Download Role of c-myc in Tamoxifen-Induced Apoptosis in Estrogen

REPORTS
Role of c-myc in TamoxifenInduced Apoptosis in
Estrogen-Independent Breast
Cancer Cells
Yuan Kang, Robert Cortina,
Roger R. Perry*
Background: The antiestrogen tamoxifen (TAM) is effective in the treatment
of estrogen receptor (ER)-positive as
well as some ER-negative breast cancers. However, the precise mechanism
of action of TAM, especially in estrogen-independent cells, remains unclear. Previous work by our laboratory
has demonstrated that TAM induces
the morphologic and biochemical changes that are characteristic of apoptosis
in both ER-positive and ER-negative
cells. Purpose: We compared the effect
of TAM at a clinically achievable concentration on cell growth and apoptosis
with the effect of TAM on c-myc (also
known as C-MYC) messenger RNA
(mRNA) and protein expression in ERnegative MDA-231 cells. Methods:
MDA-231 cells were treated for up to
72 hours with 1.0 \iM TAM alone or in
the presence of 50 \iM c-myc antisense
or nonsense oligonucleotides. c-myc
mRNA expression was determined by
northern blot analysis, protein expression by western blot analysis, cell
growth inhibition by cell counts, and
DNA cleavage by agarose gel electrophoretic analysis. Differences between
the mean values from different treatment groups were compared with the
use of the two-sided Wilcoxon ranksum test. Results: TAM treatment for
72 hours increased c-myc mRNA fivefold (from a relative radiolabeled
hybridization signal intensity of 17 ± 4
up to 93 ± 10; P<.O5) and c-Myc pro-
tein threefold (from a relative immunofluorescence signal intensity of 28 ± 7
up to 83 ± 21; P<.05). The induction of
c-myc by TAM was accompanied by
internucleosomal DNA cleavage characteristic of apoptotic cell death. Addition of c-myc antisense oligonucleotide
(5-CACGTTGAGGGGCAT-30 to MDA231 cells resulted in a nearly twofold
decrease of basal c-myc mRNA (P<.05)
and a sevenfold decrease of basal cMyc protein (/'•c.OS) expression. Addition of c-myc antisense oligomer also
antagonized the TAM-induced increase
in c-myc mRNA (P<.05) and protein
expression (/><.O5) and inhibited TAMinduced cytostasis (P<.0l) and apoptosis. In parallel experiments, addition
of the nonsense oligomer had no effect
on any of the measured parameters.
Conclusions: These results indicate
that the effects of TAM on ER-negative
MDA-231 cells may be mediated
through c-myc overexpression. c-myc
may play a critical role in the growth
and progression of MDA-231 breast
cancer cells. [J Natl Cancer Inst 1996;
88:279-84]
Breast cancer is the most common cancer among women and the leading cause
of death among nonsmoking women. The
antiestrogen tamoxifen (TAM) is widely
used for the treatment of women with
breast cancer. It appears that TAM has
both estrogen receptor (ER)-mediated and
non-ER-mediated cytostatic activity (/).
Several clinical studies [for review, see
(2)] have shown that the effectiveness of
TAM is independent of tumor ER status.
However, the mechanism of action of
TAM, particularly its apparent activity
against cells that lack the ER, is poorly
understood. Our laboratory has recently
shown that TAM induces dose- and timedependent apoptosis in ER-positive
MCF-7 and ER-negative MDA-231 human breast cancer cells (3). TAM induced
characteristic morphologic changes consistent with apoptosis, including com-
Journal of the National Cancer Institute, Vol. 88, No. 5, March 6, 1996
paction and margination of nuclear chromatin, condensation of cytoplasm, convolution of nuclear and cell outlines, and
typical biochemical changes such as internucleosomal DNA fragmentation.
Several studies (4-8) have examined
the role of the c-myc (also known as CMYC) proto-oncogene in cellular proliferation, cellular transformation and
mitogenesis, and programmed cell death
(apoptosis). Blockage of c-myc expression with c-myc antisense oligonucleotide
has confirmed that c-myc is crucial for
cell proliferation (4). In transformed cells,
c-myc expression is a prerequisite for mitogen-induced cell growth. Deregulation
of c-myc expression is associated with
apoptotic cell death (5). Expression of cMyc protein has been shown to be critical
for the growth of both hormone-dependent and hormone-independent breast cancer cells (6,7). Estrogen can stimulate
c-myc messenger RNA (mRNA) and
protein accumulation in hormone-responsive, ER-positive human breast cancer
cell lines (8). In addition, a specific
estrogen-responsive region of the c-myc
gene has been localized to a 116-basepair (bp) region located from 25 to 141
bp upstream from the PI promotor
transcriptional initiation site (8). TAM
has exhibited both estrogen antagonistic
and agonistic effects on the regulation of
c-myc expression in ER-positive human
breast cancer cells (9,10). However,
preliminary work in our laboratory
demonstrated that TAM at a \-]iM concentration, a concentration that is clinically relevant (//), had no effect on c-myc
expression in ER-positive MCF-7 cells.
