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Tufts University School of Medicine, Department of Anatomy and Cellular Biology, Boston, Massachusetts 02111
Address all correspondence and requests for reprints to: Ana M. Soto, M.D., Department of Anatomy and Cellular Biology, Tufts University School of Medicine, 136 Harrison Ave, Boston, Massachusetts 02111. E-mail: ASOTO{at}Opal.Tufts.Edu
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Introduction |
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Past contributions of our laboratory established that the proliferation of androgen and estrogen target cells (LNCaP and MCF7, respectively) is controlled by these sex hormones through a two-step mechanism. In Step-1, sex steroids would increase the proliferation of its target cells by canceling the inhibition exerted by a specific plasma-borne protein (3, 4). In Step-2, sex steroids would directly trigger the expression of yet-to-be-identified endogenous inhibitors of the proliferation of their target cells (2, 3). We showed recently that these two steps may be segregated in discrete variants of human breast tumor MCF7 and human prostate tumor LNCaP cell lines (5, 6). To elucidate the conditions necessary for sex hormone target cells to become either serum-sensitive (Step-1) or sex steroid-sensitive (Step-2), we transfected a full-length, wild-type, androgen receptor (AR) into MCF7 cells. This manipulation of the genome would offer the possibility that these cells were now endowed with a functional AR and hence, able to respond to these sex steroids as if they were genuine androgen target cells. MCF7 cells were targeted for the experiments because they carry all components necessary for the proliferative response to estrogens (7). We wished to test whether this was the case for androgen regulation of cell proliferation as well. Herein, we describe the proliferative behavior of an MCF7 cell line transfected by the AR (MCF7-AR1).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Steroids and nonsteroid agonists and antagonists
Testosterone (T) and 5
-dihydrotestosterone (DHT) were
purchased from Steraloids, Keene, NH. Mibolerone (7,
17-dimethyl-19-nortestosterone) was purchased from DuPont-New
England Nuclear (Boston, MA). 17ß-estradiol and progesterone were
from Calbiochem, Richmond, CA. Methyltrienolone (1881) was made
available to us by Roussell-UCLAF, Romainville, France. Casodex (ICI
176334) was kindly provided by Zeneca, Wilmington, DE.
Hydroxy-flutamide was graciously provided to us by Schering Corp.,
Bloomfield, NJ. The above mentioned compounds were dissolved in
absolute ethanol at a concentration of 1 mg/ml and kept at
-20 C. Before each experiment, aliquots were diluted in phenol
red-free DMEM; final ethanol concentrations in culture medium were less
than 1%. When assessing the effects of androgens, we chose to use the
synthetic androgen R1881 as the reference compound instead of DHT.
Whereas natural androgens are rapidly metabolized into inactive
compounds by many cell lines, R1881 remains unaltered for the length of
the experiment; this allows a more accurate measurement of its potency
(6).
Plasmids
The plasmid pSG-hAR (kindly supplied by Dr. Chawnshang Chang,
University of Wisconsin, Madison, WI) contains the cloned human AR
complementary DNA (cDNA) (9) under the control of a SV40 promoter. The
reporter gene pMSG-CAT has the chloramphenicol acetyltransferase gene
under the control of an inducible MMTV LTR (Pharmacia, Piscataway, NJ).
The plasmid pN2 contains the neomycin phosphotransferase gene under the
control of an HSV TK promoter and an RSV LTR enhancer (10). The plasmid
pRSV-ß-GAL (11) contains the ß-galactosidase gene under the control
of RSV LTR.
Transfection of MCF7 cells
MCF7 cells were transfected by the standard calcium-phosphate
coprecipitation technique (12) using 2 µg pN2 and 15 µg pSG-hAR
plasmids. The DNA precipitate was incubated with the cells for 3.5
h, and a 25% glycerol treatment was applied for 2 min. Two days later,
cells were subcultured into 10-cm Petri dishes; 700 µg/ml of active
G418 (GIBCO-BRL, Gaithersburg, MD) was used for selection of G418
resistance. After 3 weeks, G418-resistant colonies were propagated in
individual 25-cm2 flasks. Twenty-one colonies were tested
for AR expression using the pMSG-CAT reporter plasmid transiently
transfected by the calcium-phosphate precipitation method (4 µg
pMSG-CAT, 2 µg pRSV-ß-GAL, 4 µg salmon sperm DNA). CAT expression
was measured in the presence and absence of 30 nM R1881.
