[CANCER RESEARCH 50. 299-303, Januar) 15. 1990]
Differential Regulation of Expression of Three Transforming Growth Factor ß
Species in Human Breast Cancer Cell Lines by Estradiol
Bradley A. Arrick,1 Murray Korc,2 and Rik Derynck
Department of Developmental Biology, Genentech, Inc., South San Francisco, California 94080 IB. A. A., R. D.J; Cancer Research Institute, University of California,
San Francisco. California 94143 [B. A. A.J; and Departments of Medicine and Biochemistry, University of California, Irvine, California 92717 ¡M.K.]
negative and three estrogen receptor-positive cell lines. We have
found that estrogen decreased mRNA expression of TGF-/32
and -03 but not -01 in these cell lines. Down-regulation of TGF02 and -03 mRNA expression by estrogen in the T-47D cell
line was accompanied by decreased secretion of TGF-0 activity
into the medium.
ABSTRACT
Transforming growth factor (TGF)-0 is a potent regulator of many cell
functions and a growth inhibitor for mammary epithelial cells. We now
know of three highly homologous members of the human TGF-0 gene
family. We have studied the expression of TGF-01, -02, and -03 mRNA
in four human breast cancer cell lines. Using the RNase protection assay,
we have detected mRNA expression of TGF-01, -02, and -03 by T-47D
cells, TGF-01 and -03 by ZR-75-1 cells, and TGF-01 by MCF-7 cells.
Treatment of these estrogen receptor-positive cells with 10 nM estradiol
for 48 h resulted in decreased mRNA levels of TGF-02 and -03 but did
not affect mRNA levels of TGF-01. Expression of TGF-01 and -02
mRNA by an estrogen receptor-negative cell line, MDA-MB-231, was
not changed by estradiol treatment. Treatment of cells with the antiestrogen tamoxifen (1 MM)did not significantly alter mRNA levels for any of
the three TGF-0 species. We have further determined that estradiol
treatment of T-47D was associated with diminished secretion of TGF-0
into the medium. Both TGF-01 and -02 inhibited the proliferation of
MCF-7 cells, and neither protein affected the growth of T-47D cells.
TGF-01 was at least 10-fold more potent than TGF-02 at inhibiting the
growth of MCF-7 cells.
MATERIALS
AND METHODS
Cell Culture. All cell lines were obtained from the American Type
Culture Collection (Rockville, MD). Studies with MCF-7, T-47D, ZR75-1, and MDA-MB-231 cell lines were performed with passages 150175, 90-110, 85-110, and 30-45, respectively. Cells were routinely
cultured in phenol red-containing RPMI supplemented with 5% FBS,
2 miviglutamine, 100 units/liter penicillin, and 100 Mg/ml streptomycin.
Cells were cultured at 37°Cin 5% CO2.
Hormonal Treatments. Tamoxifen and 17ff-estradiol were obtained
from Sigma and were stored at -20°C in ethanol. Treatments were
performed in phenol red-free serum-free medium consisting of RPMI
(without phenol red) buffered with 15 mM HEPES (pH 7.2) and
supplemented with 10-20 Mg/ml transferrin (Sigma), 200-500 ng/ml
BSA, fraction V (Sigma), 2 mM glutamine, 100 units/liter penicillin,
and 100 Mg/ml streptomycin. This medium has been reported to support
the growth of breast cancer cell lines (12). Culture of the T-47D cells
in this serum-free medium longer than 48-72 h resulted in the forma
tion of cytoplasmic vacuoles.
RNA Isolation and Analysis. Cells were grown in 10-cm plastic dishes
to near confluence using RPMI-5% FBS. Following two washes with
12.5 ml of warm PBS (145 mM Na+, 4 mM K% 140 mM Cl~, 5.5 mM
INTRODUCTION
Estrogen is mitogenic for both normal and neoplastic mam
mary epithelial cells and may even facilitate carcinogenesis (1).
