New Antiprogestins with Partial Agonist Activity: Potential Selective Progesterone Receptor Modulators (SPRMs) and Probes for Receptor- and Coregulator-Induced Changes in Progesterone Receptor Induction Properties
Georgia Giannoukos1,
Daniele Szapary,
Catharine L. Smith,
James E. W. Meeker2 and
S. Stoney Simons, Jr.
Steroid Hormones Section (G.G., D.S., J.E.W.M.,
S.S.S.) Laboratory of Molecular and Cellular Biology
National Institute of Diabetes, Digestive and Kidney Diseases and
the Laboratory of Receptor Biology and Gene Expression
(C.L.S.) National Cancer Institute/Division of Basic
Sciences National Institutes of Health Bethesda, Maryland
20892-0805
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ABSTRACT
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A pharmacologically relevant property of steroid
hormone-regulated gene induction is the partial agonist activity of
antisteroid complexes. We now report that dexamethasone-mesylate
(Dex-Mes) and dexamethasone-oxetanone (Dex-Ox), each a derivative of
the glucocorticoid-selective steroid dexamethasone (Dex), are two new
antiprogestins with significant amounts of agonist activity with
both the A and B isoforms of progesterone receptor (PR),
for different progesterone-responsive elements, and in several cell
lines. These compounds continue to display activity under conditions
where another partial antiprogestin (RTI-020) is inactive. These new
antiprogestins were used to determine whether the partial agonist
activity of PR complexes can be modified by changing concentrations of
receptor or coregulator, as we have recently demonstrated for
glucocorticoid receptors (GRs). Because GR and coregulator
concentrations simultaneously altered the position of the
physiologically relevant dose-response curve, and associated
EC50, of GR-agonist complexes, we also examined
this phenomenon with PR. We find that elevated PR or transcriptional
intermediary factor 2 (TIF2) concentrations increase the partial
agonist activity of Dex-Mes and Dex-Ox, and the
EC50 of agonists, independently of changes
in total gene transactivation. Furthermore, the corepressors
SMRT (silencing mediator for retinoid and thyroid receptors) and NCoR
(nuclear receptor corepressor) each suppresses gene induction
but NCoR acts opposite to SMRT and, like the coactivator TIF2, reduces
the EC50 and increases the partial agonist
activity of antiprogestins. These comparable responses of GR and PR
suggest that variations in receptor and coregulator concentrations may
be a general mechanism for altering the induction properties of other
steroid receptors. Finally, the magnitude of coregulator effects on PR
induction properties are often not identical for agonists and the new
antagonists, suggesting subtle mechanistic differences. These
properties of Dex-Mes and Dex-Ox, plus the sensitivity of their
activity to cellular differences in PR and coregulator concentrations,
make these steroids potential new SPRMs (selective progesterone
receptor modulators) that should prove useful as probes of PR induction
properties.
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INTRODUCTION
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A common use of endocrine therapies in the clinical setting is to
block the action of an endogenous hormone in both normal and malignant
tissues. The inhibition of progesterone action in women is used to
prevent conception at a variety of stages (1, 2). The growth of many
breast cancer tumors is retarded by suppressing the action of
endogenous estrogens (3, 4). Pure antisteroids, which display no
agonist activity, represent one of the most straightforward methods of
blocking the actions of steroid hormones. The antisteroid undergoes
most of the same steps as agonist steroids, including steroid binding
to the cognate receptor, activation, nuclear translocation, and DNA
binding (5, 6). It is predominantly in the association with
coregulators and the transcriptional machinery that the antisteroid
complexes appear to differ and prevent the changes in rates of gene
transcription seen with agonist complexes (7, 8, 9). Unfortunately, the
side effects of antisteroid therapies can be quite severe. A pure
antagonist will block all of the actions of a particular receptor in
addition to the one that is targeted. Furthermore, some antisteroids,
like RU 486 (10), cross-react with other receptors to block the actions
of multiple classes of steroids (11), thereby magnifying the number of
undesired actions.
An exciting new approach to endocrine therapies has emerged with the
concept of SRMs, or selective receptor modulators. These are compounds
that are antagonists for some genes/tissues but agonists for others.
