Synergistic Enhancement of PRB-Mediated RU486 and R5020 Agonist Activities through Cyclic Adenosine 3',5'-Monophosphate Represents a Delayed Primary Response
Sabine Kahmann,
Lothar Vaßen and
Ludger Klein-Hitpass
Institut für Zellbiologie (Tumorforschung)
Universitätsklinikum D-45122 Essen, Germany
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ABSTRACT
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Activators of protein kinase A have been shown to
affect the transactivation potential of progestins and antiprogestins.
To analyze the mechanisms and factors involved, we have created HeLa
and CV1 cell clones stably expressing isoform B of progesterone
receptor. In the HeLa cell background, the progesterone antagonist
RU486 significantly induces progesterone-regulatable reporter genes,
and this agonistic effect is synergistically enhanced by elevating
cAMP or through overexpression of protein kinase A catalytic subunit.
In contrast, in CV1 cells containing functional progesterone
receptors no agonist activity of RU486 could be detected, suggesting
the involvement of cell specifically expressed factors. In a
PRB-positive HeLa cell clone containing stably integrated
copies of a thymidine kinase-luciferase reporter gene with two
progesterone response elements, we observed a complete loss of RU486
antagonist potential upon cotreatment with cAMP for 25 h while
partial antagonist potential was maintained in a 5-h experiment. This
result shows that, particularly in the presence of protein kinase A
activators, the duration of hormone treatment is a crucial parameter in
the evaluation of antagonist properties of antiprogestins. A detailed
analysis of the kinetics of the hormone effects on transcription
revealed that the onset of cAMP/RU486 synergism is delayed relative to
the responses induced by RU486 or R5020 alone. Moreover, partial
inhibition of protein synthesis by cycloheximide completely abolished
cAMP/RU486 synergism while R5020 and RU486 responses were not
inhibited. Together, these data indicate that cAMP/RU486 synergism is a
delayed primary response requiring the intermediate induction of an
essential factor.
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INTRODUCTION
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The progesterone receptor (PR) is a member of the family of
ligand-inducible nuclear receptors that regulate hormone-responsive
genes by altering the rate of transcription initiation. These proteins
share common functional domains for ligand binding, dimerization, DNA
binding, and transactivation (1). The binding of ligand to
the hormone-binding domain of PRs triggers a cascade of events that
includes dissociation of associated heat shock proteins,
conformational changes, phosphorylation, and dimerization (1, 2, 3).
Ligand-activated receptors bind with high affinity to specific DNA
sequences, termed progesterone response elements (PREs), which can also
mediate glucocorticoid induction by binding of glucocorticoid receptors
(4, 5). After binding to DNA, PRs are thought to interact with
components of the basal transcription machinery, either directly or
through coactivator proteins (6, 7), to facilitate formation of RNA
polymerase II initiation complexes (8).
In all tissues and cell lines examined, human PR is expressed in two
isoforms (PRA and PRB) of 94 and 116 kDa in
size, which arise from different messages transcribed from two
promoters in the human PR gene (9). PRB differs from
PRA only in that it contains an additional 164 amino acids
at the N terminus. The two PR isoforms display indistinguishable
hormone- and DNA-binding properties and can heterodimerize with each
other (9). However, several studies have shown that, depending on the
cell- and promoter context, PRA and PRB display
remarkably different transcriptional activities, suggesting that they
may have distinct physiological functions (9, 10, 11, 12, 13, 14, 15). Possibly, an
additional activation function that has been identified in the
PRB-unique N-terminal region is responsible for the
different transactivation potential of PRA and
PRB (14). Together, these data raise the possibility that
alterations of the PRA/PRB ratio, as have been
detected in human breast tumors (16), might profoundly affect the
progesterone responsiveness of target cells.
As progesterone is implicated in a variety of hormone-dependent
cancers, much effort has been dedicated to the development of ligands,
which display relatively little or no agonist activity at all. By
competing with progesterone for binding to the receptor, such compounds
can partially (partial antagonists) or completely (pure antagonists)
prevent induction of progesterone-inducible genes. Based on their
effects on the DNA binding of PR in the gel retardation assay, two
different types of antiprogestins have been distinguished (17).
Antiprogestins, such as ZK98299, which were classified as type I
antiprogestins, do not induce stable DNA binding of PR in
vitro, suggesting that they inhibit receptor activity at a step
before DNA binding. However, analysis of the mechanism of action in
in vivo systems has led to conflicting data (18, 19). Type
II antiprogestins, including RU486, induce stable binding of PRs to DNA
in vitro and in vivo but normally generate a
nonproductive receptor conformation that is unable to stimulate
transcription of progesterone-inducible genes (17, 20, 21, 22).
Consistently, RU486 fails to induce the interaction of the PR
ligand-binding domain with coactivators, such as steroid receptor
coactivator-1 (SRC-1) and transcriptional intermediary factor-2 (TIF2),
which have been shown to play an important role in mediating the
effects of agonist-loaded receptors (23, 24). Deletion of the 42
C-terminal amino acids or mutations within a 12-amino acid region in
the C terminus rendered PR transcriptionally active in response to
RU486, indicating that the very C-terminal end plays an important role
in preventing transcriptional activity of RU486-loaded PR (25, 26). As
overexpression of the 12-amino acid region stimulated the transcription
activation potential of RU486-liganded full-length PR, it has been
postulated that suppression of RU486 activity involves a titrable
corepressor associating with the C terminus of PR (26). Occasionally
observed agonistic effects of RU486 on transcription of specific
reporter genes have been postulated to be due to the action of
DNA-bound PRB that activates transcription via the
N-terminal activation function (20). However, a study from K.
Horwitzs group (11) presented data suggesting that
PRB-mediated activation of reporter genes by RU486 may also
occur through a yet poorly defined mechanism that does not require
binding of RU486-loaded PR to PREs.
