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Division of Endocrinology, Metabolism, and Molecular Medicine and Northwestern University Medical School, Chicago, Illinois 60611
Address all correspondence and requests for reprints to: J. Larry Jameson, M.D., Ph.D., Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, Illinois 60611.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The pure antiestrogens ICI 164384 and ICI 182780 have been shown to down-regulate ER and to block the transcription of ER-regulated genes. These antiestrogens effectively inhibit cell growth and induce apoptosis in ER-positive breast cancer MCF-7 cells (10) and GH3 pituitary mammosomatotrope tumor cells (11). Dominant negative forms of the ER have been suggested as an alternative method to inactivate the ER. Several dominant negative ER mutants have been generated (12, 13, 14): truncated receptors (ER1–530 and ER1–536, missing the last 65 or 59 amino acid residues), a point mutant (L540Q), and a frameshift mutant (S554fs). Lazennec et al. (15) demonstrated that adenovirus-directed expression of the frame-shifted ER (S554fs) suppressed the proliferation of ER-positive breast cancer cells.
GH3 cells are derived from rat pituitary tumors
that occur after long-term treatment with estrogen (16, 17) and are widely used as an in vitro model of
lactotropes or somatotropes. These cells express ER
, ERß, and the
truncated ER product lacking exons 1–4 of ER (18, 19, 20).
GH4C1 (hereafter referred
to as GH4) cells are derived from
GH3 cells and secrete less GH (21).
These cells exhibit many features of lactotropes (22, 23).
In the present study we used adenoviral vectors carrying the dominant
negative ER mutants L540Q and ER1–536 and examined their effects on
gene transcription, cell proliferation, and apoptosis in
GH4 tumor cells in vitro and in a
tumor-bearing animal model.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(hER) cDNA (provided by
Dr. Pierre Chambon, Universite Louis Pasteur, Strasbourg, France)
driven by the cytomegalovirus (CMV) promoter/enhancer with a simian
virus 40 (SV40) polyadenylation [p(A)] sequence was subcloned into an
adenoviral transfer plasmid (24) based on pCDNA3
(Invitrogen, Carlsbad, CA). The dominant negative ERs,
L540QhER and 1–536hER, were created using site-directed
mutagenesis and were exchanged for the wild-type ER (wtER) in the
adenoviral transfer plasmid. The resulting plasmids, pCwtER, pCL540Q,
and pC1–536, were used to generate recombinant adenoviruses.
Linearized transfer plasmids containing 5' 393 bp of adenoviral
sequence and expression cassette were ligated with
ClaI-digested Ad5 309/356 DNA representing map units
3.0–100. (Ad5 309/356 is a recombinant adenovirus in which the E3
region is deleted. ClaI digestion removes the E1a region,
resulting in a replication-deficient virus.) The ligation products were
transfected into 293 cells, in which cellular expression of the E1a
protein allows replication of the E1-deleted recombinant viruses. The
cloned and purified adenoviral vectors were titrated by plaque assay.
Recombinant adenoviruses carrying wild-type hER, L540QhER, and
1–536hER were designated AdwtER, AdL540Q, and Ad1–536,
respectively. AdGal, which contains ß-galactosidase driven by CMV
promoter, was used as a control.
An adenoviral reporter vector, AdERE-Luc, was created to investigate
transcriptional activity of the wild-type or dominant negative ER by
adenoviral vectors. The ERE2-TK109 promoter sequence was excised from
ERE2-tk109-luc (25) and ligated into the pGL3-promoter
plasmid (Promega Corp., Madison, WI) from which the SV40
promoter had been deleted (NheI to HindIII). A
portion of the resulting plasmid containing the upstream synthetic p(A)
signal, two consensus estrogen response elements (EREs), a 109-bp
fragment of the thymidine kinase promoter, the firefly luciferase gene,
and the downstream SV40 p(A) signal, was subcloned into the adenoviral
transfer plasmid. The resulting plasmid, pC-ERE-Luc, was used to
generate AdERE-Luc. The sequences of the expression cassettes in the
adenoviral vectors were confirmed by automated DNA sequencing.