Thus, they are not pure antagonists. This ability to display partial
agonist activity with selected reporter genes can be clinically very
beneficial. For example, the agonist activity of raloxifene and
tamoxifen in bone coupled with their antagonist activity in breast
(Ref. 12 and references therein) suggests that their use in the
treatment of breast cancer might not be accompanied by the osteoporosis
seen with other antiestrogens (13). While it is not yet possible to
predict the genes for which a particular SRM will display antagonist
vs. agonist activity, an absolute requirement is that the
steroid possesses partial agonist activity for at least one gene.
Surprisingly, there are very few antiprogestins with partial agonist
activity for any genes and thus are candidate-selective progesterone
receptor modulators, or SPRMs. RU 486 is the most commonly used
antiprogestin and displays partial agonist activity only under selected
conditions (14, 15). More recently, a closely related derivative of RU
486, called RTI 3021020 (RTI-020), was found to be a partial agonist
in T47D cells but was not active in CV-1 cells (16). Therefore, it
would be extremely helpful for theoretical and clinical studies to
identify new antiprogestins with partial agonist activity.
Another approach for modifying the activities of antisteroids involves
the coregulators that appear to be recruited by DNA-bound receptors to
help modify the rates of target gene transcription. Ligand-free nuclear
receptors, and some steroid receptors bound by antagonists (9, 15, 17),
are usually associated with the corepressors SMRT (signal mediator and
repressor of transcription) or NCoR (nuclear receptor
corepressor) (18, 19). Agonist binding is thought to cause the
release of corepressors and allow the association of coactivators
(20, 21, 22, 23, 24). While corepressors are known to influence the partial agonist
activity of antisteroid complexes (8, 9, 15, 17, 25, 26), an effect of
coactivators on the activity of receptor-antisteroid complexes has only
lately been seen with GRs (26, 27). Interestingly, different
concentrations in coregulators also alter the concentration of the
glucocorticoid dexamethasone (Dex) required for half-maximal induction,
or EC50 (26, 27). Variations in the
EC50 are highly significant for cellular
functions because physiological concentrations of steroid are
subsaturating and much closer to the EC50 than to
the saturating concentrations of steroid required for maximal induction
by receptors. Therefore, even small differences in
EC50, which are associated with shifts in the
position of the dose-response curve, can have significant consequences
for the amount of gene induction seen with endogenous levels of
steroids.
Both the EC50 for agonist complexes of
glucocorticoid receptors (GRs) and the partial agonist activity of GR
antagonist complexes have recently been shown to be additionally
influenced by changes in GR concentration (26, 27, 28). These effects were
independent of cell, enhancer, promoter, and reporter gene. In all
cases, the changes in EC50 and partial agonist
activity of antiglucocorticoids occurred in parallel. Therefore, at
least for GRs, changes in receptor and coregulator levels can affect
the induction properties of both agonist and antagonist complexes.
These observations, coupled with the documented tissue variations of
coregulator (25, 29, 30, 31, 32, 33) and GR concentrations, suggest an attractive
model: that differential control of gene expression during development,
differentiation, and homeostasis by GR may be achieved, at least in
part, by intra- and intercellular fluctuations in GR and coregulator
concentrations (26).
In this study, we report that two antiglucocorticoids,
dexamethasone-21-meslyate (Dex-Mes) (34) and dexamethasone-oxetanone
(Dex-Ox) (35), are new antiprogestins possessing partial agonist
activity. This agonist activity is observed under conditions where
other antiprogestins are inactive, thus making Dex-Mes and Dex-Ox new
potential SPRMs. Using these compounds along with the progesterone
receptor (PR) agonist R5020, we found that the partial agonist activity
of antagonist complexes, and the EC50 for agonist
complexes, could be modulated by different concentrations of receptor,
coactivator, and corepressor just like they are for GRs (26, 27, 28). These
novel methods of modifying PR activity appear to offer new tools for
studies of PR action and for endocrine treatment of clinical
disorders.