* Affiliation of authors: Division of Surgical Oncology, Eastern Virginia Medical School, Norfolk.
Correspondence to: Roger R. Perry, M.D., Eastern Virginia Medical School, 825 Fairfax Ave.,
Suite 610, Norfolk, VA 23507-1912.
See "Notes" section following "References."
REPORTS
279
Recently, we have shown that activation of the apoptotic pathway by TAM in
ER-negative breast cancer cells is independent of an ER-mediated pathway (3).
The purpose of this study was to compare
the effect of TAM at a clinically achievable concentration on cell growth and
apoptosis with the effect of TAM on cmyc mRNA and protein expression in
ER-negative MDA-231 cells.
metric analysis of the uptake of fluorescein-labeled
oligomers has shown that treatment of cells with
100 U/mL streptolysin O resulted in higher fluorescence intensity without toxicity (data not shown).
For all experiments, the untreated controls consisted of MDA-231 cells incubated in media containing the same additives for the same period of
tune as the TAM and/or oligomer-treated cells. Control and treated cells were analyzed as described
below.
Materials and Methods
Total cellular RNA was extracted from cells by
a modified one-step guanidinium isothiocyanatephenol—chloroform extraction procedure previously
described (/3). RNA (5 |ig) was denatured in 50%
formamide (Fluka Chemical Corp., Ronkonkoma,
NY) containing 2.5-mM 3-(/V-morpholino) propanesulfonic acid (pH 7.0), \-mM sodium acetate, 1-mM
EDTA, and 7.4% formaldehyde (Sigma Chemical
Co.). The denatured RNA was electrophoretically
separated in 1% agarose-formaldehyde gels. The
presence of intact and equal amounts of RNA in
each lane was confirmed by the ethidium bromide
fluorescence pattern of the gel before transfer and of
the blot after transfer. A 0.5-kilobase (kb) complementary DNA (cDNA) probe corresponding to
exon 2 of the human c-myc Pst l-Pst I fragment and
a 0.8-kb EcoR\-Hindll\ fragment of human glyceraldehyde-3-phosphate dehydrogenase
(GAPDH)
cDNA were purchased from the ATCC. The probes
were labeled with [a-32P]deoxycytidine triphosphate (specific activity, 3000 Ci/mmol; Amersham
Life Science, Inc., Arlington Heights, IL) by the use
of a random priming PRIME-IT Kit (Stratagene,
San Diego, CA) per the manufacturer's instructions.
The RNA was transferred onto a Duralon-UV
membrane (Stratagene) by capillary blotting in 20x
SSC (lx SSC = 150 mM sodium chloride and 15
mM sodium citrate) for 24 hours. The blots were
UV cross-linked (Stratalinker, Stratagene) to immobilize the nucleic acid. Prehybridization was performed for 3 hours at 42 'C in 50% formamide, 10%
dextran sulfate, lOx Denhardt's solution, 20 mM
Tris-HCI (pH 7.5), 5x SSC, 0.1% sodium dodecyl
sulfate (SDS), and 100 Hg/mL salmon sperm DNA.
Hybridization was performed with the cDNA coding
for c-myc for 20 hours at 42 'C. The membranes
were washed four times for 30 minutes at 65 *C with
0.2x SSC, and 0.1% SDS. Blots were exposed to
XAR5 film at -70 "C for 72 hours To control for
loading and transfer, we stripped the blots by washing the membrane for at least 2 hours at 75 'C in 1
mAf Tris-HCI (pH 8.0), 0.2 mM EDTA (pH 8.0),
0.05% sodium pyrophospate, and O.lx Denhardt's
solution. Rehybridization was then performed with
the use of the cDNA coding for GAPDH. The intensities of the c-myc mRNA bands were normalized to
the GAPDH mRNA bands with the use of Image
1.41 software (Division of Cancer Treatment, National Cancer Institute, Bethesda, MD).
The MDA-231 human breast cancer cell line,
which is ER negative, was obtained from the
American Type Culture Collection (ATCC, Rockville, MD). The cells were cultured in improved
minimum essential medium supplemented with 5%
fetal calf serum. Forty-eight hours before each experiment, cells were placed in phenol red-free
medium with charcoal dextran-stripped fetal calf
serum to remove endogenous steroids (12). These
are the same conditions that we used in our previous
expenmems, although not strictly necessary here,
since we have shown that estrogen does not inhibit
TAM-mduced apoptosis in MDA-231 cells (3).
TAM was provided by the Zeneca Pharmaceuticals Group (Wilmington, DE). A 1-ujM TAM dose
was chosen for these experiments because such
levels are clinically achievable with long-term TAM
treatment (//) and have been shown in preliminary
experiments to induce cell growth or apoptosis,
depending on the exposure time. MDA-231 cells
were treated for up to 72 hours with TAM alone or
with the addition of oligomers as described below.