Structures of the adenoviral vectors are shown in Fig. 1
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For infection with adenoviral vectors, cells were first depleted of estrogen for 3 d using phenol red-free DMEM/Ham’s F-12 containing 5% dextran/charcoal-stripped FBS. The transduction efficiency of the adenoviral vectors in cell lines was tested using AdGal. ß-Galactosidase expression was detected in 95–100% of GH4 cells at 48 h after infection with AdGal at a multiplicity of infection (MOI) of 5 plaque-forming units (PFU)/cell (data not shown). Therefore, subsequent experiments were performed using similar amounts (5 or 10 PFU/cell) of recombinant adenoviral vectors.
The transcriptional activities of wtER and the dominant negative mutants were assayed using an artificial estrogen-responsive reporter in a viral vector (AdERE-luc) and the naturally estrogen-responsive PRL promoter in a reporter plasmid (2.5 PRL-luciferase) (26). Briefly, 12-well plates of GH4 cells were infected overnight with 5 PFU/cell AdERE-Luc and increasing amounts (1, 5, and 10 PFU/cell) of AdwtER, AdL540Q, or Ad1–536. Fresh medium with or without estradiol (E2) was added, incubation was continued for 24 h, and luciferase activity was assayed. GH4 cells were also transfected with 500 ng/well PRL-luciferase plasmid (provided by Dr. Richard A. Maurer, Oregon Health Sciences University, Portland, OR) using Lipofectamine Plus (Life Technologies, Inc., Gaithersburg, MD), followed by infection with adenoviral vectors as described above.
Immunofluorescent detection of ER expression
GH4 cells were collected, washed twice
with PBS, and mounted on glass slides 48 h after infection with
adenoviral vectors. After 20 min of air-drying, slides were fixed in
ice-cold methanol and acetone for 10 min each. After preincubation with
serum-blocking solution (ABC kit, Vector Laboratories, Inc., Burlingame, CA) for 10 min, specimens were incubated with
mouse monoclonal antihuman ER (1:50; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 h at room temperature. After
washing with Tris-buffered saline and 0.025% Tween, staining was
performed using biotinylated secondary antibodies (ABC kit,
Vector Laboratories, Inc.) and streptavidin-FITC (1:100;
Vector Laboratories, Inc.). Cell images were analyzed
using a Carl Zeiss microscope (Axioskop, Carl Zeiss, Oberkochen, Germany) and Fuji Photo Film Co., Ltd. color film (1600 Super HG, Fuji Photo Film Co., Ltd., Tokyo, Japan).
Terminal deoxynucleotidyltransferase-mediated UTP end labeling
(TUNEL) assay
GH4 cells were infected with adenoviral
vectors (5 PFU/cell), treated with 1 nM E2 for 6 d,
then washed twice with PBS and mounted on glass slides. Cells were
fixed for 30 min in 4% paraformaldehyde and permeabilized with buffer
containing 0.1% sodium acetate and 0.4% Triton X-100 for 10 min on
ice. After washing with PBS, a modified TUNEL was performed using the
In Situ Cell Death Detection Kit, Fluorescein (Roche Molecular Biochemicals, Indianapolis, IN). Cells were visualized
and photographed as described above.
Western blot analysis of Bcl-2, BAX, and p38MAPK
expression
Cells were plated in 10-cm culture dishes at a density of 5
x 106 cells/dish. The following day, they were
infected with adenoviral vectors at an MOI of 5 PFU/cell for 5 h.
After the addition of fresh medium, the cells were incubated for 48 or
72 h with or without 1 nM E2. Cells were washed twice
with PBS, and whole cell lysates were prepared with lysis buffer [25%
glycerol, 0.5 M NaCl, 1.5 mM
MgCl2, 20 mM HEPES (pH 7.9), 1
mM phenylmethylsulfonylfluoride, 0.2 mM EDTA,
25 mM NaF, and protease inhibitor cocktail tablets
(Roche Molecular Biochemicals)]. Equal amounts of protein
(20 µg) were resolved by SDS-PAGE on 10% gel and transferred to
nitrocellulose paper. The membranes were blocked with 3% nonfat milk
in PBS for 1.5 h and then incubated overnight at 4 C with primary
antibodies. Mouse monoclonal anti-Bcl-2 (1:1000; Santa Cruz Biotechnology, Inc.) and mouse monoclonal anti-Bax (1:1000;
Santa Cruz Biotechnology, Inc.), were used for the
detection of apoptosis-associated proteins. The activated p38MAPK (the
Thr180/Tyr182-phosphorylated
p38MAPK) and p38MAPK were detected by phospho-p38MAPK (1:1000) and
p38MAPK (1:2000) polyclonal antibodies (New England Biolabs, Inc., Beverly, MA), respectively.