The transactivation potential of steroid receptors can be modulated by
simultaneous activation of various signal transduction pathways, which
transmit signals from cell surface receptors to the nucleus. Examples
of such cross-talks are the enhancement of the transactivation
potential of dexamethasone-induced glucocorticoid receptor (GR) by
12-O-tetradecanoylphorbol 13-acetate (TPA) (27), an inducer
of the protein kinase C pathway, or the potentiation of agonist-loaded
steroid receptors by activators of the protein kinase A (PKA) pathway
(28, 29, 30, 31). Interestingly, the modulating effect of cAMP is not
restricted to receptors bound to natural agonists, as cotreatment with
activators of PKA can also elicit strong transcriptional effects of
antagonist-loaded receptors (32, 33, 34, 35, 36, 37). Synergistic effects of
progesterone antagonist RU486 and cAMP on transfected reporter genes
have been shown to be mediated by GR and PRB, but not
PRA (36, 37). This phenomenon, which has also been
designated as antagonist/agonist switching, is dependent on the
promoter used for the analysis (36) and specific for type II
antiprogestins (32, 33, 34, 36). cAMP has been shown to have little effect
on the phosphorylation state of PR (32, 38). Moreover, mutations
abolishing all putative phosphorylation sites within the N-terminal 164
amino acids specific for PRB did not interfere with
cAMP/RU486 synergism (38). Thus, it appears unlikely that cAMP-mediated
phosphorylation is responsible for the increased activity of
RU486-loaded PRs (38).
For the analysis of the mechanism(s) responsible for the enhancement of
agonist and antagonist activities through activators of PKA, we have
created cell lines stably expressing PRB as well as
PRE2-TK-luc and mouse mammary tumor virus (MMTV)-luc
reporter genes, respectively. Using these model systems, we demonstrate
a role of PKA in the mechanism. Analyzing the kinetics of
agonist/antagonist induction both in the presence and absence of cAMP,
we find that the synergistic induction of reporter genes by RU486 or
R5020 and cAMP is a delayed response. Furthermore, we show that
cAMP/RU486 synergism is abolished by cycloheximide, a protein synthesis
inhibitor. Our data suggest that the synergism between cAMP-dependent
PKA and liganded PRs involves the induction of an additional factor,
which plays a crucial role in this mechanism.
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RESULTS
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Stable Cell Lines Expressing PRB
As a model for the analysis of the mechanism of cross-talk between
PKA and steroid receptor signaling pathways, we chose a HeLa cell clone
(3B2), which had been stably transfected with a SV40 early
promoter-driven expression plasmid encoding a His- and
myc-tagged human PRB (see Materials and
Methods). Using transient transfections, we previously confirmed
that the introduction of the tags, which allow Ni-NTA- and
myc-antibody affinity purification, has no influence on the
transactivation properties of PRB (data not shown).
Immunoblotting with antibodies against PR showed that the transfected
HeLa cell clone 3B2 (Fig. 1A
, lane 3)
expresses a protein that comigrates with the PRB in T47D
cells (lane 1) but is absent in the parental HeLa cells (lane 2). As
detected by immunoblotting, the level of PRB in HeLa3B2
cells is about half as high as in the T47D cell line. The DNA-binding
ability of PRB expressed in the HeLa3B2 cells was analyzed
in gel retardation experiments using whole cell extracts and a PRE as a
probe (Fig. 1B
). Extracts from HeLa3B2 cells pretreated with
progesterone formed a specific protein-PRE complex (lane 4), which was
not detected in HeLa cell extract (lane 8). Addition of PR antibodies
resulted in a further retardation of this protein-PRE complex,
demonstrating the presence of PRB (lane 6). Without
hormone, no PRB-PRE complex was formed (lane 3), indicating
that DNA binding of PRB expressed in HeLa3B2 cells is
hormone dependent. As shown in Fig. 2
, the introduced PRB
proved to be capable of mediating a significant R5020 induction of a
transiently transfected reporter gene containing two PREs in front of
the TK promoter and the luciferase gene (PRE2-TK-luc).

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Figure 1. Expression and DNA Binding Activity of
PRB in Stably Transfected HeLa and CV-1 Cell Clones
A, Whole cell extracts from parental HeLa cells (165 µg protein, lane
2), HeLa3B2 (165 µg protein, lane 3), CV-1 (42 µg, lane 4), or
CV-1.1B (42 µg, lane 5) were separated by SDS-PAGE and blotted to
nitrocellulose. Receptors were detected with a PR-specific monoclonal
antibody (Medac). Protein (165 µg) from T47D cell extract was
separated for comparison (lane 1). The positions of both isoforms of PR
detected in T47D cells are indicated. B, Whole cell extracts from the
indicated cells were preincubated in a 10 µl standard binding
reaction containing a radioactively labeled PRE probe and 1
µM progesterone (+) or control vehicle (-) for 20 min on
ice. To some of the binding reactions 1 µl of a PR-specific
monoclonal antibody was added as indicated. Specific PR-PRE complexes
and a nonspecific protein-PRE complex are noted.
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Figure 2. Effect of cAMP and Overexpression of PKA
Catalytic Subunit on RU486 Agonist Activity in PRB-Positive
HeLa3B2 Cells
HeLa3B2 cells were transiently transfected with PRE2-TK-luc
reporter plasmid and expression vectors encoding wild-type PKA (wt) or
mutated PKA (mut) catalytic subunit. Cells were incubated for 25 h
with 10 nM R5020 or RU486 in the presence or absence of 0.2
mM cAMP as indicated. Luciferase activities were normalized
to the untreated control. Bars represent the mean of four independent
experiments ± SD. Extracts from untreated controls
showed 2656 ± 73 luciferase units per 25 µl.
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Similarly, we stably transfected the PR-negative monkey kidney cell
line CV-1 with an expression vector encoding PRB.
Immunoblotting indicated that clone CV-1.1B expressed authentic
PRB, while no PR could be detected in the parental CV-1
cells (Fig. 1A
, lanes 4 and 5). On a protein basis CV-1.1B cells
expressed about 4-fold higher amounts of PRB than the
HeLa3B2 clone (Fig. 1A
). Gel retardation analysis and supershifting
with PR antibodies confirmed that the PR expressed in CV-1.1B cells
bound to PREs in a hormone-dependent manner (Fig. 1B
, lanes 913).
Transfection studies confirmed that the expressed PRB is
able to mediate R5020 induction of PRE2- and
PRE4-TK-luc reporters while no response was seen in the
parental CV-1 cells (Fig. 3
and data not shown). Thus, we have created
two model cell lines that allow us to specifically address the
influence of PKA activation on ligand-activated PRB in two
different cellular backgrounds.