The c-myc antisense oligodeoxynucleotide used
was a 15-mer phosphorothioate directed to the
region of translation initiation on the corresponding
c-myc mRNA (J'-CACGTTGAGGGGCAT-y; Bestar Technology, Cincinnati, OH). The control
oligonucleotide consisted of the same nucleotide
composition as the c-myc oligonucleotide but in
random order (5'-AGTGGCGGAGACTCT-3'). The
oligomers were synthesized on an automated solidphase synthesizer (model 380B, Applied Biosystems, Berkeley, CA), purified by reverse-phase
high-performance liquid chromatography followed
by gel electrophoresis, and precipitated twice with
ethanol. The last two phosphodiesters at each end of
the oligomer were thioated. Concentrations of oligomers were determined by densitometry. The oligomers were dissolved in phosphate-buffered saline
(PBS) and stored at -20 "C. A 50-|iM dose was
chosen on the basis on our preliminary experiments
demonstrating efficacy of this dose in inhibiting cmyc expression without significant cytotoxicity
(data not shown). Every 24 hours, the cells were
washed once with permeabilization buffer (137 mM
NaCI. 100 mM piperazine-/V7/-bis(2-ethanesulfonic
acid): 1,4-piperazine diethanesulfonic acid, pH 7.4,
56 mM glucose, 27 mM KCI, 27 mM ethyleneglycol-bis-{p-amino ethyl ether) A'//,N'JV,tetraacetic acid, 1 mM Na-adenosine triphosphate,
and 0.1% bovine serum albumin) and then treated
with 100 U/mL streptolysin O (Sigma Chemical
Co., St. Louis, MO) in permeabilization buffer for
30 minutes. Fresh medium containing antisense or
nonsense oligomers was then added. Flow cyto-
280
REPORTS
Northern Blot Analysis
SDS-PAGE and Western Blot Analysis
To detect c-Myc protein, we prepared cell
lysates, and protein content was determined by the
Bradford method (14). One hundred micrograms of
protein per lane was fractionated with the use of
SDS-10*3: polyacrylamide gel electrophoresis. The
proteins were electrophoretically transferred to a
nitrocellulose membrane (Bio-Rad Laboratories,
Mountain View, CA). The membrane was then
blocked for 4 hours with 2% nonfat milk powder in
PBS-0.2% Tween 20 and then incubated for 12
hours at 4 'C with 9E10 monoclonal antibody to
human c-myc (Oncogene Science, UniondaJe, NY)
at a concentration of 1.5 Hg/mL in blocking solution. The membrane was washed with PBS-0.2%
Tween 20 and treated with 1:5000 dilution of
alkaline phosphatase-labeled goat anti-mouse immunoglobulin G, (Sigma Chemical Co.) for 1 hour
at 24 'C. Bands were visualized by incubating the
membrane for 30 minutes in the substrate solution
containing 5-bromo-4-chloro-3-indolyl phosphatenitroblue tetrazolium (Sigma Chemical Co.). The intensities of the c-myc bands were quantitated with
densitometric scanning (SCI-scan 5000 USB densitometer, United States Biochemicals, Cleveland,
OH).
Measurement of Growth Inhibition
and Apoptosis
For analysis of the effects of c-myc antisense on
TAM-induced cell growth inhibition, MDA-231
cells were plated at 1.0 x 10 /cm and grown in T25
flasks. After 24 hours, c-myc antisense or nonsense
oligomers at a final concentration of 50 \iM were
added to the medium with or without 1 \)M TAM.
At various times, the attached cells were trypsmized
and then washed twice with cold PBS containing
1 mM EDTA. Cell number was determined by using
a Coulter counter, and cell viability from the same
diluted cell suspension was determined by trypan
blue dye exclusion with the use of a hemocytometer.
A minimum of five fields were examined by microscope for each count. Each dilution was counted at
least three times to determine each experimental
point; the experiments were independently repeated
six times. For assessment of apoptosis, DNA was
extracted from 1.0 x 106 viable cells from each
group and DNA cleavage was determined by
agarose gel electrophoresis as previously described
(3).
Statistical Methods
All results arc expressed as mean ± standard
deviation. Differences between the mean values
from different treatment groups were compared with
the use of the two-sided Wilcoxon rank-sum test.
Results
Effects of TAM and c-myc Antisense
Oligonucleotide on Expression of
c-myc mRNA
To determine the effect of TAM on the
expression of c-myc mRNA in MDA-231
cells, we performed northern blot
hybridization analysis. The c-myc mRNA
levels were adjusted for unequal loading
by rehybridizing the blots (after stripping
the labeled c-myc probe) with a labeled
GAPDH probe and then normalizing the
c-myc mRNA levels by the GAPDH
Journal of the National Cancer Institute, Vol. 88, No. 5, March 6, 1996
mRNA levels. Twenty-four-hour incubation of MDA-231 cells with TAM resulted in an increase of c-myc mRNA
expression at doses of less than 5 \iM and
a decrease of c-myc mRNA expression at
doses of greater than 10 \xM (data not
shown). We chose to focus our efforts on
levels clinically achievable with longterm TAM use (7/); hence, a \-\i\t TAM
dose was chosen to study the time-dependent effects of TAM on c-myc mRNA
and protein expression. TAM (1.0 |iAf)
induced a significant increase in c-myc
mRNA expression in MDA-231 cells,
with a maximum fivefold response noted
at 72 hours (P<.05; Fig. 1). A 72-hour
treatment of the cells with 50 \xM c-myc
antisense oligomer caused a nearly twofold decrease in the basal c-myc mRNA
level (P<.05), which could be a result of
activation of a specific RNase (75). No
decrease of c-myc mRNA was noted in cmyc nonsense oligomer-treated cells. The
c-myc antisense oligomer also antagonized the TAM-induced increase of
c-myc mRNA expression (P<.05), but the
nonsense oligomer had no effect.