After three washes in 0.1% Tween-20 in PBS, immunoreactive proteins were detected using an antimouse or rabbit horseradish peroxidase-conjugated antibody (1:5000; Promega Corp.) and the enhanced chemiluminescence system (Amersham Pharmacia Biotech, Arlington Heights, IL). Bands were detected with X-Omat film (Eastman Kodak Co., Rochester, NY).
Effect of dominant negative ERs on GH4 cell growth
in vitro
The effect of dominant negative ERs on GH4
cell growth was measured with a nonradioactive cell proliferation assay
according to the manufacturer’s protocol (Cell Titer 96 Aqueous
NonRadioactive Cell Proliferation Assay, Promega Corp.).
Cells were seeded in 96-well plates at a density of 5 x
103 cells/well and infected on the following day
with adenoviral vectors at different MOIs (0, 5, and 10 PFU/cell).
Medium was replaced at 5 h after infection and every 2 d
thereafter. To measure the effects of different doses of E2 (1, 10, and
100 nM), quadruplicate wells were assayed for viable cell
density on d 6. Relative density was calculated as the absorbance at
490 nm divided by that of the uninfected, 1 nM E2-treated
cells and expressed as a percentage (mean ± SD). In a
separate experiment cell density was assayed at 2-d intervals over an
8-d period with a fixed (1-nM) E2 concentration.
Effect of dominant negative ER on growth of GH4 cells
in nude mice
GH4 cells were infected with 5 PFU/cell of
adenoviruses and incubated at 37 C for 24 h. Cells were collected,
washed twice with PBS, resuspended in medium, and injected (2 x
106 cells) into the flanks of adult (8-wk-old)
athymic female nude mice (Harlan-Sprague Dawley, Indianapolis, IN) that
had been sc implanted with 60-d estrogen pellets (Innovative Research of America, Sarasota, FL) 7 d earlier. The mice
were divided into five groups: group A, no virus (n = 8); group B,
AdGal (n = 6); group C, AdwtER (n = 9); group D, AdL540Q
(n = 8); and group E, Ad1–536 (n = 8). Animals were examined
for tumor formation every 2 d, and the size of the tumor was
measured with calipers in three dimensions. Tumor size (cubic
millimeters) was calculated using the formula: (3.14 x
length x width x depth)/6. The experiment was terminated 2
wk after cell injection because control (no virus and AdGal) mice began
to show morbidity. All studies involving the use of nude mice were
approved by the Northwestern University Medical School animal care and
use committee.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Effect of dominant negative ER on transcriptional activity of the
endogenous ER in GH4 cells
To investigate whether expression of a dominant negative ER
affects the ability of the endogenous ER to activate an
estrogen-responsive reporter gene, AdERE-Luc was coinfected into
GH4 cells with adenoviruses carrying dominant
negative ERs. As shown in Fig. 3A, E2 (1 nM) treatment stimulated ERE reporter gene activity
6- to 8-fold over background in control (uninfected and AdGal-infected)
cells. AdwtER infection increased reporter activity 3-fold without
ligand, presumably due to residual estrogen in the medium. AdwtER
infection at an MOI of 1 PFU/cell also increased reporter response
somewhat in the presence E2. However, both unstimulated and
E2-stimulated activities declined at higher doses of the vector.
Infection with adenoviral vectors expressing dominant negative ERs also
suppressed reporter activity in both the absence and presence of E2.
The suppression was greater in cells infected with Ad1–536 (64% and
86% at 5 and 10 PFU/cell, respectively) than with AdL540Q (42% and
57%).