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Figure 3. Lack of RU486 Agonist Activity in PR-Positive
CV-1.1B Cells
CV1.1B were transfected with a PRE4-TK-luc reporter and PKA
expression vectors and treated with hormones as described in the legend
of Fig. 2 . Bars represent the mean of two independent
experiments. Controls showed 480 luciferase units after substraction of
background activity.
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PRB-Mediated cAMP/RU486 Synergism Is a Cell-Specific
Phenomenon and Involves PKA
First we analyzed the effects of the antiprogestin RU486 and
8-Br-cAMP (cAMP), a membrane-permeable activator of PKA, on
transcription of a PR target promoter in HeLa clone 3B2. For this
purpose HeLa3B2 cells were transiently transfected with the
PRE2-TK-luc reporter plasmid. Cells were incubated in the
absence or presence of ligands with or without cAMP for 25 h.
Treatment with R5020 induced reporter gene activity about 9-fold. cAMP
alone stimulated luciferase activity about 3-fold while the
antiprogestin RU486 induced luciferase expression about 16-fold (Fig. 2
). Cotreatment with cAMP and RU486
resulted in 130-fold induction. Similar results were obtained using a
PRE4-TK-luc reporter or the MMTV promoter (data not shown).
Thus, in this transient model RU486 displays substantial agonist
activity even in the absence of exogenous cAMP, and this agonist
activity is synergistically enhanced by addition of cAMP.
Since cAMP is an activator of PKA, we asked whether directly increasing
the intracellular level of PKA catalytic subunits (isoform ß) might
also bring about enhancement of RU486 agonist activity (Fig. 2
). In the
absence of RU486, transient transfection of an expression vector
encoding wild-type catalytic subunit (wt) of PKA stimulated
PRE2-TK-luc activity about 12-fold. However, in the
presence of RU486, overexpression of catalytic subunit led to about
15-fold potentiation of the RU486 activity that was even higher in
magnitude than the potentiation evoked by cAMP (8-fold). In contrast,
coexpression of a mutated catalytic subunit (mut), which lacks kinase
activity due to a point mutation within the catalytic site, did not
significantly enhance the RU486 response, proving that the catalytic
activity of PKA is essential for the effect. Together, these results
clearly suggest that the stimulatory effect of cAMP on the RU486
agonist activity is mediated via activation of PKA.
A similar transfection experiment was performed with a TK-luc reporter
containing four PREs (PRE4-TK-luc) in the
PRB-positive clone (CV-1.1B) derived from the monkey kidney
cell line CV-1. As shown in Fig. 3
, treatment with R5020 for 25 h strongly induced luciferase activity
in CV-1.1B cells, indicating that the introduced receptor is
functional. cAMP alone had no detectable effect on the relative low
basal promoter activity. In contrast to the results observed in HeLa3B2
cells, both in the absence and presence of cAMP, RU486 displayed no
agonist activity at all. Like cAMP, cotransfection of the expression
vector encoding the wild-type catalytic subunit of PKA (isoform ß)
did not elicit any agonist activity of RU486 (Fig. 3
). Therefore, it
seems unlikely that the failure to observe cAMP/RU486 synergism in
CV-1.1B cells is due to insufficient amounts of endogenous PKA. The
remarkable difference in the ability of PRB-positive HeLa
and CV-1 cells to mediate a synergistic response of cAMP or PKA and
RU486 suggests that a cell specifically expressed factor(s), possibly
acting downstream of PKA, might play an essential role in the
mechanism.
HeLa3B2 Derivatives Containing Chromosomally Integrated Copies of
PRE2-TK-luc or MMTV-luc Reporter Genes
Most studies analyzing cross-talk between steroids and PKA
activators employed transiently transfected receptor expression vectors
and reporter genes. However, transient transfections have several
important disadvantages: 1) they are laborious and expensive; 2) levels
of transiently transfected reporter genes as well as receptor levels
can vary from experiment to experiment; 3) they may not correctly
reflect the regulation of endogenous genes due to the lack of chromatin
structure on the promoter of interest; and 4) they do not allow precise
kinetic analysis in long-term experiments, as the amount of available
expression vector and reporter gene may decrease with time. To avoid
these possible drawbacks, we stably transfected HeLa3B2 cells either
with a reporter plasmid containing two PREs in front of a TK promoter
and the luciferase gene (PRE2-TK-luc) or with a luciferase
reporter containing the MMTV promoter (MMTV-luc). For further analysis
we selected representative PRE2-TK-luc (3B2.TK) and
MMTV-luc (3B2.M) clones expressing luciferase activities clearly above
background level, ensuring an accurate estimation of fold induction
values. Regulatory phenomenons observed in the transient transfections
were reproduced in several independent stable transformants analyzed
(Figs. 2
and 4A
). However, fold
stimulation values as determined in cell lines containing stably
integrated copies of the reporter genes were generally lower than those
observed with transiently transfected reporters. The reason for the
decreased responsiveness of stably integrated reporter genes remains
unclear.

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Figure 4. Effect of cAMP Cotreatment on the Time
Courses of R5020 and RU486 Responses of a PRE2-TK-luc
Reporter Gene
A, HeLa3B2.TK cells stably expressing PRB and containing
stably integrated copies of the PRE2-TK-luc reporter
plasmid were incubated with 10 nM R5020 or 10
nM RU486 in the presence or absence of 0.2 mM
cAMP. At different times, cells were harvested and assayed for
luciferase activity. Luciferase activities were normalized to untreated
controls. Points represent the mean of two independent experiments with
less than 10% deviation. Control showed 25,884 luciferase units per 25
µl of extract. B, HeLa.TK cells (PRB-negative) containing
stably integrated copies of PRE2-TK-luc were cultured in
medium containing 10 nM dexamethasone, 10 nM
RU486, or 0.2 mM cAMP as indicated and analyzed as
described in panel A. Controls showed 3,258 luciferase units per 25
µl of extract.