Effects of TAM and c-myc Antisense
Oligonucleotide on c-Myc Protein
To investigate the effect of TAM on cMyc protein expression in MDA-231
cells, we performed a western blot
analysis. TAM stimulated a steady increase in c-Myc protein expression during
the 72-hour treatment period, with a maximum threefold increase at 72 hours
(P<.05; Fig. 2). This corresponded with
the observed accumulation of c-myc
mRNA at 72 hours (Fig. 1). A specific
sevenfold inhibition of basal c-Myc
protein expression by c-myc antisense
oligomer was noted (P<.05; Fig. 2). The
c-myc antisense oligomer also antagonized the TAM-induced increase of c-Myc
protein expression (P<.05). The c-myc
nonsense oligomer had no effect on basal
c-Myc protein levels or on the levels induced by TAM.
Growth Response to TAM and c-myc
Antisense Oligonucleotide
The growth response data for MDA231 cells treated with 1.0 \iM TAM are
summarized in Fig. 3. Slight stimulation
of cell growth by 1.6-fold (/><.01) was
observed when cells were treated with
TAM for 24 hours. TAM treatment for
Fig. 1. Effects of tamoxifen (TAM) and c-myc antisense oligodeoxynucleotide on c-myc messenger
RNA (mRNA) expression. MDA-231 breast
cancer cells were treated
for up to 72 hours with 1
\iM TAM alone or in the
presence of 50 \iM c-myc
antisense or nonsense
ohgonucleotides. Cellular
RNA was then extracted
and subjected to northern
blot analysis. Results
shown are representative
of the four experiments
performed. Lane: 1 =
control (media plus additives for 72 hours); 2 =
TAM for 24 hours; 3 = TAM for 48 hours; 4 = TAM for 72 hours; 5 = c-myc antisense alone for 72 hours; 6
= c-myc antisense + TAM for 72 hours; 7 = c-myc nonsense for 72 hours; and 8 = c-myc nonsense + TAM
for 72 hours. Relative intensities of the c-myc mRNA band in each lane from four experiments were quantitated and normalized as described in the "Materials and Methods" section with results (mean ± standard
deviation) as follows: 1 = 17 ± 4 ; 2 = 29 ± 6; 3 = 82 ± 13; 4 = 93 ± 10; 5 = 9 ± 5; 6 = 24 ± 12; 7 = 34 ± 11;
and 8 = 81 ± 17.
longer intervals resulted in growth inhibition as compared with untreated control
cells. TAM resulted in a twofold decrease
(f<.01) at 48 hours and a nearly fivefold
decrease (/><.01) at 72 hours in cell numbers compared with untreated control
cells. The c-myc antisense oligomer alone
inhibited cell growth at 48 hours by
twofold (P<.0l) and at 72 hours by more
than twofold (P<.01), suggesting the importance of c-myc expression in cell
growth. Co-incubation of the cells with cmyc antisense oligomer also partially
reversed the TAM-induced cytostatic effect at 48 hours (P<.0\) and at 72 hours
(P<.0\). These results indicate that overexpression of c-myc could be responsible
for both cell cycle progression and TAMinduced cytostasis. The c-myc nonsense
oligonucleotide had no effect on cell
1 2
growth or on the response of cells to
TAM (Fig. 3, B).
Effect of c-myc Antisense
Oligonucleotide on TAM-induced
Apoptosis
Continuous incubation of MDA-231
cells with 1.0 \xM TAM induced apoptosis with kinetics as shown in Fig. 4. Intemucleosomal DNA ladders were absent
in untreated cells and in cells treated by
1.0 \iM TAM for 24 hours. DNA ladders
were clearly present in cells treated with
1.0 ]iM TAM for 48-72 hours, corresponding with TAM-induced inhibition
of cell growth (Fig. 3). To determine if
induction of c-Myc protein by TAM is
the cause, rather than the effect, of TAMinduced apoptosis, we tested the cells
3
4
5
6
7
8
Fig. 2. Effects of tamoxifen (TAM) and c-myc antisense oliogodeoxynucleotide on c-Myc protein expression. MDA-231 breast cancer cells were treated, for up to 72 hours with 1 \iM TAM alone or in the presence
of 50 \lM c-myc antisense or nonsense oligodeoxynucleotides. Cellular protein was then extracted and subjected to western blot analysis. The results shown are typical of the four experiments performed. Lane: 1 =
control (media + additives for 72 hours); 2 = TAM for 24 hours; 3 = TAM for 48 hours; 4 = TAM for 72
hours; 5 = c-myc antisense alone for 72 hours; 6 = c-myc antisense + TAM for 72 hours; 7 = c-myc nonsense
for 72 hours; and 8 = c-myc nonsense + TAM for 72 hours. Relative intensities of the c-Myc protein band in
each lane from four experiments were quantitated as described in the "Materials and Methods" section with
results (mean ± standard deviation) as follows: l = 2 8 ± 7 ; 2 = 3 4 ± 9 ; 3 = 6 O ± l l ; 4 = 8 3 ± 2 1 ; 5 = 4 ± 2 ; 6
= 31 ± 13; 7 = 26 ± 8 ; and 8 = 88 ±14.