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). E2 treatment stimulated PRL promoter activity 6- to
8-fold in control (uninfected and AdGal-infected) cells. AdwtER
infection increased PRL reporter response by 20-fold over background
without ligand. However, higher doses of AdwtER diminished reporter
activity. In cells infected with AdwtER, reporter activity was
independent of or slightly inhibited by treatment with E2. This may be
due to cooperative activation by endogenous Pit-1 and unliganded
wild-type ER (27) or enhanced sensitivity to residual
traces of E2 in the stripped serum when wild-type ER is overexpressed.
In contrast to AdwtER, infection with adenoviral vectors expressing
dominant negative ERs suppressed PRL promoter activity in a
dose-dependent manner, with or without E2. This suppression was greater
in cells infected with AdL540Q (69% and 70% at 5 and 10 PFU/cell,
respectively) than in those infected with Ad1–536 (54% and 65%).
Effect of dominant negative ER on GH4 cell growth in
vitro
To investigate whether the disruption of ER signaling by dominant
negative ER expression could influence cell growth, we analyzed the
proliferation of GH4 cells infected with two
different doses (5 and 10 PFU/cell) of adenoviral vectors. As shown in
Fig. 4, A and B,
GH4 cell growth was stimulated by 6-d E2
treatment (1–100 nM) in uninfected cells. AdL540Q or
Ad1–536 infection suppressed the growth of E2-treated
GH4 cells in a pattern dependent on viral dose
(65–75% with 5 PFU/cell, 75–85% with 10 PFU/cell). AdGal had little
effect on growth of GH4 cells. Infection with 5
PFU/cell of AdwtER caused minimal growth inhibition, but 10 PFU/cell of
AdwtER also induced 60–70% growth inhibition in the presence of E2.
Although their effects were most pronounced in the presence of E2,
AdL540Q, and Ad1–536, but not wtER, produced growth inhibition in the
absence of added hormone (48% and 70%, respectively, at 10
PFU/cell).
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, C and D). Growth inhibition by Ad1–536 or AdL540Q was
apparent by d 4 or 6. AdwtER had little effect on cell growth at 5
PFU/cell, but 10 PFU/cell inhibited growth significantly. The growth
rate of GH4 cells was not affected by the AdGal
virus, indicating that growth inhibition was not due to nonspecific
effects of viral infection.
Effect of dominant negative ER on induction of apoptosis, BAX and
Bcl-2 expression, and p38 MAPK activation in GH4 cells
The TUNEL reaction was used to investigate whether dominant
negative ERs induce apoptosis. GH4 cells were
infected with adenoviral vectors, treated with E2, and assayed as
described in Materials and Methods. A positive TUNEL
reaction was obtained in about 40–50% of cells infected with AdL540Q
and Ad1–535, but in only 5–10% of cells infected with AdwtER (Fig. 5, B and C, D). The TUNEL reaction was
negative in AdGal-infected cells (Fig. 5A) and uninfected cells
regardless of estrogen treatment (data not shown).
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, A and B). The levels of
Bax expression were markedly increased in Ad1–536-infected cells.
AdwtER-infected cells also showed decreased Bcl-2 expression.
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Effect of dominant negative ER on growth of GH4 cells
in nude mice
Based on the findings that dominant negative ERs inhibit cell
growth and induce apoptosis in vitro, we hypothesized that
expression of a dominant negative ER might inhibit tumor formation by
pituitary prolactinoma cells in nude mice (see Fig. 7).
GH4 cells were injected sc into estrogen-treated
female athymic mice as described in Materials and Methods.
Tumors developed within 6 d in mice injected with uninfected or
AdGal-infected cells. These tumors grew very rapidly and reached half
the size of the mouse at the end of 2 wk. In mice injected with cells
infected with AdwtER, tumor formation was delayed until 10 d after
injection. The rate of growth and the size of the tumors were greatly
reduced compared with those in control groups (no virus and AdGal).