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RESULTS
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Derivatives of the Glucocorticoid Dexamethasone Are New Ligands for
PR with Partial Agonist Activity
The synthetic glucocorticoid dexamethasone (Dex, Fig. 1
) has very low affinity for, and no
biological activity with, PR (36, 37) (data not shown). It was,
therefore, very unexpected when two derivatives of Dex, Dex-Mes and
Dex-Ox (Fig. 1
), exhibited significant quantities of agonist activity
with transfected PR in CV-1 cells (Fig. 2A
). Dex-Mes
consistently displayed appreciable levels of partial agonist activity
with both human PR isoforms (PR-A and PR-B) with two different reporter
constructs (GREtkCAT and MMTVLUC). RTI-020, a derivative of RU 486
(Fig. 1
), has been reported to possess partial agonist activity with PR
under some conditions but not in CV-1 cells (16). Our experiments
confirmed this while showing that Dex-Mes and Dex-Ox manifest
significant amounts of activity under the same conditions (see PR-B
with GREtkCAT in Fig. 2A
).

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Figure 2. Activity of Dex Derivatives with Transfected and
Endogenous PR
A, Transiently transfected PR-A or PR-B with GREtkCAT or MMTVLUC
reporter in CV-1 cells. Triplicate cultures were transiently
transfected with GREtkCAT, or MMTVLUC, plus the indicated amounts of
hPR cDNA plasmid, induced with either 30 nM R5020 or 1
µM of the indicated steroids, and assayed for luciferase
activity as described in Materials and Methods. The
total activity was normalized for total protein and plotted
as fold induction above the EtOH control (open bar).
Error bars represent SEM (n = 5) for
R5020 and Dex-Mes with GREtkCAT and range (n = 2) for all data for
PR-A with MMTVLUC. Other data are averages of triplicates from one
experiment. B, GREtkLUC reporter in T47D cells. Triplicate cultures
were transiently transfected as in panel A with 1 µg of GREtkLUC,
treated with either 30 nM R5020 or 1 µM of
the designated steroids, and assayed for luciferase activity as
described in Materials and Methods. The data were
normalized for total protein and plotted as fold induction above the
EtOH control (open bar). Error bars
indicate the range of two experiments except for RU 486, which is the
average of triplicates from one experiment.
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T47D human breast cancer cells contain both PR isoforms in addition to
GRs (38). When these cells were transiently transfected with the
GREtkLUC reporter, sizable amounts of activity were afforded by the
endogenous receptors for all of the added steroids except RU 486 (Fig. 2B
). Under these conditions, Dex-Ox was much less effective than R5020,
consistent with Dex-Ox being a partial agonist for PR. Dex-Ox is also a
partial agonist for GR (35, 39). However, very little of the Dex-Ox
agonist activity in T47D cells can be attributed to the endogenous GR
as the total activity with Dex-Ox is much greater than that seen for
the full glucocorticoid Dex (Fig. 2B
). Dex-Mes is also a partial GR
agonist (26, 28, 34, 35, 39). As the total Dex-Mes activity is less
than that for Dex, we can not immediately determine which receptor(s)
is responsible for the activity of Dex-Mes in T47D cells.
The above fold inductions in CV-1 cells by transiently transfected PR-A
or PR-B with saturating concentrations of R5020 were low except for
PR-A with the mouse mammary tumor virus (MMTV) reporter (Fig. 2A
). To
further investigate the properties of Dex-Mes and Dex-Ox under
conditions providing more robust responses, we looked for an
alternative cell line. We decided to concentrate on the larger PR
isoform, PR-B, because it has been the focus of many previous studies
on PR action (14, 16, 40). 1470.2 mouse mammary adenocarcinoma cells
contain very little endogenous PR (38) but were found to display steady
increases in total transactivation and fold induction over a range of
transiently transfected human PR-B cDNA (Fig. 3A
). Therefore, under these conditions,
PR is limiting for transactivation. 1470.2 cells do contain endogenous
GR, which binds Dex with the normal affinity of 23 nM
(data not shown) (41, 42). These GRs are inducible by added Dex but not
by any of the other steroids examined (Fig. 3B
). Therefore, any partial
agonist activity displayed by Dex-Mes, Dex-Ox, or RTI-020 in 1470.2
cells that are transiently transfected with PR-B would be due to PR
complexes.

