M B - 2 3 1 (c) breast c a n c e r cells cultivated in
m e d i u m c o n t a i n i n g 5% of c h a r c o a l stripped fetal calf s e r u m
in the a b s e n c e of p h e n o l - r e d . Cells were s e e d e d (1 x 104 cells
p e r well) and treated in (a) with triidothyronine (Ts) at the
concentrations indicated. Control flasks r e c e i v e d 0.01% etha-
nol (vehicle) without the addition of the h o r m o n e s (CT). In
(b) and (c) the following h o r m o n e c o n c e n t r a t i o n s were uti-
lized: estradiol (E2)10 -7 M; T s 10-SM and tamoxifen (TAM)
10 ~ s M . Results are m e a n s of four e x p e r i m e n t s in
triplicates + S D p e r f o r m e d s i m u l t a n e o u s l y . * P < 0.005 (the P
values are in relation to control).
 T3 Effects in Breast Cancer Cells 275

Table 3. Effects of 7"3 on progesterone receptors (PR) levels in (a) kb

MCF-7 breast cancer cells
Treatments PR K d (nM) 9.5
Control 4 9 9 1 3 + 3046 '~ 6.7 ± 1.7
10 -8 M T3 (24 h) 74:287 _+ 15733 b 5.5 _.+ 1.9 6.7 --~
10 - s M T 3 (72 h) 166555 __ 54283 ~ 5.4 + 1.0
10-s M T3 + 10-6 M
49680+ 9046 5.4 + 1.4 4.5 --~
TAM (72 11)
2.3 --~
Cells were cultured as described in T a b l e 1 and treated with either
T3 or T3 plus T A M in ethanol, or with vehicle alone (control). 1.9 --~
T o t a l cellular P R was measured utilizing the whole cell tech-
nique an d Scatchard analysis and expressed in sites/cell. Values
are m e a n + SD of three different experiments in triplicates. (a) 18S
vs Co), P < 0.05; (a) vs (c), P < 0.05.

associated to 10-6/~[ T A M the induction was (b) 4.0

repressed. When M C F - 7 were exposed to 10-7 M E2,

PR levels of 200000 siites/cell were obtained in 24 h.
This value was no further modified after 72 h of treat- 3.0
ment. PR-mRNA, quantitated by Northern analysis
in M C F - 7 cells appears as five species (9.5, 6.7, 4.5,
2.3 and 1.9 Kb). PR mRNA levels increased approxi- c-
O
mately by 75% after E2 treatment ( P < 0.005) and by O
® 2.0