Even more striking, there was almost complete suppression of tumor
formation in the animals injected with cells infected with AdL540Q or
Ad1–536.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As dominant negative ER mutants are known to suppress the transcription of genes regulated by wtER, it has been suggested that such mutants could influence the proliferation of ER-positive tumor cells. An efficient gene delivery system is required to achieve high levels of expression of ER mutants in target cells. A recent report (15) demonstrated that adenovirus-directed expression of the frame-shifted ER mutant S554fs, a dominant negative ER, induced apoptosis of ER-positive breast cancer cells. A similar strategy might be applied to pituitary lactotrope adenoma cells. In this study, using adenoviral vectors carrying different dominant negative ER mutants (L540Q and 1–536), we demonstrated the induction of apoptosis in ER-positive lactotrope GH4 cells and suppression of GH4 tumor growth in nude mice.
Pituitary lactotrope cells express ER and ERß
(18, 19, 20), both of which regulate PRL gene transcription
(2, 27). Due to heterodimerization, dominant negative
mutants of each are able to inhibit the activity of both isoforms
(28). We confirmed that L540QhER and 1–536hER
suppress ERß transcriptional activity effectively in transiently
transfected TSA cells (data not shown). When expressed by adenoviral
vectors, both mutants suppressed the transcriptional activity of
endogenous ERs in GH4 cells, as assessed using
ERE reporter genes or the rat PRL promoter (Fig. 3B). Interestingly,
Ad1–536 was a more effective inhibitor of the artificial
estrogen-responsive reporter gene, whereas AdL540Q was more effective
against the natural PRL promoter. Thus, the relative efficacy of the
dominant negative mutants appears to vary with respect to different
reporter genes.
Apoptosis, or programmed cell death, plays an important role in maintaining cellular homeostasis to ensure the balance between the rates of cellular proliferation and cell loss. Apoptosis is inhibited by the Bcl-2/Ced-9 family of proteins (29). The bcl-2 gene is overexpressed in many tumors, including breast cancers (30, 31). This gene is also expressed in about 60% of prolactinomas, a higher expression rate than in any other subset of pituitary tumors (32). A positive correlation of Bcl-2 expression with markers of angiogenesis was also demonstrated in prolactinomas (33). The induction of apoptosis in GH3 cells by bromocriptine is accompanied by decreased Bcl-2 expression (34). These results suggest that bcl-2 gene expression is an important factor in the survival of pituitary lactotrope tumor cells. Estrogen is known to up-regulate bcl-2 transcription in ER-positive MCF-7 and T47D human breast cancer cells (31, 35, 36). A recent report revealed that estrogen induction is mediated by two EREs present in the bcl-2-coding region (37). Consistent with these findings, we show that infection of AdL540Q and Ad1–536 decreased estrogen-induced Bcl-2 expression. However, AdwtER had a similar effect, suggesting that the ER regulation of this antiapoptotic gene may involve a nonclassical transcriptional mechanism.
The dominant negative ER mutants also increased expression of the proapoptotic Bax protein. These results indicate that in GH4 cells, apoptosis induced by dominant negative ERs is associated with down-regulation of Bcl-2 and up-regulation of Bax. In many cancer cells, Bax overexpression produces increased sensitivity to stressful stimuli, resulting in decreased cell survival and increased apoptosis. The ratio of Bcl-2 to Bax, rather than the absolute level of either protein, may therefore determine the sensitivity to apoptosis (38). However, in the present experiments this ratio did not correlate to apoptosis by AdL540Q or Ad1–536, suggesting that another pathway might be involved in apoptosis induced by dominant negative ERs in GH4 cells.
AdwtER-infected cells showed growth inhibition and induction of
apoptosis when treated with estrogen. These results were not entirely
unexpected, because growth inhibition has been reported previously in
cells transiently or stably transfected with the ER
(39, 40, 41, 42). In addition, we observed similar results in
ER-positive T47D breast cancer cells infected with AdwtER or adenovirus
encoding mouse ER (unpublished results). Although the mechanism of
cell death remains unknown, it is possible that high levels of ER
expression titrate transcription factors that are necessary for cell
proliferation or induce the expression of estrogen- regulated
growth inhibitory/cytotoxic genes. Of note, the AdwtER was less
effective than the dominant negative mutants for inhibiting in
vitro cell proliferation, inducing DNA fragmentation, and
suppressing tumor growth, suggesting that different mechanisms may be
involved in the induction of apoptosis by the AdwtER and mutants
(43).