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cAMP/RU486 Synergism Is Delayed with Respect to the Onset of the
R5020 and RU486 Responses on PRE2-TK-luc
Using the HeLa3B2.TK clone containing chromosomally integrated
copies of PRE2-TK-luc, we next analyzed the time course of
the responses to R5020 and RU486 in the presence and absence of cAMP in
detail (Fig. 4A
). Treatment with cAMP alone had little effect on
transcription at all times investigated. Transcriptional stimulation by
R5020 was clearly detectable after 5 h, reached a maximum of
11-fold at 10 h, and decreased to about 5-fold induction values at
25 h. As already observed in the transient transfection experiment
(Fig. 2
), RU486 alone led to an induction (5-fold) of reporter gene
activity. The RU486 induction occurred as rapidly as the R5020
response, but remained more or less constant up to 35 h of
incubation. Importantly, cotreatment with cAMP for 5 h had no
significant effect on the activity of RU486 while longer treatments
resulted in increasing synergistic enhancement. This delayed onset of
the cAMP/RU486 synergism relative to the RU486 response suggests that
de novo protein synthesis of an involved factor is required
for the mechanism. Interestingly, cAMP slightly increased the R5020
response only at earlier time points. Thus, cAMP modulates extent and
kinetics of R5020 and RU486 responses on PRE2-TK-luc in a
distinct manner.
Synergistic Induction of PRE2-TK-luc by cAMP and RU486
Is Mediated by PRB
As HeLa cells contain functional GRs, which possess high affinity
for RU486 and bind to the same response elements as PRB (4, 5), it was important to determine whether the RU486 effects observed in
HeLa3B2 cells were indeed mediated by the introduced PRB.
We thus created HeLa cell clones containing chromosomally integrated
copies of the PRE2-TK-luc reporter and determined the
effect of RU486 in the presence and absence of cAMP in a representative
clone (HeLa.TK). In this PRB-negative clone, dexamethasone
treatment caused about 6-fold induction of luciferase activity, proving
the presence of functional GRs (Fig. 4B
). Compared to HeLa3B2 cells,
HeLa.TK cells showed a relatively strong response to cAMP, which
increased up to 6-fold at 35 h. Possibly, this increased cAMP
responsiveness reflects a clonal effect. However, in contrast to the
results observed in HeLa3B2 cells (Fig. 4A
), RU486 alone exhibited no
agonist activity at all and no potentiation of RU486 activity was
observed upon cotreatment with cAMP, suggesting that the RU486-loaded
GRs are not capable of mediating agonistic effects on
PRE2-TK-luc in HeLa.TK cells. Similar results were obtained
in transient transfections using the parental HeLa cell line (data not
shown), excluding the possibility that the lack of GR-mediated
cAMP/RU486 synergism in the HeLa.TK clone might represent a
clone-specific defect. We conclude that the RU486 agonist effects on
PRE2-TK-luc transcription, which we observed in HeLa3B2
cells in the absence or presence of cAMP, are both mediated exclusively
by the introduced PRB.
Length of Treatment Largely Affects the Ability of RU486 to
Antagonize R5020 Induction of PRE2-TK-luc
As shown in Fig. 4A
, upon cotreatment with cAMP R5020 is a more
potent inducer of PRE2-TK-luc transcription than RU486 at
5- and 10-h time points while the order is reversed at 25- and 35-h
time points. Thus one would predict that, in the presence of cAMP,
RU486 might still be able to partially antagonize R5020 induction of
PRE2-TK-luc in a competition experiment employing 510 h
of treatment, but completely lack antagonist potential in 25- to 35-h
experiments. To test this prediction directly, we performed R5020/RU486
competition experiments with 5 and 25 h of treatment in the
presence of cAMP (Fig. 5
). A saturating
concentration of R5020 (10-8 M) and a 100-fold
molar excess of RU486 were used. After 5 h of treatment,
luciferase activity was higher with R5020 (10-fold, left
panel) than with RU486 (4-fold). In the competition experiment
(R5020 + RU486) excess of RU486 blocked the R5020 stimulation down
to the value observed with RU486 alone. Thus, despite the presence of
cAMP, in this short-term experiment RU486 still displays partial
antagonist activity. As already shown in Fig. 4A
, cells exposed for
25 h showed a R5020 response (4-fold) that was lower than that
observed with RU486 (15-fold). Addition of excess RU486 did not result
in inhibition of the R5020 response, but rather an increase in
luciferase activity up to the level observed with RU486 alone was
observed. Thus, in a 25-h experiment RU486 no longer exhibits any
antagonist activity. These data illustrate that cotreatment with an
activator of PKA can indeed result in a complete loss of antagonist
activity of RU486. Moreover, our experiments reveal that the duration
of hormone treatment is a crucial parameter in the evaluation of
antagonist properties, as complete loss of antagonist potential of
RU486 is only observed in the experiment involving 25 h of
incubation.

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Figure 5. Time-Dependent Reversal of Antagonist/Agonist
Properties of RU486 in the Presence of cAMP
HeLa3B2.TK cells stably expressing PRB and containing
stably integrated copies of the PRE2-TK-luc reporter
plasmid were incubated for 5 and 25 h with 0.2 mM cAMP
and 10 nM R5020 or 1 µM RU486. Cells were
harvested and assayed as described in Fig. 2 .
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cAMP Acts Synergistically Both with Progesterone Agonists and
Antagonists on Transcription of an MMTV-promoter
In the studies described above, we have analyzed the hormonal
effects on transcription using the artificial PRE2-TK-luc
reporter gene. To examine a different and more complex promoter, we
determined the kinetics of cAMP/ligand responses using a
HeLa3B2-derived clone (3B2.M) stably transfected with a reporter
containing MMTV-long terminal repeat (LTR) sequences from position
-235 to +122 in front of the luciferase gene (MMTV-luc). cAMP by
itself gave a modest induction of MMTV-luc transcription (Fig. 6
). RU486 induced luciferase activity
about 3-fold at maximum. This RU486 response was already detectable
after 5 h of incubation and remained constant over the time period
investigated. Cotreatment with cAMP and RU486 for 25 and 35 h
resulted in a strong and synergistic enhancement of MMTV-luc
transcription (18- and 53-fold induction, respectively) while synergism
was not evident at 5, 10, and 15 h. Thus, cAMP/RU486 synergism on
MMTV-luc is a delayed response, confirming the results obtained with
the PRE2-TK-luc reporter (Fig. 4A
). Furthermore, our data
demonstrate that cAMP/RU486 synergism is not a
PRE2-TK-luc-specific phenomenon. Induction of the MMTV
reporter by the progestin R5020 became detectable at the earliest time
point (5 h) analyzed and increased continuously up to 85-fold at
35 h. At 5 to 15 h of incubation, cotreatment with R5020 and
cAMP had just an additive effect on MMTV-luc activity while at 25 and
35 h synergism became evident. Thus, our experiments show that
cAMP/R5020 as well as cAMP/RU486 synergism occur with a pronounced
delay relative to the onset of R5020 and RU486 responses. This
similarity suggests that enhancement of R5020 and RU486 agonist
activities through activation of PKA involves related mechanisms.