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Figure 3. Gene Activation with Transiently Transfected PR in
1470.2 Cells
A, Effect of PR concentration on total transactivation and fold
induction with R5020. Triplicate cultures were transfected with MMTVLuc
and Renilla null luciferase reporters plus the indicated
amounts of hPR-B cDNA plasmid, induced with 30 nM R5020,
and assayed for luciferase and Renilla activities as
described in Materials and Methods. The average values,
normalized for cotransfected Renilla, were then plotted.
Similar results were obtained in at least two other experiments. B,
Activity of various steroids with endogenous GR of 1470.2 cells.
Triplicate cultures were transfected with GREtkLUC reporter as in panel
A and induced with the indicated steroids. The levels of luciferase
activity, normalized for cotransfected Renilla, were
then expressed as fold induction above basal levels. Similar results
were obtained in a second experiment. C and D, Activity of Dex
derivatives with transiently transfected PR in 1470.2 cells. Triplicate
cultures were transfected with 3 ng PR cDNA plasmid and GREtkLUC
reporter as in panel A and induced with the indicated steroids. The
levels of luciferase activity, normalized for cotransfected
Renilla, were then expressed as fold induction above
basal levels in panel C. The same data were replotted in panel D as
percent of the maximal induction (30 nM R5020) above basal
levels (open bar in panel C) to show directly the
percent partial agonist activity. The error bars in
panels C and D indicate the SD of triplicate values.
Similar results were obtained in a second experiment.
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When 1470.2 cells are transfected with low amounts of PR-B (3 ng) and
GREtkLUC reporter, significant inductions are observed for Dex-Mes and
Dex-Ox, but both are less than that for the full progestin R5020 (Fig. 3C
). As was suggested by the data in CV-1 cells (Fig. 2A
), the partial
agonist activity of Dex-Ox is greater than that of Dex-Mes, and both
are greater than that of RTI-020, which is almost inactive. This is
shown more clearly when the data of Fig. 3C
are expressed as percent of
maximal activity by saturating concentrations of R5020 to display
directly the percent partial agonist activity of Dex-Mes, Dex-Ox, and
RTI-020 (Fig. 3D
). Under these conditions in 1470.2 cells, Dex-Mes and
Dex-Ox have 20% and 45% partial agonist activity, respectively,
compared with less than 5% activity for RTI-020. Collectively, these
data indicate that Dex-Mes and Dex-Ox are new partial agonists for PR
in a manner that is independent of receptor form (PR-A or -B), of
enhancer, promoter, and gene (GREtkCAT, GREtkLUC, and MMTVLuc), and of
cell type (1470.2, T47D, and CV-1 cells).
Dex-Mes and Dex-Ox Are Antiprogestins That Interact with PR
The fact that Dex-Mes and Dex-Ox usually elicit less total
activity than R5020 under the variety of above conditions suggested
that these Dex derivatives are antiprogestins with partial agonist
activity. To determine whether these effects resulted from steroid
binding to PR, we examined the ability of each steroid to bind to PR
and to competitively inhibit the whole cell activity of PR-agonist
complexes. In a cell-free competition assay, the relative affinity for
PR is found to be R5020 >> Dex-Ox > Dex-Mes (Fig. 4A
). From these data, one can determine
that the apparent affinity of Dex-Ox for PR is at least 10-fold higher
than Dex-Mes and about 100-fold lower than R5020.

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Figure 4. Antiprogestin Properties of Dex-Mes and Dex-Ox
A, Competition of R5020 binding to cell-free receptors. Duplicate
samples of 2.5 nM [3H]R5020 plus the
indicated excesses of nonlabeled steroids were assayed for the total
binding of [3H]R5020 to cell-free preparations of
overexpressed hPR-B at 0 C, as described in Methods and
Materials. The range of duplicate values is indicated by the
error bars. Similar results were obtained in a second
experiment. B, Competition of PR-mediated induction by R5020 in intact
cells. Triplicate cultures were transfected with 3 ng hPR-B cDNA
plasmid and 1 µg MMTVLUC as in Fig. 3A and induced with the indicated
concentrations of R5020 ± Dex-Mes or Dex-Ox. The absolute levels
of luciferase activity, normalized for cotransfected
Renilla (± SD of triplicate values), were
plotted.