Reporter gene assays were done by standard protocols (12). Transfection
efficiency was assessed by ß-galactosidase activity; CAT activity was
corrected for transfection efficiency.
Gel electrophoresis and Western blot analysis of androgen-receptor
expression
Cells were plated in 75-cm2 flasks at 2 x
106 cells/flask and cultured in 5% FBS for 4 days. Next,
they were washed twice with cold PBS, scraped off the flask, and
collected in PBS containing 2 mM phenylmethylsulfonyl
fluoride. Cells were pelleted and resuspended in lysis buffer (125
mM Tris, 2% SDS, 5% Triton X-100, 2 mM
phenylmethylsulfonyl fluoride, pH = 6.8). The extracts were
centrifuged again; aliquots of the supernatant were used for protein
determination, and the remainder was diluted 1:1 in loading buffer (125
mM Tris buffer, 4% SDS, 20% glycerol, 5%
mercaptoethanol, 0.01% bromophenol blue, pH 6.8). After boiling, the
samples were separated by SDS-PAGE. Seventy micrograms of total protein
were loaded in each lane. Electrophoresis was carried out in 12%
polyacrylamide mini-gels (Bio-Rad Laboratories, Inc., Richmond, CA) at
100 V and 250 mA for 1.5 h. High molecular mass markers (Rainbow
Markers, Amersham Corp., Arlington Heights, IL) were applied to one of
the lanes. Proteins were electroblotted onto polyvinylidene difluoride
membranes (Millipore, Bedford, MA). The membranes were blocked in 5%
nonfat powder milk (Carnation Co., Los Angeles, CA) and incubated
overnight with a polyclonal antibody against the N-terminal 21 amino
acids of the human AR (rabbit, clone PG-21, Affinity Bioreagents, Inc.,
Neshanic Station, NJ) at 5 µg/ml in Tris-buffered saline (10
mM Tris buffer, 150 mM NaCl, 0.05% Tween 20).
After washing with Tris-buffered saline, the membranes were incubated
in peroxidase-linked secondary antibody (goat antirabbit IgG) for
1 h, washed again, and developed using a chemiluminescence method
(Amersham). Autoradiogram images were digitalized and analyzed
using the BioImage’s Whole Band software package (BioImage, Ann Arbor,
MI).
Proliferation yield and rate experiments
Proliferation yield and proliferation rate experiments were
conducted in 6- and 12-well multiplate plastic dishes (Costar,
Cambridge, MA). Between 40,000 and 50,000 cells were seeded in each
well of the 12-well plates used for these experiments. The parental
MCF7 and the androgen-receptor stably transfected cells (MCF7-AR1) were
seeded in 5% FBS; after 24 h, when cells became attached to the
plastic surface, the seeding medium was removed, cells were rinsed once
with phenol red-free DMEM, and experimental media were added.
Experiments addressing the effect of serum on cell proliferation were
done with ITDME supplemented with 0 and 10% CDHuS. Once sex hormones
or their putative antagonists were added to cells, cultures were kept
undisturbed until harvesting; however, comparable results were obtained
regardless of whether medium was changed every other day. For yield
experiments, cells were lysed during the late exponential phase
following a protocol described in detail elsewhere (6). Nuclei were
counted in a Coulter Counter Model ZM (Coulter Corp., Hialeah, FL). For
proliferation rate experiments, cells were lysed and nuclei counted
daily.