In recent years, human breast cancer cells have been shown to
produce and respond to a variety of growth factors. Estrogen
stimulates the production of TGF3-«,insulin-like growth factor
II, and platelet-derived growth factor by some hormone-respon
sive cell lines (2-4). Conversely, the production of TGF-0 by
the estrogen receptor-positive cell line MCF-7 has been re
ported to be most affected by antiestrogens (5). Tamoxifen
caused a 5-fold increase in production of TGF-0 by MCF-7
cells. Hormonal regulation of TGF-0 production was felt to be
posttranscriptional, since the level of mRNA did not change
with tamoxifen treatment (5). Following the publication ofthat
report, the existence of a second and third human TGF-0
species was revealed. These three human types of TGF-0 are
70-80% identical at the amino acid level (6-9). TGF-/31 and
-02 share many but not all biological effects, while the biological
activities of TGF-03 have as yet not been described. TGF-01 is
a potent growth inhibitor for both normal and transformed
mammary epithelial cells (10, 11).
phosphate), the cells were incubated in 10-11 ml of serum-free medium
containing estradiol, tamoxifen, or ethanol vehicle (0.1%) for varying
times. RNA was prepared following lysis of cells in a hypotonie solution
with the nonionic detergent Nonidet P-40 (13) supplemented with 200
Mg/ml heparin (Sigma) as an RNase inhibitor. RNA was quantitated by
absorbance at 260 nm. Analysis of total RNA by the RNase protection
assay was performed according to published protocol, with some opti
mizing changes ( 13). Briefly, 10 ^g cellular RNA plus 20 ng tRNA was
combined with 32P-labeled cRNA (1.5-2 x IO5 cpm) in 30 ^1 hybridi
zation buffer, heated to 85°Cfor 5 min, and then incubated at 53°C
overnight. RNase digestion was carried out by the addition of 350 i¿\
of ice-cold digest buffer, containing 0.24 units/ml RNase A and 80
units/ml RNase T,, and proceeded for 30 min at 35°C.RNase A,
In this report, we expanded the evaluation
of hormonal
regulation of TGF-0 production by human breast cancer cell
lines. Using the RNase protection assay, we have analyzed the
expression of TGF-01, -02, and -03 in an estrogen receptorReceived5/22/89; revised9/13/89; accepted 10/12/89.
The costs of publicationof this article were defrayedin part by the payment
of page charges. This article must therefore be hereby marked advertisementin
accordancewith 18 U.S.C. Section 1734solelyto indicatethis fact.
1To whom requests for reprints should be addressed, at Department of
Developmental Biology, Genentech, Inc., 460 Point San Bruno Blvd., South San
Francisco. CA 94080.
2 Supported by National Cancer Institute Grant CA 40162. On sabbatical leave
from the University of Arizona.
3 The abbreviations used are: TGF, transforming growth factor; FBS, fetal
bovine serum; HEPES, 4-(2-hydroxyethyl)-I-piperazineethanesulfonicacid;
BSA,
bovine serum albumin; PBS. phosphate-buffered saline; RPM1, RPMI 1640.
RNase T,, and tRNA were from Boehringer Mannheim (Indianapolis,
IN). Samples were then treated with protcinase K, extracted with
phenol/chloroform, and precipitated with ethanol. Samples were dis
solved in gel-loading buffer and fractionated by electrophoresis with
4.5% polyacrylamide-6 M urea sequencing gels, each lane containing
the equivalent of 6 /¿gof initial cellular RNA. Autoradiograms were
analyzed by laser densitometric scanning of the probe fragments pro
tected from RNase digestion. Control samples, processed concurrent
with authentic samples, contained tRNA alone (negative control) or
tRNA plus a mixture of sense cRNAs prepared by in vitro transcription
of cDNA for TGF-01, -02, and -03 positioned downstream of the SP6
promoter (positive control). The use of sense cRNAs containing fulllength coding sequences for each of the three human TGF-0S demon
strated the lack of cross-hybridization between the three TGF-0 probes
(data not shown). Dilutions of this mixture of cRNAs were tested over
a 10-fold range, and densitometric quantitation of the specific bands
on the resulting autoradiogram was linear.
Preparation of Probes for RNase Protection Assay. The plasmid for
299
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HORMONAL REGULATION
antisense TGF-/31 cRNA synthesis was constructed by cloning a 280base pair EcoRl-Kpn\ fragment, corresponding to nucleotides 997 to
1277 of the human TGF-/J cDNA sequence (6), into the pSP72 vector.
A similar plasmid for the detection of human TGF-/52 mRNA was
constructed by inserting the 600-base pair Hind\\\-Pst\ fragment of a
human TGF-02 cDNA,4 corresponding to nucleotides 253 through 853
OF TGF-/J EXPRESSION
calculate the percentage of inhibition of cell proliferation for each
concentration of TGF-0.