Journal of the National Cancer Institute, Vol. 88, No. 5, March 6, 1996
REPORTS
281
Fig. 3. Effects of c-myc antisense oligodeoxynucleotide on tamoxifen (TAM)-induced growth inhibition. MDA-231 breast cancer cells were grown in 25-cm flasks.
At various times the cells were trypsinized and counted. Results are shown in two parts to assist with clarity: A) Cells incubated with media + additives alone (control, O), 1 |JJW TAM (•), 50 \lM c-myc antisense oligodeoxynucleotide (A), or TAM + c-myc antisense oligodeoxynucleotide (A); and B) cells incubated with
media + additives alone (control, O), ' ]iM TAM ( • ) , 50 \iM c-myc nonsense oligodeoxynucleotide ( • ) , or TAM + c-myc nonsense oligodeoxynucleotide (•).
Data from six separate experiments. Point = mean viable cell number, bar = standard deviation.
with 1.0 \LM TAM for 72 hours in the
presence of 50 \iM c-myc antisense oligonucleotide. As shown, c-myc antisense
oligomer provided protection against
TAM-induced apoptosis (Fig. 4). By contrast, c-myc nonsense oligonucleotide
was unable to prevent cells from undergoing TAM-induced apoptosis. Antisense
or nonsense oligomers alone did not induce DNA cleavage. Thus, the ability of
c-myc antisense oligomer to protect
against TAM-induced apoptosis appears
to be a specific effect of down-regulating
1 2
3
4
5
6
7
(i.e., decreasing) the expression of c-myc
mRNA and protein (Figs. 1 and 2).
Discussion
The antiestrogen TAM is commonly
used in the treatment of postmenopausal
women with ER-positive breast cancer.
The effect of this agent is not limited to
competitive inhibition of estrogen binding to its receptors. The results of growthinhibition experiments have shown that
the mechanism of action of TAM is
8
Fig. 4. Effects of c-myc antisense oligodeoxynucleotide on tamoxifen (TAM)-induced DNA
cleavage. MDA-231 breast cancer cells were
treated for up to 72 hours with 1 \iM TAM
alone or with the addition of 50 \iM c-myc
antisense or nonsense oligodeoxynucleotides.
DNA was isolated and cleavage determined
with the use of agarose gel electrophoresis.
Gel shown is representative of the four assays
performed. Lane: I = control (media + additives for 72 hours); 2 = TAM for 24 hours; 3 =
TAM for 48 hours; 4 = TAM for 72 hours; 5 =
c-myc antisense alone for 72 hours: 6 = c-myc
nonsense alone for 72 hours: 7 = c-myc antisense + TAM for 72 hours: and 8 = c-myc
nonsense + TAM for 72 hours.
282
REPORTS
somewhat different in ER-positive cells
compared with ER-negative cells (3).
TAM-induced cytostasis and apoptosis
occur via an ER-mediated process in
ER-positive cells and via an estrogen-independent mechanism in ER-negative
cells. Other antiestrogens, including 4hydroxytamoxifen (76) and toremifene
{17), also induce apoptosis in breast cancer cells.
Usually, TAM inhibition of cell proliferation is associated with a transition
delay or block in the early to mid-Gj
phase of the cell cycle (18). In addition to
inhibiting cell growth, data from our
laboratory (3) and others (19) have shown
that TAM has a stimulatory effect on cell
growth in both ER-positive and ER-negative cells at low doses or at higher doses
given for short periods of time. The
stimulatory effect of TAM on ER-positive cells may be because of its partial
estrogen-like activity (20). Marked increases of insulin-like growth factor binding proteins by antiestrogens could also
explain the stimulatory effect of TAM on
ER-positive cells (21). However, the precise mechanism whereby TAM promotes
cell growth or inhibits cell growth and induces apoptosis in ER-negative cells
remains unclear.
Several distinct but coupled functions
have been ascribed to c-myc expression.
Expression of the c-myc proto-oncogene
is closely correlated with cell prolifera-
Journal of the National Cancer Institute, Vol. 88, No. 5, March 6, 1996
tion and differentiation as well as programmed cell death. In ER-positive cells,
the c-myc gene is estrogen-inducible and
the c-Myc protein appears to be responsible for the effects of ER stimulation on
cell growth (6). Transient ER-related increases in the expression of the c-myc
gene have been observed in a variety of
estrogen-responsive tissues (22). In addition, inhibition of the estrogen-induced
expression of c-Myc protein by an antisense oligonucleotide resulted in the arrest
of estrogen-stimulated cell proliferation
(4). In contrast, c-myc appears to have a
different role in ER-negative breast cancer cells, where constitutive expression of
the c-myc gene has been noted (23).