50% after T3 treatment ( P < 0.025). 10-6M T A M

suppressed the effects of both hormones (Fig. 3).
Effect of E2 and T~ on LDH5
M C F - 7 cells express only one L D H isoenzyme
(LDH5) whereas in M D A - M B - 2 3 1 cytosols all five
forms were detected (data not shown). M D A - M B -
231 cells show a doubling of total L D H levels com-
pared to M C F - 7 ( P < 0.005, Table 4). Exposure of
M C F - 7 cells to T3 (10 -8 M) and E2 ( 1 0 - 7 M) lead to
an increase of LDH5 activity ( P < 0.01). In contrast
LDH~ activity of M D A - M B - 2 3 1 cells did not change
with the same treatments.
Effects of E2 and 7"3 on TGF~t and TGFfl m R N A levels
Previous studies have shown that estrogenic regu-
lation of proliferation in breast cancer appears to be
related to the modulation of transforming growth fac-
tors TGFct and TGFfil [14, 15]. Northern blot analy-
sis revealed in MCF--7 cells, various transcripts for
TGFfl (4.5, 2.5, 0.56 and 0.13 Kb), whereas only one
4.5 Kb transcript was detected with the TGFct probe.
Exposure of M C F - 7 cells to Ee (10-7M) or T3
(10-SM) caused T G F ~ mRNA levels to increase
four-fold and three-fi~ld over controls, respectively.
T A M (10 -6 M) reverted this stimulation (Fig. 4). On
the other hand E2 and T3 depressed TGFfl mRNA
expression to 30% a~d 40% of the control, respect-
ively. TGFfl RNA levels were incompletely restored
by 10-6M T A M (Fig:. 5). M D A - M B - 2 3 1 expressed
higher levels of T G F ~ mRNA and lower levels of
TGFfl mRNA compared to M C F - 7 cells and T3 or
E2 did not evoke any alterations in T G F a and TGFfl
message levels (data not shown).
1.0
}-- 04
+ +
t'q t~
tU P-
Fig. 3. P R m R N A i n d u c t i o n in M C F - 7 cells. (a) T o t a l RNA
(20 pg) was isolated from control cells (CT) and cells treated
for 72 h w i t h 10-7M E 2 or 10-SM T3, without or plus 10-6 M
T A M and hybridized w i t h a 32P-labelled P R p r o b e . (b)
Graphic representation o f the m e a n relative c o n t e n t (+SD) o f
P R m R N A m e a s u r e d in five separate experiments after nor-
realization to the internal reference s i g n a l (18S). *E2
P < 0.005 a n d T3 P < 0.025.
Unlabelled 7"3 displaced binding of 3H-estradiol to the es-
trogen receptor (ER)
When M C F - 7 cells were incubated with 5 nM 3H-
estradiol in the presence or absence of increasing con-
centrations of radio inert T3 or E2, it was observed
that T3 significantly displaced the binding of labelled
E2 to ER, although the displacement was weaker than
that observed with E2 (Fig. 6). By comparing the con-
centrations of E 2 and T3 at half 3HE2 we verified that
T3 shows a binding relative affinity approximately
100-fold lower than the binding affinity of E2 for ER.
Binding of (OH) 17[3 estradiol and (1251) T3 to the ER
Our results in Fig. 7 show that a nuclear extract
component(s) from M C F - 7 cells labelled with 12sI
T3 was specifically immunoprecipitated by an ER-
276 C. R. N o g u e i r a a n d M . M . B r e n t a n i

Table 4. Effect of 7"3 and Ez on lactate dehydrogenase (LDH) activity expressed by breast cancer cells
Cells

MCF-7 MDA-MB-231
LDHs LDH %LDH5

Control 1.03 + 0.26 a 2.09 + 0.34 37

Ez (10 -7 M) 1.81 + 0.50 b 2.18 + 0.48 34
T~ (10 -s M) 1.63 + 0.27 c 2.12 __+0.30 35

Total L D H activity and isoenzyme patterns were determined in cell cytosols obtained as described in Materials and methods. Data are in
terms of micromoles of pyridine nucleotide altered per min per mg of cytosolic protein and refer to mean + SD for three different exper-
iments, (a) vs (b) P < 0.01; (a) vs (c) P < 0.005.

specific monoclonal antibody (H226) which is DISCUSSION

believed to interact with the DNA-binding domain of The main objective of the present work was to
the receptor [23]. Results were similar to the immu- compare the effects of T3 and E2 o n two human
noprecipitation observed with (3H) E2 and statistically breast cancer cell lines: M C F - 7 and M D A - M B - 2 3 1 .
different from the control (irrelevant IgG) According to our determinations of ER, PR, c-erb-
( P < 0 . 0 0 5 ) . Values obtained with M D A - M B - 2 3 1 A~I and c-erbAfll M C F - 7 can be classified as
cells were equivalent to those verified using an irrele-
vant IgG (non-specific binding). Equivalent results (a) kb
were obtained with the D A K O - E R 1D 5 monoclonal 4.5 .--~
antibody, directed against the N-terminal region of 2.5 --4,
the ER (data not shown).
0.56 - - ~
0.13 " b

18S - - ~

ilii
(a) 4.5kb - - b (b) 2o

18S --4,
(b)