RESULTS
in the published sequence (7), into the pSP72 vector. Likewise, the
125-base pair Xba\-Bgl\\ fragment of TGF-/33, beginning at nucleotide
917 (8), was cloned into the pSP72 plasmid. The TGF-/31 and -ß2
plasmids were linearized with Hpal and transcribed with SP6 polymerase in the presence of "P-labeled CTP, to yield RNA probes comple
mentary to mRNA for TGF-/31 and -02. The TGF-/Ì3cDNA was
inserted in the opposite orientation of transcription and was therefore
linearized with Nde\ and transcribed with T7 polymerase. Transcription
buffers, nonradioactive nucleotides, enzymes, and the pSP72 plasmid
were from Promega (Madison, WI). RNA probes were stored in water
at -70"C and used within 4-7 days of preparation. For RNase protec
tion assays, probes for TGF-/31 and -ß2
were mixed together (1.5 x IO5
cpm each/sample) since the diagnostic '2P-labeled fragments could be
easily distinguished by size. The probe for TGF-/53 was hybridized with
samples separately (2 x IO5cpm/sample).
Preparation of Conditioned Medium. Cells were grown to near conflu
ence in T-150 plastic flasks with RPMI-5% FBS, washed twice with
30-40 ml warm PBS, and then incubated with 40-45 ml serum-free
medium containing 10 n\i 17rf-cstradiol. 1 //\i tamoxifen, or ethanol
diluent (0.1%). The medium was replaced after 48 h with fresh serumfree medium containing the appropriate drug. After a subsequent 40 h
incubation, conditioned medium was collected and the cells were har
vested and counted. Aprotinin (final concentration, 0.04 trypsin inhib
itor units/ml; Sigma) was added to the conditioned medium. Cell debris
was removed by centrifugation at 1000 x g for 15 min. Conditioned
medium was concentrated 20- to 30-fold using Centricon-10 concentra
tors with a M, 10,000 exclusion limit (Amicon). Concentrated condi
tioned medium was mixed with 0.1 volume of 1.2 N HC1, left at room
temperature for 20-30 min, and then neutralized by the addition of 0.1
volume of 1.44 N NaOH in l M HEPES, pH 7.2, to activate latent
TGF-0.
TGF-0 Bioassay. Six serial dilutions were prepared for each sample
in 96-well plates. To each well was added I x I()' mink lung epithelial
cells in a final volume of 0.15 ml minimum essential medium-5% FBS.
After 18-24 h at 37°C,20 Mlof 50 ^Ci/rnl [3H]TdR was added to each
Expression of Different TGF-0 mRNA Species in Mammary
Carcinoma Cell Lines. We have determined the expression of
TGF-01, -02, and -03 mRNA in four human breast cancer cell
lines using an RNase protection assay. Three of the cell lines,
T-47D, ZR-75-1, and MCF-7, are estrogen receptor-positive
and thus are hormone responsive. The T-47D cell line has
been reported to lack detectable levels of TGF-0 mRNA, using
a probe specific for TGF-01, while the MCF-7 and ZR-75-1
cell lines and three estrogen receptor-negative lines, including
MDA-MB-231, were shown to express TGF-/31 mRNA at
roughly equivalent levels ( 15). Other investigators have reported
that MCF-7 contained mRNA for TGF-/32 (16). We have
detected mRNA for TGF-02 and -03 in T-47D cells, -01 and
-03 in ZR-75-1 cells, -01 and -02 in MDA-MB-231 cells, and
-01 in MCF-7 cells (Figs. 1 and 2). Our T-47D cells may, under
some conditions, contain low levels of mRNA for TGF-01,
since prolonged exposure of autoradiograms occasionally re
sulted in a faintly visible band at the expected position (Fig. 1).
Expression of TGF-01 mRNA by T-47D cells at a much lower
level than in MCF-7, ZR-75-1, and MDA-MB-231 cells has
recently been confirmed (17). Our earliest experiments with
MCF-7 revealed the presence of both TGF-01 mRNA and a
low level of TGF-02 mRNA. During the subsequent course of
our investigations, however, we have been unable to detect
TGF-02 mRNA in MCF-7 cells. Interestingly, each of the four
cell lines we have evaluated has demonstrated a different pattern
of TGF-0 expression, often co-expressing more than one TGF0 species.
Effect of Estradici on TGF-0 mRNA Expression. The effects
Exp1
well. The plates were then incubated for an additional 4 h, at which
time the cells were harvested and the incorporation of radioactivity was
measured. A standard dilution curve using recombinant human K.I
ß\was prepared with each assay, and the activity of each sample was
calculated using dilutions which resulted in inhibition close to 50%.