TAM has rapid and transient effects on cmyc expression in ER-positive breast
cancer cells (8) compared with the longer
time period required in ER-negative
MDA-231 cells (Figs. 1 and 2).
Our data have demonstrated that a
clinically relevant concentration of TAM
induces overexpression of c-myc mRNA
and protein in MDA-231 cells in a timedependent manner (Figs. 1 and 2). The increase in c-myc mRNA corresponded
with peak translational induction of cMyc protein at 72 hours, indicating that
TAM regulates c-myc expression at the
transcriptional level. TAM incubation for
less than 24 hours resulted in stimulation
of cell growth, which corresponded with
the TAM-induced increase in c-myc expression. In contrast, further exposure of
the cells to TAM induced cytostasis via
apoptosis (Fig. 4) accompanied by a further increase of c-myc expression. These
data clearly suggest that the levels of cmyc expression determined whether
stimulation of cell growth or the induction of cytostasis and apoptosis would
occur. This conclusion is supported by
the antisense experiments where 50 |iAf
c-myc antisense oligonucleotide was able
to partially reverse the cytostatic and
apoptotic effects of TAM. This effect appears to be specific, since c-myc nonsense oligonucleotide had no effect on
basal c-myc levels or on the response of
cells to TAM.
This study did not address the precise
mechanism of c-myc induction, but other
studies (2425) indicate that there is considerable variation in the mechanism of cmyc activation, depending on the mitogen
and cell type being studied. Our data
show that TAM affects c-myc expression
at the transcriptional level, but how this
occurs is uncertain. Because these cells
are ER negative, the mechanism of TAM
induction or inhibition of c-myc gene expression through direct ER-related transactivation does not apply (7). TAM may
deregulate expression of transforming
growth factors (TGFs), a or p\ in ERpositive and ER-negative human breast
cancer cells (2627). Since TGFs may
directly regulate c-myc expression at the
level of transcriptional initiation in cells
that constitutively express c-myc (25), it
is possible that the effect of TAM on
TGFs may result in overexpression of cmyc. Another possibility is that TAM
may affect expression of bcl-2 (29) or the
p53 tumor suppressor gene (30), resulting
in complementary alteration of c-myc expression (3J J2). These areas are currently being investigated by our laboratory.
Because TAM-induced c-myc expression
has been shown to be associated with
either cell growth stimulation or apoptosis, which are two distinct c-myc actions, it is unlikely that TAM interferes
with or promotes c-myc functional activity related to Myc-Max heterodimer
formation (55).
Our data in this model system have
shown that the induction of c-myc mRNA
and protein expression by TAM leads to
apoptosis. Other data from our laboratory
(5) and the work of others (18) have
shown that TAM also induces a significant G|/Go cell cycle arrest. Thus,
TAM treatment of MDA-231 cells may
lead to intracellular signals with opposite
effects, depending on the TAM dose or
treatment time: signals that induce G|/Go
arrest and other signals, such as increased
c-myc, which promote cellular proliferation. Similar situations have been identified in other models that undergo
apoptosis, such as fibroblasts with dysregulated c-myc exposed to serum starvation (5). Our results suggest that there is a
threshold concentration of c-Myc protein
for the induction of apoptosis to occur.
In summary, we have shown that TAM
at a clinically achievable concentration
can induce apoptosis in ER-negative
MDA-231 cells. TAM-induced apoptosis
in these cells was accompanied by overexpression of c-myc. The effects of TAM
on c-myc expression and apoptosis can be
inhibited in a specific manner by a c-myc
Journal of the National Cancer Institute, Vol. 88, No. 5, March 6, 1996
antisense oligonucleotide but not by a
c-myc nonsense oligonucleotide. These
results indicate that the effects of TAM
on estrogen-independent cell growth may
be mediated through regulation of c-myc
expression.
References
(/) Reddel RR, Murphy LC, HaJI RE, Sutherland
RL. Differential sensitivity of human breast
cancer cell lines to the growth-inhibitory effects of tamoxifen. Cancer Res 1985:45:152531.
(2) Jaiyesimi IA, Buzdar AU, Decker DA.
Hortobagyi GN. Use of tamoxifen for breast
cancer: twenty-eight years later [see comment
citation in Medline]. J Clin Oncol 1995;13:
513-29.
(3) Perry RR, Kang Y, Greaves B. Effects of
tamoxifen on growth and apoptosis of estrogen-dependent and -independent human breast
cancer cells. Ann Surg Oncol 1995:2:238-45.
(4) Watson PH, Pon RT, Shiu RP. Inhibition of
c-myc expression by phosphorothioate antisense oligonucleotide identifies a critical role
for c-myc in the growth of human breast cancer. Cancer Res 1991:51:3996-4000.
(5) Evan GI, Wyllie AH, Gilbert CS, Littlewood
TD, Land H, Brooks M, et al. Introduction of
apoptosis in fibroblasts by c-myc protein. Cell
1992;59:119-28.