0.8

O
0.6 o® 10

c-
o
0

---~ 0.4

0.2

o + +

LU

4- 4-
¢~ o4
I-- LM
Fig. 4. N o r t h e r n a n a l y s i s o f TGF= m R N A e x p r e s s i o n i n
M C F - 7 cells. (a) R N A (20/~g) w a s o b t a i n e d f r o m c o n t r o l cells
(CT) a n d cells t r e a t e d for 72 h with 10-7M E2 o r 10- s M T3
a l o n e o r in a s s o c i a t i o n w i t h 10~SM T A M . (b) G r a p h i c r e p -
r e s e n t a t i o n o f t h e m e a n relative c o n t e n t (_+SD) o f TGF~
m R N A m e a s u r e d i n two s e p a r a t e e x p e r i m e n t s , n o r m a l i z e d to
t h e i n t e r n a l r e f e r e n c e s i g n a l (18S).
Fig. 5. TGFfl m R N A e x p r e s s i o n in M C F - 7 cells. (a) Total
R N A (20/Jg) w a s o b t a i n e d f r o m c o n t r o l cells (CT) a n d c e l l s
t r e a t e d for 72 h e i t h e r with 10-7M E2 o r 10-SM T3 in t h e
a b s e n c e o r p r e s e n c e o f 10-6 M T A M a n d N o r t h e r n blot a n a l y -
sis w a s p e r f o r m e d . Co) G r a p h i c r e p r e s e n t a t i o n o f t h e m e a n
relative c o n t e n t ( _ S D ) o f TGFfl m R N A m e a s u r e d in t w o s e p -
arate experiments. Autoradiograms were scanned densitome-
t r i c a l l y a n d t h e a r e a h y b r i d i z i n g with t h e TGFfl c D N A p r o b e
was n o r m a l i z e d to t h e i n t e r n a l r e f e r e n c e s i g n a l (18S).
 T3
2.2
2.1
2.0
°._ 1.9
)<
1.8
P..
(~ 1.7
i
tO
t~ 1.6
1.5
1,,
1.4
1.3
0 10 9 8 7 6
- log [M] ligand
Fig. 6. D i s p l a c e m e n t cu:rves o f radiolabelled E2 f r o m its
r e c e p t o r in M C F - 7 cells. Whole cell b i n d i n g assay was p e r -
f o r m e d with a fixed c o n c e n t r a t i o n o f labelled estradiol (5 nM)
in the p r e s e n c e o r absen(:e o f i n c r e a s i n g m o u n t s (10 -l° M -
10-s M) o f r a d i o i n e r t Ha or T 3. The a m o u n t o f residual (3H)
estradiol b i n d i n g was plol~tcd in the o r d i n a t e against the log
o f - n l a b e l l e d h o r m o n e c o n c e n t r a t i o n in the abscissa. Data
are m e a n s f r o m two i n d e p e n d e n t e x p e r i m e n t s , each c o u n t e d
in triplicates.
ER÷PR+TR + and M D A - M B - 2 3 1 as E R - P R - T R +.
T h e expression of T R mRNAs in M C F - 7 cells con-
firmed previous determinations using a radioligand
binding assay [8]. T h e inducibility of the mitochon-
drial enzyme ~-glycerophosphate dehydrogenase
pointed out the presence of a functional T R in both
cell lines and excluded the possibility that T A M may
act through T R , as basal enzyme levels were not
modified by T A M administration.
Our results showed that in M C F - 7 cells, T3
mimicked the effects of E2 stimulating growth, m o d u -
lating m R N A transcription of growth factors and
inducing the expression and activity of estrogen-indu-
cible proteins. An increase in S + G2 + M fraction by
T3 in the same cells was previously reported [30]. In
addition, these T3 effects were antagonized by the
simultaneous addition of T A M . Similar effects, how-
ever, were not observed in M D A - M B - 2 3 1 cells in
spite of the presence of high T R amounts, suggesting
that in M C F - 7 cells both ligands share a c o m m o n
signalling pathway via ER.
As E R and T R display cross-recognition of hor-
mone responsive elements, one plausible mechanism
to explain such data could be the direct binding of
T R to ERE. Glass et al. [31] recently reported that
T R competed with E R to bind the palindromic ERE
and inhibited the latter's transcriptional activity.
Another possibility could be the involvement of a
heterodimer formed between E R and T R [32]. An
Effects in Breast Cancer Ceils 277
l I IMOF-7BMDA-MB-231J
1"3+ IgG
E2 + IgG
T3 + anti ER
E2 + anti ER
i I I I l
0 20 40 60 80
fM/mg protein