Cell cycle kinetics
Cell cycle kinetics were studied by flow cytometry. About 5
x 105 MCF7 and MCF7-AR1 cells were seeded into
75-cm2 flasks in 5% FBS. Cells were then exposed to
different experimental media. At chosen intervals cells were
trypsinized, pelleted by centrifugation at 100 x g for
3 min, and resuspended in 10% dimethyl sulfoxide-10% CDHuS and
snap-frozen. Cells were kept at -20 C for up to 7 days. Cells were
quickly thawed at 37 C, centrifuged, and resuspended at a density of
106 cells/ml of a solution containing 0.1% Triton X-100,
0.1 mg/ml propidium iodide (Sigma Chemical Co., St. Louis, MO) in DMEM.
Total DNA was quantified by propidium iodide binding. The ribonuclease
treatment used in the original method to hydrolyze double-stranded RNA
did not significantly affect the DNA fluorescence and was omitted.
Cells were analyzed in a Beckton-Dickinson FACSCAN flow cytometer (San
José, CA). Ten thousand cells were collected for each point. DNA
content was determined, and the resulting two dimensional (X-axis
fluorescence, Y-axis cell number) flow cytometric dot plot results were
analyzed; 3 fractions (G0-G1, G2-M and S) were quantified using a
Hewlett-Packard 346 (San José, CA) computer equipped with the
Beckton-Dickinson Lysis II and Cell Fit software.
Selection of shutoff-negative variants from MCF7-AR1
cells
MCF7-AR1 cells were cultured in DMEM with 5% FBS plus 30
nM R1881 supplemented with 0.6 mg/ml G418. The medium was
changed every 4 days; cells did not proliferate in this medium.
However, there was no cell death observed. After more than a month,
some cells formed discrete colonies on the plastic surface of each
25-cm2 flask. Cells from the arising colonies were cloned
and kept in 30 nM R1881-containing medium until further
studies were performed with them.
Receptor studies
[3H]Mibolerone (specific activity 82.5 Ci/mmol)
was purchased from DuPont-New England Nuclear. To estimate the Kd and
concentration of ARs, binding assays were performed in whole cell
extracts. Extracts were obtained from centrifuged fractions
(105,000 x g, 45 min) of a sonicated cellular
suspension in 10 mM Tris, 500 mM KCl, 0.5
mM EDTA buffer, pH 7.4. Aliquots of these extracts were
incubated for 18 h at 4 C with increasing concentrations of
labeled mibolerone (0.03–30 nM) in the presence and
absence of 100-fold excess of the unlabeled ligand. Bound and free
fractions were separated by dextran-coated charcoal adsorption (3).
Statistics
Transient transfection experiments were repeated at least four
times. Proliferation yield experiments conducted in duplicate wells
were repeated a minimum of three times. Mean cell numbers from each
experiment were normalized to the steroid-free control to correct for
differences in the initial plating density. Flow cytometry experiments
were run in single points and repeated three times. Proliferation rate
experiments were conducted four times. Data were analyzed by ANOVA
using the STATQUIK program (Lundon Software, Chagrin Falls, OH).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Maximal expression of
pMSG-CAT was reached at 0.3 nM R1881 (Fig. 2). The 5 clones overexpressing AR were tested for their
ability to express the androgen-induced inhibition of cell
proliferation. Three nM R1881 in ITDME decreased cell
yields to between 16 and 75% of the values obtained with ITDME alone
(not shown). No inhibitory effect of androgens was noted in clones that
did not express increased AR levels. Clone MCF7-AR1 was consistently
inhibited by R1881 to 16–25% of the proliferative yields obtained
with ITDME alone; because of this reason, this clone was used in
subsequent experiments.
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). These data concur with those obtained on the
expression of the reporter gene. AR concentration in MCF7-AR1 cell
extracts was 92 ± 3 fmol/mg cell protein (68,000 sites/cell) when
measured by Scatchard analysis of 3H mibolerone binding
data.
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); this feature
remained unaltered when compared with the pattern observed in the
parental MCF7 cells (7, 13, 14). DHT (not shown) and R1881 did not
affect the proliferation yield of these cells in 10% CDHuS (Fig. 4A).