(6) Dubik D, Shiu RP. Transcriptional regulation
of c-myc oncogene expression by estrogen in
hormone-responsive human breast cancer
cells. J Biol Chem 1988;263:12705-8.
(7) Dubik D, Dembinski TC, Shiu RP. Stimulation of c-myc oncogene expression associated
with estrogen-induced proliferation of human
breast cancer cells. Cancer Res 1987;47:6517-21.
(8) Dubik D, Shiu RP. Mechanism of estrogen activation of c-myc oncogene expression. Oncogene 1992;7:1587-94.
(9) Wosikowski K, Kung W, Hasmann M, Loser
R, Eppenberger U. Inhibition of growth-factoractivation proliferation by anti-estrogens and
effects on early gene expression of MCF-7
cells. Int J Cancer 1993;53:29O-7.
(10) Vink-van Wijngaarden T, Pols HA, Buurman
CJ, van den Bemd GJ, Dorssers LC, Birkenhager JC, et al. Inhibition of breast cancer cell
growth by combined treatment with vitamin
D3 analogues and tamoxifen. Cancer Res
1994;54:5711-7.
(//) Lien EA, Solheim E, Lea OA, Lundgren S,
Kvinnsland S, Ueland PM. Distribution of 4hydroxy-A/-desmethyltamoxifen and other
tamoxifen metabolites in human biological
fluids during tamoxifen treatment. Cancer Res
1989;49:2175-83.
(12) Reddel RR, Murphy LC, Sutherland RL. Factors affecting the sensitivity of T-47D human
breast cancer cells to tamoxifen. Cancer Res
1984;44:2398-4O5.
(13) Kang Y, Perry RR. Effect of alpha-interferon
on P-glycoprotein expression and function and
on verapamil modulation of doxorabicin resistance. Cancer Res 1994;54:2952-8.
(14) Bradford MM. A rapid and sensitive method
for the quantitation of microgram quantities of
protein utilizing the principle of protein-dye
binding. Anal Biochem 1976;72:248-54.
(15) Giles RV, Spiller DG, Tidd DM. Detection of
ribonuclease H-generated mRNA fragments in
human leukemia cells following reversible
REPORTS
283
(16)
(17)
(18)
(19)
(20)
(2/)
(22)
(23)
(24)
(25)
(26)
(27)
(28)
(29)
(30)
(31)
(32)
284
membrane permeabilization in the presence of
antisense oligodeoxynucleotides. Antisense
ResDev 1995:5:23-31.
Bardon S, Vignon F, Montcoumer P,
Rochefort H. Steroid receptor-mediated cytotoxicity of an antiestrogen and an antiprogestin
in breast cancer cells. Cancer Res 1987;47:
1441-8.
Warri AM, Huovinen RL, Laine AM,
Manikainen PM, Harkonen PL. Apoptosis in
toremifene-induced growth inhibition of
human breast cancer cells in vivo and in vitro.
J Natl Cancer Inst 1993:85:1412-8.
Osbome CK, Boldt DH, Estrada P. Human
breast cancer cell cycle synchronization by
estrogens and anliestrogens in culture. Cancer
Res 1984:44:1433-9.
Hug V, Hortobagyi GN, Drewinko B, Finders
M. Tamoxifen-citrate counteracts the antitumor effects of cytotoxic drugs in vitro. J Clin
Oncol 1985:3:1672-7.
Katzenellenbogen BS, Norman MJ, Eckert
RL, Peltz SW, Mangel WF. Bioactivities, estrogen receptor interactions, and plasminogen
activator-inducing activities of tamoxifen and
hydroxy-tamoxifen isomers in MCF-7 human
breast cancer cells. Cancer Res 1984:44:112-9.
Pratt SE, Pollak MN. Estrogen and antiestrogen modulation of MCF7 human breast
cancer cell proliferation is associated with
specific alterations in accumulation of insulinlike growth factor-binding proteins in conditioned media. Cancer Res 1993,53:5193-8.
Rempel SA, Johnston RN. Steroid-induced
cell proliferation in vivo is associated with increased c-myc proto-oncogene transcript abundance. Development 1988; 104:87-95.
Shiu RP, Watson PH, Dubik D. C-myc oncogene expression in estrogen-dependent and
-independent breast cancer. Clin Chem
1993:39:353-5.
Leder A, Pattengale PK, Kuo A, Stewart TA,
Leder P. Consequences of widespread deregulation of the c-myc gene in transgenic
mice: multiple neoplasms and normal development. Cell l986;45:485-95.
Chemey BW, Bhatia K, Tosato G. A role for
deregulated c-Myc expression in apoptosis of
Epstein-Barr virus-immortalized B cells. Proc
Natl Acad Sci U S A 1994;91:12967-71.
Jeng MH, ten Dijke P, Iwata KK, Jordan VC.
Regulation of the levels of three transforming
growth factor beta mRNAs by estrogen and their
effects on the proliferation of human breast cancer cells. Mol Cell Endocrinol 1993:97:115-23.