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Figure 6. Effect of cAMP on the Time Courses of R5020
and RU486 Responses of a Stably Integrated MMTV-luc Reporter Gene
HeLa3B2.M cells stably expressing PRB and containing stably
integrated copies of the MMTV-luc reporter plasmid were incubated with
10 nM R5020 or 10 nM RU486 in the presence or
absence of 0.2 mM cAMP. At the indicated times, cells were
harvested and assayed for luciferase activity. Luciferase activities
were normalized to untreated controls and represent the mean of two
independent experiments. Controls showed 33487 luciferase units in 25
µl of extract.
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cAMP Does Not Alter the Subcellular Distribution or Increase
Expression of PRB
A possible mechanism that could account for the stimulating effect
of cAMP on transcription of PR-dependent reporter genes would be for
cAMP to promote nuclear localization of the receptor or to increase the
PR content in the cell. To investigate an effect on subcellular
localization, immunofluorescence studies with HeLa3B2.TK cells were
performed. Using antibody 9E10 directed against the myc-tag
present in the transfected PRB, we observed a strong
nuclear staining with HeLa3B2.TK cells in untreated controls (Fig. 7
, lower panel). In contrast,
no such signal was observed with the parental HeLa cells, confirming
the specificity of the antibody (Fig. 7
, upper panel).
Clearly, treatment with R5020, RU486, and cAMP, alone or in
combinations, for up to 25 h did not alter the subcellular
distribution significantly. Thus, the cAMP effects on
PRB-mediated transcription are not due to an effect on PR
localization. However, prolonged treatment with R5020 significantly
decreased the nuclear PR staining. To investigate the effect of
hormones on the PR content more precisely, extracts from HeLa3B2.TK
cells treated with the various hormones for 5 to 35 h were
subjected to Western blotting with a PR-specific antibody. As shown in
Fig. 8
, R5020 mediated a strong
down-regulation of PR levels, which became clearly evident after
15 h of incubation. In contrast, RU486 alone had relatively little
effect on the PR content of the cells while cAMP treatment alone
resulted in a modest reduction of PR. Cotreatment with cAMP seemed to
enhance down-regulation observed by both R5020 and RU486. Because the
effect of cAMP on PR levels is contrary to its effect on RU486- or
R5020-dependent transcription, we conclude that the transcriptional
enhancement resulting from cotreatment with cAMP cannot be ascribed to
changes in PR content.

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Figure 7. Lack of an Effect of cAMP on the Subcellular
Localization of PRB in HeLa3B2.TK Cells
HeLa3B2.TK cells (lower panel) were treated with hormones
(0.2 mM cAMP, 10 nM RU486, 10 nM
R5020) as indicated and analyzed by immunofluorescence using antibody
9E10. The immunofluorescence picture of untreated control cells
(HeLa3B2.TK) is shown together with the corresponding phase contrast
picture. PRB-negative HeLa cells are shown for comparison
(upper panel).
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Figure 8. Effect of cAMP and Steroids on
PRB Levels
HeLa3B2.TK cells were treated with cAMP (0.2 mM), RU486 (10
nM), R5020 (10 nM), or cycloheximide (chx, 40
µM) as indicated. Cycloheximide treatment was started 40
min before the addition of hormones. Whole cell extracts were assayed
for PR content by Western blotting with a PR-specific antibody (B30).
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Interestingly, concomitant treatment of cells with cycloheximide, an
inhibitor of protein synthesis, completely prevented down-regulation of
PR under all conditions (Fig. 8
). This could indicate the
hormone-mediated down-regulation of PR involves a factor that is
subject to a fast turnover.
cAMP/RU486 Synergism Requires Protein de Novo
Synthesis
Our analysis of the cAMP-mediated modulation of RU486 and R5020
responses revealed that the synergistic increase of ligand induction
through cAMP represents a delayed response relative to the effects of
the ligands alone (Figs. 4A
and 6
). This delay could indicate that
de novo synthesis of an essential factor(s) is required for
this synergism. To study the requirement of ongoing protein synthesis,
we investigated the effect of cycloheximide, an inhibitor of protein
synthesis, on the regulation of luciferase mRNA in HeLa3B2.TK cells
by RNase protection analysis (39). A cycloheximide concentration was
used that caused about 70% inhibition of the luciferase enzyme
activity in untreated controls but had little effect on cell viability
(data not shown). Luciferase RNA was determined using an in
vitro synthesized antisense RNA of 331 nucleotides in length that
yielded a protected RNA of 276 nucleotides, as expected (Fig. 9
). As internal control,
-actin
antisense RNA resulting in three major signals of 125 to 130
nucleotides was used. Although fold induction values as measured by RNA
analysis were generally lower than those obtained in luciferase assays,
RNA analysis results qualitatively confirmed the effects of steroids
and cAMP/RU486 synergism (Fig. 9
). Consistent with a direct
PRB-mediated mechanism, luciferase mRNA induction elicited
by R5020 was not decreased, but elevated by cycloheximide (compare
lanes 1 and 6 with 5 and 10, respectively). Similarly, the RU486
response proved to be insensitive to cycloheximide (lanes 3 and 8),
suggesting that it also represents a primary PRB-mediated
transcriptional response. Importantly, the cAMP/RU486 synergism that
was evident in the absence of protein synthesis inhibitor (lanes 24)
was completely abolished by cycloheximide treatment (lanes 79). This
result, which was consistently observed in three independent
experiments, indicates that cAMP/RU486 synergism requires de
novo synthesis of an involved factor.