Like the parental cell line, MCF7-AR1 proliferated maximally in ITDME,
regardless of the presence of estrogens (Fig. 5). DHT,
R1881, and T added to ITDME decreased MCF7-AR1 cell yields in a
dose-dependent manner; DHT and R1881 were equally potent (Fig. 5)
whereas T was 10-fold less potent (not shown). Cell yields in ITDME + 1
nM R1881 were comparable with those reached in 10% CDHuS
alone or 10% CDHuS + R1881 (Fig. 4A and 5). In addition, proliferation
of MCF7-AR1 cells, grown in the presence of 10% CDHuS + 0.1 (or 1
nM estradiol), was inhibited by R1881 (Fig. 4B).
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).
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).
Effects of steroidal and nonsteroidal antiandrogens
The antiandrogens Casodex and hydroxy-flutamide did not
significantly affect cell proliferation patterns of MCF7-AR1 cells when
added alone to ITDME (Fig. 7). However, both
antiandrogens blocked the androgen-induced inhibition of cell
proliferation generated by R1881 in ITDME in a dose-dependent manner
(Fig. 7).
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), whereas only 6% of the cells exposed
for 24 h to ITDME plus 3 nM R1881 were in S phase
(Fig. 8C). A concurrent increase of the percent of cells in the
G1 phase was observed (from 53.8%–88%) (Fig. 8C). Cells
exposed to 10% CDHuS also underwent a Go/G1
arrest; only 5% of cells were in S phase (Fig. 8B). At 48 h, the
percent of cells in S decreased further in cultures treated with ITDME
plus R1881 and in cells treated with 10% CDHuS (2.4% and 2.8%,
respectively).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have proposed that sex hormone target cells are subject to proliferative regulation by sex hormones through a two-step mechanism (2, 18). The main purpose of this article is to explore the role of ARs in mediating the effect of androgens on cell proliferation.
Sex steroid effects on the proliferation of MCF7-AR1 cells
MCF7 cells are known to express low, consistent levels of
androgen, glucocorticoid, and progesterone receptors (20, 21, 22, 23).
Nevertheless, at physiological concentration, only 17ß-estradiol
reversed the inhibition mediated by CD serum on these cells. Also,
these steroids (androgens, estrogens, progestagens and,
glucocorticoids) did not inhibit the proliferation of MCF7 cells when
grown in ITDME. 17ß-estradiol elicited a monophasic proliferative
response when MCF7-AR1 cells were grown in CDHuS-supplemented medium
(7, 13) (Fig. 4A). Androgens and progestins added to CDHuS-supplemented
medium did not elicit a proliferative response in either the parental
or the MCF7-AR1 cells (not shown). Thus, the stable expression of a
full-length, active AR in MCF7-AR1 cells did not modify their
proliferative response to estrogens when compared with the response of
parental MCF7 cells.
Requirements of androgen target cells for proliferation
The failure of stable transfectants expressing functional estrogen
or ARs to evoke the proliferative response to sex steroids has been
extensively documented (24, 25, 26, 27, 28, 29, 30, 31, 32, 33). In the animal and in culture
conditions, sex steroid-target cells seem to be sensitive to a
serum-borne component that conveys an inhibitory signal when they enter
the cycle [estrocolyone-I for estrogen-target cells, and
androcolyone-I for androgen-target cells (3, 4, 7)]. In culture
conditions, the inhibitory effect of CD-serum over MCF7-AR1 cells was
reversed by either placing cells in serumless conditions (Fig. 5) or by
adding 17ß-estradiol to CDHuS (Fig. 4A). Estrogen administration
triggered the expression of Step-1 in MCF7-AR1 cells; however,
transfection of a full-length AR did not confer to these cells the
ability to respond to androgens by overcoming the serum-induced
inhibition. An explanation for this phenomenon is that, as it was
before AR transfection, the serum inhibition of these cells is caused
by estrocolyone-I; this inhibition can be reversed only by estrogens.
The serum-sensitivity of androgen-target cells, such as that shown by
LNCaP-FGC cells, may instead be caused by androcolyone-I (6).