TGF-01 and -02 have been reported to be equipotent in this assay (14).
TGF-0 Radioreceptor Assay. A549 cells were incubated overnight in
Dulbecco's modified Eagle's medium with 5% calf serum in 24-well
plates (2 x 10* cells/well) and then washed twice with assay diluent
(50:50 Ham's F12:Dulbecco's modified Eagle's medium, 0.1% BSA,
C 5 25 48
25 mM HEPES, 42.5 mM NaCI). Cell monolayers were then incubated
for 2 h at ambient temperature, in 0.2 ml assay diluent with dilutions
of concentrated conditioned medium or recombinant human TGF-01
standard to which '"I-labeled TGF-01 had been added (2 x 10" cpm,
approximately 0.13 ng TGF-01 protein). Each well was then washed 4
times with cold PBS containing 0.1% BSA. Well contents were solubilized with 0.75 ml oflO% glycerol, 10% Triton X-100,25 mM HEPES
(30 min at 37°C).TGF-0 activity in the conditioned medium was
calculated from the standard curve using a nonlinear curve-fitting
program and was expressed as TGF-01 equivalents.
Cell Proliferation Assay. MCF-7 and T-47D cells were seeded at 4 x
lO'/well in 1 ml RPMI-5% FBS in 24-well plates. The next day,
medium was replaced with fresh medium containing dilutions of recom
binant human TGF-01 (Genentech, Inc.) or porcine platelet TGF-02
(R & D Systems, Inc., Minneapolis, MN). After 3 days, medium and
TGF-0 were replaced and, after 2 additional days of culture, monolayers
were harvested with trypsin and cells were counted using a Coulter
counter. Cell counts from wells not exposed to TGF-0 were used to
Exp2
2448
ß:
ß
Fig. 1. Effect of estradici on expression of TGF-fi in T47-D cells: time course.
In Experiment 1, cells were incubated with 100 n\i estradici in serum-free medium
for 5, 25, or 48 h prior to RNA isolation, as indicated. Lane C represents a
control sample of RNA from cells incubated with ethanol (0.1 ^c) in serum-free
medium for 25 h. Experiment 2 gives results for RNA from cells incubated in
serum-free medium plus ethanol (0.1%) for 1 and 48 h (lanes C/and Ca) or 10
nM estradici for 24 and 48 h. RNase protection assays were performed as described
in "Materials and Methods." The faint TGF-fil signals shown in Experiment 2
were obtained by prolonged exposure of the autoradiogram, followed by modified
photographic technique. The positions of the protected fragments of the TGF-/32
and -/33 probes are indicated along the left.
4J. Tamm, A. Lee, and R. Derynck, unpublished observations.
300
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HORMONAL REGULATION
of estrogen treatment on the levels of the different TGF-0
mRNA species were evaluated in T-47D cells. Estradiol, at 10
and 100 nM, caused a gradual decrease of mRNA content for
both TGF-02 and -/33 over a 48-h time course (Fig. 1). Downregulation was also observed with 1 n\i estradici (not shown).
Incubation of T-47D cells in serum-free medium resulted in
increased levels of mRNA for TGF-02 and -03 over time (Fig.
1). Whether this is due to the absence of estrogenic activity
contained in serum or phenol red (18) or to some other differ
ence in culture conditions is not known.
Treatment with estradici resulted in a decrease in TGF-03
mRNA expression by another estrogen receptor-positive cell
line, ZR-75-1, while expression of TGF-01 mRNA in these
same cells was not affected by estradiol (Fig. 2). The level of
mRNA for TGF-01 was also not changed by estradiol treatment
of MCF-7 cells (Fig. 2). In contrast to the observed downregulation of TGF-02 mRNA expression in estrogen receptorpositive cell lines, the level of TGF-02 mRNA was unaffected
by estradiol treatment of the estrogen receptor-negative cell
line MDA-MB-231 (Fig. 2). Table 1 contains a compilation of
data obtained from experiments in which the relative abundance
of the three TGF-0 mRNA species was determined by the
RNase protection assay, as in Figs. 1 and 2. The expression
levels are shown as the relative ratios for estradiol-treated and
untreated control cells. Estradiol consistently resulted in downregulation of TGF-02 and -03 in the estrogen receptor-positive
cells but not in the estrogen receptor-negative cell line MDAMB-231. In contrast, the mRNA level for TGF-/31 was not
regulated by estradiol in either the estrogen receptor-positive
or estrogen receptor-negative cells. T-47D cells demonstrated
the greatest degree of TGF-0 down-regulation by estradiol, such
that by 48 h levels had dropped to just under 50% of control.