 |
DISCUSSION
|
---|
Several studies using different cellular models have demonstrated
that cAMP can dramatically increase the agonist potential of
progesterone antagonists (32, 33, 34, 36, 37). By stable transfection of a
PRB cDNA expression vector, we have created novel cell
culture models that allowed us to specifically address the regulation
of PRB through ligands and activators of PKA in the HeLa
and CV-1 cell background. In a second step, we stably transfected
HeLa3B2 cells with two different hormone-responsive reporter genes,
namely PRE2-TK-luc and MMTV-luc, to create model systems
that combine the advantages of stable transfectants with the superior
sensitivity of the luciferase reporter system. As shown by
immunoblotting and gel retardation, HeLa3B2 cells express slightly
lower levels of PRB as compared to T47D cells (Fig. 1A
),
which have been the established cell culture model for the analysis of
the mechanism of progestin and antiprogestin action. It is important to
note that the PRB-positive HeLa cell clone expresses
functional endogenous GRs, which bind RU486 with high affinity and can
recognize the PREs present in the PRE2-TK- and MMTV-luc
reporters. Using the PRE2-TK-luc reporter, we observed
significant cAMP-independent agonist activity of RU486 in
GR/PRB-positive, but not GR-only cells, indicating that
this is a PRB-specific phenomenon (Fig. 4
). This finding
confirms a previous study from K. Horwitzs group (11) in which a
TK-chloramphenicol acetyltransferase (CAT) reporter with a single PRE
was used. A synergistic stimulation of PRE2-TK-luc
transcription by RU486 and an activator of PKA was also detected
exclusively with the PRB-positive HeLa cell clone,
suggesting that only RU486-loaded PRB, but not
RU486-occupied GR, is sensitive to activation of PKA. This conclusion
apparently contradicts results of Nordeen and co-workers (36), who
reported a GR-mediated synergism of cAMP and RU486 on
GRE2E1b- and MMTV-CAT reporter genes in two different
GR-positive fibroblast cell lines. Whether this discrepancy is due to
the different reporter genes used remains to be established.
Previous reports analyzing modulation of RU486 and R5020 agonist
activities already indicated that PKA activators preferentially
increase RU486 agonist activity and therefore partially decrease the
antagonist potential (33, 34, 36). Here, in HeLa3B2 cells containing
chromosomally integrated copies of PRE2-TK-luc, we observed
that upon incubation with cAMP for 25 h, RU486 can acquire much
stronger agonist potential than R5020, resulting in a complete loss of
its antagonist potential (Fig. 5
). To our knowledge such a complete
reversal of the agonist/antagonist roles of RU486 and R5020, leading to
the paradoxical situation in which the agonist R5020 has the potential
to act as a partial antagonist of the antagonist RU486, has not been
observed in previous studies. The complete loss of RU486 antagonist
potential, as observed with the PRE2-TK-luc reporter gene,
is partly due to the cAMP-mediated enhancement of the RU486 agonist
activity beginning after 10 h of incubation and partly due to the
concomitant decrease of the R5020 response (Fig. 4A
). As the decrease
of the R5020 response of PRE2-TK-luc observed during
incubation periods exceeding 10 h is also observed in the absence
of the PKA activator, we conclude that it is not an effect of cAMP
(Fig. 4A
). Rather, we believe that this decrease of R5020
responsiveness is due to the R5020-mediated down-regulation of
PRB levels (Figs. 7
and 8
). With the MMTV-luc reporter we
also observed a strong enhancement of RU486 agonist activity upon
cotreatment with cAMP (Fig. 6
). However, partial RU486 antagonist
potential was maintained at all time points investigated, since the
R5020 response of this promoter did not decrease during long incubation
periods. This is quite astonishing as the PRB levels in the
stable HeLa cell clone analyzed were down-regulated through R5020
treatment to a similar extent as in the PRE2-TK-luc
expressing HeLa cell clone showing a dramatic loss of responsiveness to
R5020. We speculate that the ability of MMTV-luc to maintain high
responsiveness at lower PRB concentrations could be due to
the extreme cooperativity of the PREs of the MMTV promoter, which may
ensure sufficient PRE occupancy even at low receptor levels.
Another novel aspect of our studies that has not been elaborated in
previous papers is that, in the presence of cAMP, the ratio of the
agonist activities of R5020 to RU486 and consequently also the
antagonist potential of RU486 vary considerably with the duration of
the hormone treatment (Figs. 4A
, 5
, and 6
). As can be deduced from the
time courses of agonist responses on PRE2-TK-luc and
MMTV-luc transcription, RU486 (as the weaker agonist) maintains
relatively high antagonist potential during short incubation periods;
with longer incubation time, an increasing loss of antagonist potential
of RU486 can be observed. This time-dependent variation of the RU486
antagonist potential was evident both with the PRE2-TK-luc
and MMTV-luc reporter and thus appears not to represent a
promoter-specific phenomenon.
In agreement with studies on T47D cells (40), immunocytofluorescence
studies indicated that the unliganded PRB of HeLa3B2 cells
is predominantly located in the nucleus and that R5020, RU486, and
cAMP, alone or in combinations, do not alter the intracellular
distribution (Fig. 7
). By immunoblotting we show that PRB
levels in HeLa3B2.TK cells are substantially down-regulated by R5020
treatment (Fig. 8
), confirming similar results of a study that used a
stable transfectant expressing PRB in the T47D background
(37). Compared to R5020, both RU486 and cAMP had relatively little
effect on PR expression. Clearly, cotreatment with cAMP did not prevent
RU486- and R5020-mediated down-regulation of PR (Fig. 8
), but rather an
enhancement of ligand-induced downregulation was observed. Together,
these data indicate that the enhancement of PRB-mediated
agonist activities of RU486 and R5020 by activators of PKA cannot be
ascribed to effects on PRB levels or subcellular
localization.
We demonstrated that in PRB expressing HeLa3B2 cells the
effect of cAMP could be mimicked by overexpression of PKA catalytic
subunit (Fig. 2
). This result indicates that the cAMP effect is not due
to some nonspecific effects of this compound, but attributable to
activation of PKA via an intracellular rise in cAMP. A PKA mutant
without the catalytic activity had no effect on RU486 agonist activity
(Fig. 2
). This result proves directly that PKA-mediated phosphorylation
represents an essential step in the mechanism. Since the PR is a
phosphoprotein, it has been assumed that PKA-mediated alteration of the
phosphorylation state of the PR itself might play an important role in
the mechanism. However, several studies attempting to address this
question did not provide direct evidence for this concept (32, 38).