The androgen-mediated proliferative shutoff
The prostate of castrated rats subjected to steady androgen
administration responds with a biphasic proliferative pattern. First,
cells proliferate, and once a few rounds of proliferation replenish the
cell numbers in this organ, a second, proliferative shutoff phase
ensues (1, 3, 18). The data in
Figs. 4–6 and 8 suggest that androgens
trigger a proliferative shutoff in AR-positive, stably-transfected
MCF7-AR1 cells. A comparable pattern was generated also by
administration of androgens to human prostate LNCaP-FGC and LNCaP-LNO
cells (34). It is worth noting that AR levels in MCF7-AR1 cells are
comparable with those present in FGC cells and are 3-fold lower than in
LNO cells. This argues against the proliferative shutoff being caused
by toxicity by squelching. Segregation of Step-1 and Step-2 were
observed also in LNCaP variants (6). From these data, we conclude that
Step-1 and Step-2 are discrete entities that are controlled through
different pathways.
Flow cytometric analysis revealed that androgens in ITDME dramatically
increased the number of MCF7-AR1 cells in
Go/G1. This phenomenon is comparable with the
proliferative shutoff triggered by androgens in LNCaP-FGC and LNO cells
(6). Antiandrogens antagonized the androgen-induced proliferative
shutoff in MCF7-AR1 cells (Fig. 7) and LNCaP-LNO cells (6). The
proliferation of two AR-positive human breast cancer cell lines, T47-D
and ZR-75–1, was slightly inhibited by androgens (35). AR antisense
oligonucleotide treatment completely reversed the inhibitory effects of
1 nM mibolerone on ZR-75–1 cells, suggesting an
AR-mediated phenomena (35). Androgens may act by specifically inducing
the synthesis of one or more gene products whose function is to prevent
the entry of these target cells into the next cycle. We have called
this protein(s) androcolyone-II to distinguish it from the serum-borne
inhibitor of the proliferation of androgen target cells, androcolyone-I
(18). Evidence gathered in LNCaP variants suggests that the
proliferative shutoff is caused by the direct effect of androgens on
their target cells rather than by an autocrine or paracrine mechanism
triggered by these hormones (6). Others have proposed instead that the
androgen-triggered shutoff in LNCaP cells was mediated by TGF-ß1
(36); however, data incompatible with this interpretation have been
disclosed recently by the same group (37).
Continuous exposure to R1881 generated the emergence of resistant clones that lack the ability to express the androgen-induced proliferative shutoff. Androgen-induction of the pMSG-CAT reporter gene in R1881-resistant clones was comparable with that obtained with parental MCF7 cells (not shown). This process mimics that described for shutoff-positive LNCaP-LNO cells, which under comparable selective pressure, evolve into a shutoff-negative variant (6).
In conclusion, stably transfected cells expressing AR acquire the ability to respond to androgen by evoking a proliferative shutoff. This phenomenon occurs at high frequency (5 out of 5 AR-positive clones analyzed in this series). The identification of genes involved in the expression of this phenotype is currently under way (38).
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Acknowledgments |
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Footnotes |
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2 Recipient of a postdoctoral fellowship from Fundacion Ramon Areces
(Madrid, Spain).
Received September 16, 1996.
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) or 10% CDHuS plus 30 nM R1881 (
) for
42 h. CAT activity is expressed as mean ± SD.
-), and to R1881 (-•-); cells
were grown for 5 days in 10% CDHuS. Panel B, Dose-response curve to
R1881; cells were grown for 5 days in 10% CDHuS supplemented with 0.1
nM (-
-). Cells were grown for 5 days.
Ordinate, cell number/well (mean ±
SD); abscissa, hormone concentration
(nM).
-) ITDME plus 1 pM R1881; and (-
-)
ITDME plus 100 pM R1881. Ordinate, cell
number/well is expressed as mean ± SD;
abscissa, time (days).
-) at the concentrations indicated in the
abscissa or 10 pM R1881 plus concentrations
of Casodex (•) or hydroxy-flutamide (
). Ordinate, % cells
(mean ± SD); abscissa; time (h).