Treatment of the cells with the antiestrogen tamoxifen (1
ßM)did not consistently result in a significant effect on the
mRNA levels for any of the three types of TGF-0 in any of the
cells studied (Fig. 2), although in a few experiments TGF-02
mRNA in T-47D was slightly elevated by treatment with ta
moxifen (less than 2-fold after 48 h) (not shown). Concentra
tions of tamoxifen greater than l /UMor incubation times longer
than 48 h were not tested.
OF TGF-rf EXPRESSION
Influence of Estradiol on TGF-0 Production by T-47D Cells.
Hormonal regulation of expression of TGF-0 at the protein
level was evaluated using a mink lung cell growth inhibition
bioassay and a radioreceptor assay specific for TGF-0. Unlike
the RNase protection assay of mRNA levels, both the bioassay
and the radioreceptor assay are incapable of distinguishing
between TGF-01 and -02, and neither assay has been evaluated
for its ability to detect TGF-03 activity. Despite these limita
tions, we have observed significant and reproducible reduction
in the TGF-/3 content of conditioned medium of T-47D cells
treated with estradiol (Table 2). Without activation of latent
TGF-ß,conditioned media exhibited 5-15% of the activity of
acid-activated conditioned media, and this percentage did not
change with estrogen or tamoxifen treatment (not shown). In
two separate experiments, the ratio of TGF-0 bioactivity in the
conditioned medium of estrogen-treated and control cells was
0.52 and 0.57.
Effect of TGF-01 and -ß2on Cell Proliferation. The prolifer
ation of MCF-7 cells, growing as a monolayer on plastic, was
inhibited by human TGF-01 and porcine TGF-02. TGF-01
resulted in 50% inhibition of growth, assessed by cell counts,
at approximately 1 ng/ml (40 pM). Equivalent growth inhibition
with TGF-02 required 10 ng/ml (Fig. 3). Inadvertent loss of
bioactivity and dilutional errors were controlled for by concur
rent analysis of the medium containing TGF-ßby the mink
lung cell bioassay. When compared on the basis of the bioassay
units instead of the protein concentrations used above, TGF01 was 30- to 40-fold more potent than -02 in inhibiting the
proliferation of MCF-7 cells. Under similar conditions, T-47D
cell proliferation was unaffected by either TGF-/31 or -02, at
concentrations as high as 10 ng/ml (not shown).
DISCUSSION
Growth factors and their receptors are felt to exert critical
control over carcinogenesis and the malignant phenotype (19).
The availability of human breast cancer cell lines with and
without functional estrogen receptors has provided investiga
tors with a useful system to study hormonal regulation of cancer
(1). TGF-0 is one of many growth factors produced by and/or
affecting breast cancer cells. In recent years, we have learned
ZR-75-1
Fig. 2. Expression of TGF-01, -02, and -03
by three breast cancer cell lines: effect of estra
diol and tamoxifen. Two estrogen receptor-pos
itive cell lines (ZR-75-1 and MCF-7) and one
estrogen receptor-negative cell line (MDA-MB231) were incubated with 10 nM estradiol (lane
E), 1 JIMtamoxifen (lane T), or ethanol vehicle
(lane C) in serum-free medium for 48 h prior to
RNA isolation and analysis by the RNase pro
tection assay. The two left lanes are results from
control samples containing tRNA alone (-) or
tRNA plus in vitro transcribed cRNAs contain
ing sequences for the three TGF-0 mRNAs (+).
As in Fig. 1, the positions of the protected frag
ments specific for TGF-01, -02, and -03 are
indicated. ND, not detected.
CET
-ND-
ß;
MCF-7
C E T
C E T
-ND-
-ND-
ß3
MDA-MB231
-ND-
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HORMONAL REGULATION
Table 1 Effect of estradiol on expression ofTGF-fil, -$2, and -Ã i mRNA by
breast cancer cell lines
RNA samples prepared from near confluent monolayers of cells, after incu
bation for 48 h at 37°Cwith serum-free medium containing estradiol (10 n.M)or
ethanol vehicle, were assayed by RNase protection assays using probes specific
for each of the three forms of TGF->1 Quantitation of relative expression was by
laser densitometry of the predicted protected fragments, and values reported are
means ±SD for the ratio of two to seven pairs of samples from estradiol-treated
and control cells.