Fig. 7. I m m u n o p r e c i p i t a t i o n o f n u c l e a r ER. Aliquots o f the
5 nuclear extracts o f M C F - 7 or M D A - M B - 2 3 1 cells were
labelled with 100 n M [3H] E2 or 12sI "T3. E R m o n o c l o n a l a n t i -
body or n o n - i m m u n e m o u s e IgG was a d d e d to the n u c l e a r
m i x t u r e at 5% o f final volume. E a c h m i x t u r e , after i n c u -
b a t i o n was a d s o r b e d to p r o t e i n A - s e p h a r o s e w h i c h w a s then
pelleted by centrlfugation, the s u p e r n a t a n t was r e m o v e d a n d
radioactivity was d e t e r m i n e d in the pellet after t h r e e w a s h e s .
B a r s c o r r e s p o n d to the b i n d i n g o f [3H]17fl estradiol a n d 125I
"1"3 to the E R i m m u n o p r e c i p i t a t e d by an ER-specific m o n o -
clonal antibody as c o m p a r e d to non-specific b i n d i n g using an
irrelevant IgG. ,Values are the m e a n ___SD o f t h r e e i n d e p e n -
d e n t e x p e r i m e n t s , e a c h p e r f o r m e d in triplicate. Values were
expressed as fimol p e r m g protein, (a) vs. (c) P < 0.005; (b) vs.
(d) P < 0.005.
effort was made to minimize a possible interplay
between the two receptors, using T3 in the absence of
E2 in our model.
We favour the hypothesis that the effects reported
are due to the binding of T3 to E R for the following
reasons: unlabelled T3 competed for 3H-estradiol
receptor binding sites, albeit to a lesser extent than
E2. In addition, a nuclear extract c o m p o n e n t from
M C F - 7 cells prebound with 1251 T3, was immuno-
precipitated by an ER-specific monoclonal antibody.
An alteration of the ER-binding domain permitting
the binding of T3 could account for our results.
Variant messenger RNAs have been detected in breast
cancer cell lines and in clinical breast cancer speci-
mens [33]. We detected E R m R N A in M C F - 7 cells
as a single m R N A species around 6.4 Kb in agree-
ment with the transcript size expected [34]. Certain
point mutations in the ligand-binding domain may,
however, alter the responsiveness of mutated ERs
[35]. As it was previously suggested that a lower
population of M C F - 7 cells contain such mutations
[36] we can not formally exclude this possibility.
On the other hand, such effects seem not to be a
peculiarity of M C F - 7 cells. Zou-Li et al. [12] have
shown that antiestrogens are inhibitors of T3 action
on the proliferation of pituitary cells which also con-
tain E R and express c-erb A~I and fll transcripts and
278 C. R. Nogueira and M. M. Brentani
excluded the possibily that T A M could be acting
through TR. Koga et al. [37] also demonstrated that
the levels of PR were up-regulated by T3 in a rat pitu-
itary tumour cell line and G H induction by T3 can be
inhibited by tamoxifen in an ovariectomized-thyroi-
dectomized rat model [38].
Other studies reported an ER specificity spill-over.
In breast cancer cells in culture, 5-androstene-3/~,17/~-
diol and testosterone in high doses are able to induce
estrogenic responses via an ER-mediated mechanism
[39-41]. Our work expands the findings on the inter-
ference between steroid hormones controlling cell
proliferation and gene expression, and provide evi-
dence that breast cancer may be under thyroid hor-
mone control.
Acknowledgements---The authors thank Drs M. Waterfield, M. A.
Lazar, C. Heldin, P. Chambon for generously providing the probes
and cells used in this work, Dr A. C. Bianco (I.C.B., USP, Brazil)
for assistance in setting up aGPD activity and Ms F. S. Passini for
assistance with LDH determinations. This research was supported
by a CNPq grant (350188). Part of this work was presented at the
International Congress on Hormones and Cancer (Quebec, 1995).
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