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Despite these low binding affinities of Dex-Mes and Dex-Ox for PR, both
compounds were found to be good inhibitors of R5020 activity in intact
cells. In view of the lower affinity of Dex-Mes vs. Dex-Ox
for PR in Fig. 4A
, we used 1 and 10 µM Dex-Mes,
but only 1 µM Dex-Ox, to inhibit the action of
300 pM R5020 with 3 ng of transfected PR. With
high enough concentrations, each Dex derivative reduces the activity of
300 pM R5020 to that of the competing steroid
alone, with Dex-Ox being at least 10-fold more potent than Dex-Mes
(Fig. 4B
). Therefore, Dex-Mes and Dex-Ox are antiprogestins with
potencies that appear to parallel their affinities for PR. This
correspondence suggests that it is not a metabolite of Dex-Mes, or
Dex-Ox, that is the active antiprogestin steroid. The ability of 0.3
nM R5020 to partially overcome the inactivity of
1 µM, but not 10 µM,
Dex-Mes also argues that Dex-Mes is exerting its antiprogestin activity
by binding directly, albeit weakly, to PR as opposed to acting via a
non-PR-mediated pathway.
Effect of Changing PR Concentration on R5020 Dose-Response
Curve
We previously reported that changing concentrations of GRs
modulate the partial agonist activity of several antiglucocorticoids
(26, 27, 28). This property appears to be intimately associated with
concomitant changes in the dose-response curve of glucocorticoid
agonists. Thus, conditions that cause a shift of the Dex dose-response
curve to lower EC50s are always accompanied by an
increased partial agonist activity of the antagonists. With the
discovery of new antiprogestins with partial PR agonist activity, we
wanted to know whether the activity of these compounds could similarly
be modified in the presence of different PR concentrations. In
preparation for this study, we first inquired whether the dose-response
curve of PR agonists could be shifted to lower
EC50s in the presence of higher amounts of PR. As
shown in Fig. 5
, the dose-response curve
of PR-agonist complexes is progressively shifted to lower
EC50s with higher PR concentrations. The average
left shift in the dose-response curve when going from 3 to 30 ng of
PR cDNA is 5.2 ± 1.1-fold (SD, n = 5,
P = 0.001). The dashed vertical lines in
Fig. 5
show how the activity of a single subsaturating concentration of
R5020 varies with different PR concentrations. Thus, as for GR
(26, 27, 28), the dose-response curve for PR-mediated transactivation is
not invariant but can be altered simply by varying the concentration of
PR.
Effect of Changing PR Concentration on Dose-Response Curves of New
Partial PR Agonists
The precise mechanism by which antisteroids exhibit partial
agonist activity is not well understood but is thought to proceed via
the same steps as used by full agonists. For this reason, we expected
that the magnitude of change in the left shift of the dose-response
curve for the PR agonist R5020 with elevated PR (Fig. 5
) would be
reproduced for the dose-response curves of the partial agonists Dex-Mes
and Dex-Ox. Interestingly, this is not the case. With 3 ng of
transfected PR-B, the EC50 for Dex-Mes is about
350 nM while that for Dex-Ox is a factor of 10 lower at 30
nM (Fig. 6
). However, there
is only a marginal effect of increased PR on the position of the
dose-response curve for Dex-Mes (Fig. 6A
: 1.49 ± 0.03-fold;
SD, n = 3, P = 0.0014) under
conditions where there is a 7-fold left shift of the R5020
dose-response curve (Fig. 6A
, insert). Similarly, the shift
in the dose-response curve for Dex-Ox with added PR is much less
(2.6 ± 0.4-fold; n = 2; Fig. 6B
) than that for R5020
(6.8 ± 1.6-fold, n = 2; see insert). These
differences in the magnitude of left shift of R5020 vs.
Dex-Mes and Dex-Ox suggest that the mechanistic details by which higher
levels of PR can reposition the dose-response curve are not the same
for PR-bound agonists vs. antagonists/partial agonists.