(estradiol/control)Cell
Ratio of mRNA expression
lineT47D
±0.24
±0.19
MCF-7
1.01 ±0.29
ND
ND
ZR-75-1
0.64 ±0.03
1.1 ±0.03
ND
MDA-MB-231TGF-/J1NO"
1.19±0.12TGF-020.47
1.04 ±0.01TGF-030.39
ND
" ND, not routinely detected.
Table 2 Effect of estradiol and tamoxifen on secretion ofTGF-fj activity by T47D breast cancer cells
Conditioned media were collected after a 40-h incubation of T47-D cells with
estradiol (10 mil. tamoxifen (1 J.MI. or ethanol vehicle alone. Cell monolayers
had already been previously incubated for 48 h in serum-free medium with the
indicated treatments. Conditioned media were concentrated, acid activated, and
assayed for I <.1 bioactivity by inhibition of |3H]TdR uptake by mink lung cells
and by a '"I-TGF-ii radioreceptor assay. TGF-(i activity is expressed as TGF-/31
equivalents, since TGF-J1 was used as standard in both assays. Values reported
are means ±SD of four determinations of bioactivity and eight replicates in the
radioreceptor assay.
TreatmentControl
binding
(ng/106
cells)1.24
(ng/106
cells)12.4
±1.9
±0.14
7.12 ±0.76°
0.67 ±0.11*
Estradiol
TamoxifenBioactivity 11.4 ±0.98Receptor
1.21 ±0.27
" Significantly decreased compared to untreated controls (P < 0.005).
* Significantly decreased compared to untreated controls (P < 0.0001 ).
80
40
o
Q-
20
.1
1
10
TGF-p- (ng/ml)
Fig. 3. Growth inhibition of MCF-7 by TGF-fil and -02. Cells were cultured
in serum-containing medium with a range of concentrations of TGF-/J1 (D),
), or medium alone, as described in "Materials and Methods."
that TGF-/Õcan affect a wide range of cell functions and is a
potent inhibitor of proliferation for many cell types. It has
recently become evident that early in evolution the ancestral
TGF-/3 gene was copied, resulting in multiple genes for different
types of TGF-/3 in current-day vertebrate genomes. The exist
ence of three types of human TGF-0 has now been documented,
TGF-01 (6), TGF-02 (7), and TGF-03 (8, 9). Published studies
of hormonal regulation of TGF-0 expression by MCF-7 cells
were performed prior to the isolation of cDNAs for TGF-02
and -03 (5). The experiments described in this report were
designed to reexamine this issue, using a sensitive assay capable
of detecting and distinguishing all three known types of human
TGF-/3 mRNA.
We have found that expression of TGF-02 mRNA by T-47D
cells and TGF-03 mRNA by T-47D and ZR-75-1 cells was
decreased by estradiol, whereas expression of TGF-01 mRNA
remained unchanged. The RNase protection assays measure
only the mRNA levels and thus do not distinguish between
changes in rates of RNA transcription and degradation. The
OF TGF-d EXPRESSION
levels of TGF-0 secreted into the medium were also decreased
after estradiol treatment of T-47D cells, as has been reported
(20). We have observed that 5-15% of the TGF-0 in conditioned
medium from T-47D cells was biologically active without acid
activation. Knabbe et al. (5) have previously reported that, in
unacidified conditioned medium from untreated MCF-7 cells,
a similar fraction of the TGF-0 was active.
Our observations that levels of TGF-/Ì1mRNA were unaf
fected by estrogen may apply only to the specific cell lines or
tumor types studied. Other cell lines have yielded different
results. For instance, expression of TGF-01 mRNA in an
estrogen receptor-containing osteosarcoma cell line was in
creased 2.5-fold by estradiol (21). Recently, Murphy and Dotzlaw have reported that treatment of T-47D cells with medroxyprogesterone resulted in a 3-fold reduction of TGF-01 mRNA
levels (17).