The p38 MAPK pathway is also known to be strongly correlated to
apoptosis, although the underlying mechanisms are not well understood.
p38 MAPK is activated by several environmental stresses, such as UV
light, heat shock, and osmotic shock. p38 MAPK is also activated by the
proinflammatory cytokines IL-1 and TNF-. These stressful stimuli
induce growth inhibition or apoptosis in cells, and SB203580, a
selective p38 MAPK inhibitor, abolishes these effects (44, 45), suggesting that the activation of p38 MAPK plays an
important role in apoptosis. A similar result was observed in
bromocriptine-induced apoptosis of pituitary
GH3 cells (46). A recent report
showed that activation of the p38 MAPK pathway is involved in E2
induction of apoptosis in HeLa cells stably expressing significant
levels of ER (47). In our study overexpression of wtER
increased p38 MAPK activation in estrogen-treated
GH4 cells. Expression of dominant negative ERs
had a similar effect. The activation of p38 MAPK by UV light or
genotoxic stress is known to phosphorylate the p53 tumor suppressor
(45, 48), which results in increased transcription of
genes involved in apoptosis. The present study also demonstrates that
induction of apoptosis by dominant negative ERs in
GH4 cells is associated with up-regulation of the
proapoptotic protein Bax, which is regulated positively by wild-type
p53 (49). However, at present it is unknown whether Bax
induction is related to p53 phosphorylation through the activation of
p38 MAPK.
The GH4 cell tumors in control groups of nude mice (uninfected or AdGal-infected) grew very rapidly and reached half the size of the mice by the end of 2 wk, suggesting that GH4 cells are highly malignant. These rapidly growing tumors may not be an appropriate model for human pituitary prolactinomas, which are usually benign and slow-growing. The rate of tumor growth may affect the choice of the appropriate gene for gene therapy; for example, the herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) suicide system is mitosis dependent. In a previous study using the HSV-TK/GCV system we observed a highly cytotoxic effect on rapidly growing tumors in nude mice (24). In contrast, the HSV-TK/GCV system under control of the PRL promoter was not effective for lowering PRL levels in a rat model of pituitary lactotrope hyperplasia, which may more closely resemble human prolactinomas (50). Presumably this is due to the decreased effectiveness of GCV in slowly dividing cells. Delivery of apoptosis-inducing or directly toxic genes may therefore be more effective for slowly growing tumors, and it will be interesting to examine the AdL540Q and Ad1–536 dominant negative mutants in the rat lactotrope-hyperplasia model. The clinical application of this strategy must await further analyses of efficacy and safety of the recombinant adenoviruses.
In conclusion, we have demonstrated that adenovirus-directed expression of dominant negative ERs induces growth suppression and apoptosis in pituitary lactotrope adenoma cell lines in vitro and inhibits tumor growth in vivo in nude mice. These results suggest that dominant negative ER mutants have the potential to suppress growth or induce apoptosis of ER-positive tumor cells, and that the delivery of dominant negative ERs by adenoviral vectors may be an alternative modality for the targeted therapy of pituitary lactotrope adenomas.
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Acknowledgments |
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, Dr. Richard A. Maurer for providing 2.5 PRL-luciferase reporter
gene plasmid, and Tom Kotlar for critical reading and discussion. |
Footnotes |
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Abbreviations: CMV, Cytomegalovirus; ERE, estrogen response
element; hER, human ER; HSV-TK/GCV, herpes simplex virus
thymidine kinase/ganciclovir; MOI, multiplicity of infection; p(A),
polyadenylation; PFU, plaque-forming units; SV40, simian virus 40;
TUNEL, terminal deoxynucleotidyltransferase-mediated UTP end labeling;
wtER, wild-type ER.
Received December 21, 2000.
Accepted for publication May 11, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß in GH3 cells. Endocrinology 140:2651–2658 and ß
pathways by each dominant negative mutant. FEBS Lett 423:129–132[CrossRef][Medline]
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