Interestingly, in a CV1 cell clone stably expressing functional
PRB, RU486 showed no agonist activity at all, even when a
PKA catalytic subunit was cotransfected (Fig. 3
). Our finding that
overexpression of the catalytic subunit of PKA does not suffice to
elicit RU486 agonist activity in CV-1.1B cells also argues against a
simple model, in which direct phosphorylation of PRB
through PKA triggers the increased transcriptional activity of
RU486-liganded receptors. Furthermore, these data suggest that in
addition to catalytically active PKA and RU486-loaded PRB,
the mechanism requires another cellular factor(s), which appear(s) to
be expressed in a cell-specific manner.
The perhaps most important contribution of this study to the
understanding of cAMP/RU486 synergism is our finding that the mechanism
requires the intermediate induction of an essential factor and
therefore represents a delayed primary response (41). Two different
experiments support this notion. First, we demonstrated that
synergistic induction of PRE2-TK-luc by cAMP and RU486 is
sensitive to partial inhibition of protein synthesis by cycloheximide
(Fig. 9
). The effect of cycloheximide on the synergism was specific,
since neither R5020 induction nor the cAMP-independent RU486 induction
of PRE2-TK-luc were decreased by the inhibitor. Second, our
analysis of the time courses of the hormonal responses on both
PRE2-TK-luc and MMTV-luc promoters revealed that the onset
of synergism is delayed by 5 to 10 h relative to the
cAMP-independent RU486 response (Figs. 4A
and 6
). Together, these data
are consistent with a model in which cAMP/RU486 treatment induces a
factor(s) that is necessary for synergism. Interestingly, the synergism
between the agonist R5020 and cAMP, which was specific for the MMTV-luc
reporter gene, also required at least 10 h to develop (Fig. 6
).
This similarity suggests that PKA-mediated enhancement of both R5020
and RU486 agonist activities occur through the same mechanism involving
the intermediate synthesis of an essential factor.
The functional role of the postulated intermediately induced factor
enabling synergism between PKA and RU486-loaded PRB on
PRE-containing reporter genes remains completely unclear at present.
However, as originally proposed by Nordeen et al. (36), this
factor could be a protein that mediates protein-protein interactions
between RU486-loaded PR and the initiation complex and thereby enhances
transcription initiation by RNA-polymerase II. Alternatively, the
induced factor could be a kinase that modifies a preexisting
transcriptional coactivator of PRB, e.g. SRC-1
or TIF-2 (23, 24), in a way that allows it to interact with
antagonist-loaded PRB. Recently, Jackson and co-workers
(42) provided evidence that the partial agonist activity of
RU486-loaded PR is inhibited by nuclear receptor corepressor (N-CoR)
and silencing mediator of retinoic acid and thyroid hormone receptors
(SMRT), two related nuclear proteins previously characterized as
corepressors of unliganded thyroid and retinoid acid receptors, but
stimulated through L7/SPA, a factor that interacts with the hinge
region of PR. Importantly, both corepressors were able to reverse the
stimulatory effect of L7/SPA on RU486 agonist activity, suggesting that
the ratio of coactivators and corepressors is a crucial parameter in
regulating the activity of RU486-loaded PR. Thus, it is an attractive
possibility that cAMP exerts its stimulatory effect on RU486 agonist
activity through altering the balance between positively and negatively
acting cofactors. Consistent with the requirement for intermediate
protein synthesis, this could be achieved either through increased
synthesis of a positive cofactor facilitating agonist activity or
through a more indirect mechanism that involves the induction of a
factor, e.g. a protein kinase or specific protease, that
ultimately reduces the expression or inactivates corepressors, thereby
leading to increased transcriptional activity of RU486-loaded PR bound
to DNA. It has been postulated that corepressor could also be
associated with unliganded PRs, but dissociate from the receptors upon
binding of progesterone (26). Assuming that free and corepressor-bound
PRs might be in equilibrium in the agonist-loaded state, our model
could also account for the stimulating effect of cAMP on R5020-loaded
PRs, as cAMP-mediated down-regulation of corepressors would shift the
equilibrium toward the free and active form.
 |
MATERIALS AND METHODS
|
---|
Plasmids
pSGN-His6-myc-hPR0 is a eukaryotic SV40 early
promoter-driven expression vector encoding human PR isoform B with
N-terminal his- and myc-tags (further information is available upon
inquiry). The luciferase reporter plasmid PRE2-TK-luc
contains two PRE consensus sequences inserted into the SacI
site of pT81luc (43) in front of the TK kinase promoter (-81 to +52)
upstream of the luciferase gene. The MMTV-luc reporter plasmid
(pHCwtluc, Ref.44) contains MMTV-LTR sequences from
position -235 to +122 linked to the luciferase cassette of pXP1 (43)
at the XhoI site. RSV-CHO-PKA-Cß and RSV-CHO-PKA-Cßmut
are mammalian expression vectors for the catalytic subunit ß or the
mutant subunit ßmut of the cAMP-dependent protein kinase of chinese
hamster ovary cells (45).
Cell Culture and Transfection
HeLa and CV1 cells were grown in DMEM supplemented with 1
mM glutamine, 100 U/ml penicillin, 100 U/ml streptomycin,
and 10% FBS (Cytogen). Puromycin (0.5 µg/ml) or G418 (0.5 mg/ml) was
added to the medium for stably transfected cells containing pPUR or
pSV2neo selection markers, respectively. HeLa cells were cotransfected
with pSGN-His6-myc-hPR0 and pSV2neo and selected for G418
resistance. Similarly, CV1 cells were cotransfected with a
PRB expression vector and pPUR (CLONTECH, Palo Alto, CA)
and selected for puromycin resistance. PRB expressing
clones (HeLa3B2 and CV1.1B) were identified by immunocytofluorescence
with PR antibodies (Medac, Hamburg, Germany) and further characterized
by Western blot analysis. HeLa3B2.TK cells were created by stable
transfection of HeLa3B2 cells with PRE2-TK-luc and pPUR,
followed by selection in medium containing G418 and puromycin.