Noguchi S, Motomura K, Inaji H, Imaoka S,
Koyama H. Down-regulation of transforming
growth factor-alpha by tamoxifen in human
breast cancer [see comment citation in Medline]. Cancer 1993:72:131-6.
Alexandrow MG. Kawabata M, Aakre M,
Moses HL. Overexpression of the c-Myc oncoprotein blocks the growth-inhibitory response but is required for the mitogenic effects
of transforming growth factor beta 1. Proc
Natl Acad Sci U S A 1995:92:3239-43.
Wang TT, Phang JM. Effects of estrogen on
apoptotic pathways in human breast cancer
cell line MCF-7. Cancer Res 1995:55:2487-9.
Vancutsem PM, Lazarus P, Williams GM.
Frequent and specific mutations of the rat p53
gene in hepatocarcinomas induced by tamoxifen. Cancer Res 1994;54:3864-7.
Fanidi A, Harrington EA, Evan GI. Cooperative interaction between c-myc and bcl-2
proto-oncogenes. Nature 1992:359:554-6.
Wagner AJ, Kokontis JM, Hay N. Mycmediated apoptosis requires wild-type p53 in a
REPORTS
manner independent of cell cycle arrest and
the ability of p53 to induce p21wafl/cipl.
Genes Dev 1994;8:2817-30.
(33) Amati B, Brooks MW, Levy N, Littlewood
TD, Evan GI, Land H. Oncogenic activity of
the c-Myc protein requires dimerization with
Max. Cell 1993:72:233-45.
Notes
Supported in part by American Cancer Society
grant CDA 93-283 and by the Medical Society of
Virginia Alliance.
Manuscript received July 11, 1995; revised
November 16, 1995; accepted November 29, 1995.
Estrogen Receptor Variants
in Normal Human
Mammary Tissue
Etienne R. Leygue, Peter H.
Watson, Leigh C. Murphy*
Background: Several estrogen receptor
(ER)
variant
messenger
RNAs
(mRNAs) have been identified previously in human breast cancer biopsy
samples and cell lines. The relative
levels of certain ER variant mRNAs
have been observed to increase with
breast tumor progression. In vitro assays of the function of polypeptides encoded by some of these variant mRNAs
have led to speculation that ER
variants may be involved in the progression from hormone dependence to
independence in breast cancer. Purpose: We set out to establish if ER
variant mRNAs are present in normal
human breast tissues and, if so, to compare levels of these variants between
normal and neoplastic human breast
tissues. Methods: Four human breast
tissue samples from reduction mammoplasties and five samples from tissue
adjacent to breast tumors were analyzed. The tissue samples were confirmed to be normal (i.e., not malignant) by histopathologic analysis. RNA
was extracted immediately from adjacent frozen sections. Human breast
tumor specimens originally resected
from 19 patients were acquired from a
tumor bank and processed in the same
way as the normal tissue samples. The
RNAs were then reverse transcribed
and subsequently amplified with the
use of the polymerase chain reaction
(PCR). PCR primer sets were designed
to detect several different exon-deleted
ER variants and a truncated ER
variant (i.e., clone 4). A semiquantitative PCR-based method was used to
determine the relative expression of
exon 5- and exon 7-deleted variants to
wild-type ER mRNAs in the nine normal breast tissues and in 19 ER-positive breast tumor tissues. The MannWhitney rank sum test (two-sided) was
used to determine P values. Results: ER
variant mRNAs corresponding to the
clone 4 ER truncated variant and to
variants deleted in either exon 2, exon
3, exons 2-3, exon 5, or exon 7 were
detected in all normal samples. The
results were confirmed by restriction
enzyme analyses and sequencing of the
PCR products. The expression of exon
5-deleted ER variant relative to the
wild-type ER mRNA was significantly
lower (P<.001) in normal tissue than
in tumor tissue. A similar trend was
noted for expression of the exon 7deleted ER variant mRNA; however,
the difference did not achieve statistical
significance (P - .476). Conclusion: Several ER variant mRNAs are present in
normal human breast tissue, but the
level of expression of some of these variants may be lower in normal tissue than
in tumor tissue. Implication: These data
suggest that the mechanisms generating
ER variant mRNAs exist in normal
breast tissue and may be deregulated in
breast cancer tissues. Further investigation of the role of variant ER expression in development and progression of
human breast cancer appears warranted.
[J Natl Cancer Inst 1996; 88:284-90]
The estrogen receptor (ER), which
belongs to the superfamily of steroid—
thyroid-retinoic acid receptors (/), is an
important regulator of growth and dif-
*Afflliations of authors: E. R. Leygue, L. C. Murphy (Department of Biochemistry and Molecular
Biology), P. H. Watson (Department of Pathology),
University of Manitoba, Winnipeg, Canada.
Correspondence to: E. R Leygue, Ph.D., Department of Biochemistry and Molecular Biology.
University of Manitoba, Winnipeg. Manitoba,
Canada R3E OW3.
See "Notes" section following "References."
Journal of the National Cancer Institute, Vol. 88, No. 5, March 6, 1996