Estrogen is commonly thought to regulate gene expression
by binding to its receptor, followed by interaction of the hor
mone receptor complex with regulatory sequences of DNA
leading to enhanced transcription (22). There are, however,
many examples of down-regulation of gene expression by estro
gen, in addition to this report. Estradiol has been reported to
decrease the mRNA levels of the estrogen receptor and of the
HER-2/m>«protein in MCF-7 cells (23,24). Similarly, the level
of prolactin mRNA in a rat pituitary tumor model and of
albumin mRNA in Xenopus hepatocytes has been shown to be
decreased by estradiol (25, 26).
The bioactivities of TGF-01 and -/J2 have been compared in
various assays. In many systems, TGF-01 and -02 have been
reported to be equipotent. Thus, both proteins inhibited interleukin 1 induced murine thymocyte proliferation (27), enhanced
expression of adhesion protein receptors by murine fibroblasts
(28), inhibited DNA synthesis in mink lung epithelial cells (14),
inhibited human hematopoietic cell proliferation (29), inhibited
hepatocyte proliferation in vivo following partial hepatectomy
(30), and modulated gene expression of differentiated adipocytes (31) with roughly equivalent potency. On the other hand,
when compared with TGF-02, TGF-01 has been reported to be
8-fold more potent at inhibition of hydrogen peroxide release
by activated macrophages (32), 10-fold more potent at inhibi
tion of mitogen-stimulated proliferation of murine B-cells (33),
60-fold more potent at inhibition of DNA synthesis in bovine
aortic endothelial cells (14), and 100-fold more potent at inhi
bition of interleukin 3 stimulated proliferation of a murine
hematopoietic cell line (34). Of note, TGF-02, but not -01, has
demonstrated activity in a Xenopus mesoderm induction assay
(35).
We compared the bioactivities of TGF-01 and -02 by their
effects on the proliferation of MCF-7 and T-47D cells. Growth
inhibition of MCF-7 was observed with both TGF-01 and -02,
whereas T-47D cell growth was unaffected by either protein.
We have determined that TGF-01 was at least 10-fold more
potent than TGF-02 in inhibiting proliferation of MCF-7 cells.
Other investigators have reported that TGF-01 inhibits the
anchorage-independent growth of MCF-7 (5, 10). In contrast,
Karey and Sirbasku (36) have reported that MCF-7 cells were
not growth inhibited by TGF-01, but the serum-free growth
medium alone supported only one cell doubling in their un
treated cells over the 8-day assay period. Arteaga et al. (37)
have reported that none of the three estrogen receptor-positive
cell lines used in this study, including MCF-7, were growth
inhibited by TGF-01 in serum-containing medium but that
MDA-MB-231 cells were. Evidently, the ability to demonstrate
growth inhibition of human breast cancer cells depends on
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HORMONAL
REGULATION OF TGF-.J EXPRESSION
many factors and may vary with different subclones, culture
conditions, and growth assays.
In summary, we have demonstrated that hormonal regulation
of gene expression can differ among the three members of the
human TGF-/3 family. Our results clearly indicate the need for
future analyses to evaluate the expression of the different TGFßspecies and not only TGF-/31. Examination of TGF-01
mRNA expression alone, especially in cells with significant
levels of TGF-02 or -ß3mRNA, may yield incomplete and
potentially misleading conclusions. The existence of multiple
species of TGF-ß,each with independent regulation of mRNA
expression, may reflect differences in function. Future studies
comparing the activities of TGF-01, -/32, and -/33 will help
clarify this issue.
ACKNOWLEDGMENTS
The authors wish to express their gratitude to Ann Rowland for her
assistance with the TGF-0 radioreceptor assays.
Note Added in Proof
We have isolated clones of MCF-7 transfected with a plasmid car
rying a neomycin resistance gene (pRSV-neo) that contain mRNA for
TGF-02 and/or -03 in addition to TGF-01 mRNA. The effect of
estradiol on TGF-0 mRNA levels was evaluated as in Table 1 using a
clone which expressed all three species of TGF-0. The means ±SD for
the ratios of four pairs of estradiol-treated and control cells were 0.99±
0.11, 0.64±0.16,and 0.61 ±0.16for TGF-01, -ß2,
and -03, respectively.
Thus, data from these cells are consistent with our observation that
estradiol down-regulated levels of TGF-02 and -03 mRNA but had no
effect on mRNA levels of TGF-01.
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Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1990 American Association for Cancer Research.
Differential Regulation of Expression of Three Transforming
Growth Factor β Species in Human Breast Cancer Cell Lines by
Estradiol
Bradley A. Arrick, Murray Korc and Rik Derynck
Cancer Res 1990;50:299-303.
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