Similarly, HeLa3B2.M transformants containing an MMTV-luc reporter
(pHCwt-luc) were generated by cotransfection with pPUR.
HeLa.TK (PR-) cells were obtained by cotransfection of
HeLa cells with PRE2-TK-luc and pPUR and puromycin
selection. For stable transfection of CV1 cells, Lipofectamin (GIBCO
BRL, Gaithersburg, MD) was used while HeLa were transfected by
lipofection (GIBCO BRL).
For transient transfection, 6 x 105 HeLa3B2 or 4
x 105 CV1.1B cells were plated per well of a six-well dish
(Falcon, Becton-Dickinson, Bedford, MA) and grown for 1 day. Opti-MEM
(200 µl) containing 1 µg PRE2-TK-luc, 100 ng
RSV-CHO-PKA-Cß or RSV-CHO-Cßmut, 900 ng pBluescript, and 5 µg
Lipofectin were incubated for 30 min at 20 C. After addition of 1.3 ml
Opti-MEM, the DNA/lipofectin mixture was added to the cells, which had
previously been washed two times with Opti-MEM. After 5 h, cells
were transferred to DMEM medium containing 10% FCS and incubated
overnight. Hormone treatment was started about 5 h after the
addition of DNA. After a further incubation for 25 h, cells were
harvested and analyzed for luciferase expression.
HeLa cell clones containing stably integrated luciferase reporter genes
were plated in six-well dishes at a density of 6 x
105 cells per well and grown for 1 day. Then cells were
incubated in medium containing hormones as given in the figure legends.
Control samples received appropriate amounts of vehicle. Harvested
cells were washed twice in PBS and lysed by the addition of 100 µl of
lysis buffer (25 mM Tris-phosphate pH 7.8, 2 mM
dithiothreitol, 2 mM 1,2-cyclohexanediamine
NNN'N'-tetraacetic acid, 10% glycerol, 1% Triton X-100).
After removal of cell debris by centrifugation, luciferase assays were
performed on 25 µl of extracts using a Lumat LB9501 luminometer
(Berthold). In each experiment cell culture treatment groups were in
duplicate.
SDS-PAGE, Western Blot, and Gel Retardation Assay
For the preparation of whole cell extracts, cells were washed
with PBS, centrifuged and resuspended in whole cell extract buffer
(10% glycerol, 20 mM HEPES, pH 7.9, 1 mM EDTA,
600 mM NaCl, 1 mM dithiothreitol, and protease
inhibitors). Cells were lysed by three cycles of freeze-thawing. After
centrifugation, supernatants were saved as extracts. Protein
concentrations were determined with the Bio-Rad protein assay. SDS-PAGE
and transfer to nitrocellulose was performed as described by Sambrook
et al. (46). Blots were incubated with monoclonal antibodies
against human PR (Medac) and rabbit anti-mouse IgG antibodies linked to
peroxidase. PR immunoreactivity was revealed using a chemiluminescence
method (ECL, Amersham, Arlington Heights, IL).
The DNA-binding activity of PR was determined in a gel-shift assay
using a 32P-labeled PRE-oligonucleotide and preincubated
with whole cell extract 1 µM progesterone or ethanol in a
10 µl standard binding reaction for 20 min on ice (17). Monoclonal PR
antibodies (B30) were added to the binding reaction as indicated.
Samples were subjected to electrophoresis on a nondenaturing 4%
polyacrylamide gel. After fixing and drying of the gel, protein
PRE-complexes were visualized by autoradiography.
Immunocytofluorescence
Cells were cultured on cover slides in 24-well dishes for 525
h in the presence or absence of the indicated hormones. Samples were
fixed and permeabilized with ice-cold methanol for 10 min. After
blocking in PBS containing 0.5% BSA for 10 min, cells were incubated
with antibody 9E10 against the myc-tag (47) for 90 min at 37
C, washed three times with blocking buffer, and incubated with
FITC-conjugated antimouse antibody (Dianova, Hamburg, Germany) for 60
min at 37 C.
RNase Protection Analysis
To generate an RNase protection probe for luciferase RNA, a
276-bp EcoRI/RsaI fragment of the luciferase gene
of pTK81luc (43) was cloned between the EcoRI and
EcoRV sites of pBluescript SK+. Radiolabeled antisense RNA
of 331 nucleotides in length was synthesized from this plasmid
(EcoRI linearized) by T7 polymerase in vitro. As
an internal control, a
-actin antisense RNA synthesized by SP6
polymerase from a HinfI digested plasmid (kindly provided by
Ian Kerr, London) was included in all hybridization reactions. Specific
activity of
-actin antisense RNA was reduced by including unlabeled
UTP in the SP6 polymerase reaction.
For isolation of total RNA, HeLa3B2.TK cells were treated for 15 h
in medium containing the indicated hormones or vehicles. Hormone
treatment of cells receiving cycloheximide (40 µM) was
initiated 40 min after addition of the protein synthesis inhibitor.
Total RNA was isolated using Trizol reagent (Gibco BRL). Hybridization
of 40 µg cellular RNA with luciferase (200,000 cpm) and
-actin
(50,000 cpm) antisense RNAs and RNase digestion was performed as
described (48). Protected transcripts were separated on sequencing
gels. After fixing and drying, protected bands were quantified on a
bio-imaging analyser (BAS1500, Fuji).
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to W. Schulz (Düsseldorf, Germany), A.C.B.
Cato (Karlsruhe, Germany), R. A. Maurer (Iowa City, IA), Ian Kerr
(London, U.K.), and H. Gronemeyer (Strasbourg, France) for
providing plasmids. We thank D. P. Edwards (Denver, CO) for providing
the monoclonal antibody B30 and Roussel-Uclaf (France) for providing
RU486. We also thank V. Ulber for excellent technical
assistance and C. Schwerk and D. Michels for a critical reading of the
manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Ludger Klein-Hitpass, Institut für Zellbiologie (Tumorforschung), Universitätsklinikum, Virchowstr. 173, D-45122 Essen, Germany.
This work was supported by a joint grant of Schering AG and BMBF (to
L. K.-H.).
Received for publication June 5, 1997.
Revision received October 30, 1997.
Accepted for publication November 14, 1997.
 |
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