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Figure 6. Effect of PR Concentration on the Dose-Response
Curve and EC50 of (A) Dex-Mes and (B) Dex-Ox in 1470.2
Cells
After transfecting triplicate cultures with 3 or 30 ng of hPR-B cDNA
plasmid as in Fig. 3A , the luciferase values induced by the indicated
concentrations of Dex-Mes (A) or Dex-Ox (B) were normalized for
cotransfected Renilla and plotted as percent of the maximal values for
the same steroid, as in Fig. 5 . Insets present, for
comparison, the dose-response curves for R5020 with the different PR
concentrations in the same experiment. The SD of triplicate
values is indicated by the error bars. Similar results
were obtained in at least one additional experiment.
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Effect of Changing PR Concentration on the Partial Agonist Activity
of Dex-Mes and Dex-Ox
Although there is little effect of changing PR concentration on
the dose-response curves of Dex-Mes or Dex-Ox (Fig. 6
), it can not be
predicted what the effect will be on the partial agonist activity of
each steroid with PR. This is because there is no known relationship
between the EC50 for steroid induction of a
responsive gene and the total level of transactivation for the same
gene. To address this question, we performed experiments such as in
Fig. 3C
to determine the partial activity of Dex-Mes and Dex-Ox at two
different PR concentrations. However, for ease of interpretation, the
data were replotted as percent of maximal induction by saturating
concentrations of R5020, as in Fig. 3D
. This is done because the total
amount of transactivation by R5020 increases at higher levels of PR
(see Fig. 3A
), thus making it difficult to directly assess changes in
percent agonist activity simply by looking at the raw data. When the
results are expressed as percent of maximal induction by R5020,
however, it is clear that added PR has a dramatic effect (Fig. 7
). The increased activity of
subsaturating concentrations of R5020 (30 and 90 pM) is
what is expected from a left shift in the dose-response curve for R5020
(see Fig. 5
). For Dex-Mes and Dex-Ox, conditions that have little
effect on the dose-response curves of these steroids (see Fig. 6
) have
a very large effect on the partial agonist activity relative to
saturating concentrations of R5020. Dex-Mes has about 20% activity
with 3 ng PR but 60% activity with 30 ng PR. Dex-Ox, which displays
around 50% agonist activity with 3 ng PR, is almost a full agonist
with 30 ng PR. These dramatic increases in partial agonist activity can
occur in the absence of appreciable changes in
EC50 because they depend upon different
parameters. The partial agonist activity of the antiprogestins is
determined by the total transactivation with 1 µM
antagonist as a percent of the total transactivation by saturating
concentrations of R5020 at each PR concentration. On the other hand,
the EC50 for the antagonist with 3 and 30 ng PR
is independent of the activity of R5020 or the absolute amount of
transactivation by 1 µM antagonist. In summary, it is
clear that the partial agonist activity of Dex-Mes and of Dex-Ox with
PR is not constant but increases in proportion to the amount of PR
present in the cell.

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Figure 7. Variation of Partial Agonist Activity of
Antiprogestins with Increasing PR in 1470.2 Cells
Triplicate cultures were transfected with 3 or 30 ng of PR cDNA and 1
µg MMTVLUC as in Fig. 3A , followed by treatment with the indicated
steroids. The induced luciferase values were determined, normalized for
cotransfected Renilla, and plotted as percent of maximal
induction by 30 nM R5020, as in Fig. 3D . The SD
of triplicate values is indicated by the error bars.
Similar results were obtained in a second experiment.
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It is interesting that the partial agonist activity of RTI-020 does not
increase under conditions that augment the activity of Dex-Mes and
Dex-Ox (Fig. 7
). Thus, while RTI is a partial agonist under some
conditions (16), Dex-Mes and Dex-Ox retain partial agonist activity
under a possibly wider variety of conditions.
Effects of Coactivators and Corepressors on PR Induction
Properties
The total transactivation of responsive genes by PR has been found
to increase in the presence of cotransfected coactivators (43, 44)
while corepressors can decrease receptor transactivation (8, 18, 19, 26). Recently, coactivators and corepressors have also been shown to
modify the dose-response curve of GR-agonist complexes and the partial
agonist activity of GR-antagonist complexes (26, 27). Given the high
homology between GR and PR, we asked whether similar effects might be
seen with PR complexes. Such a study was difficult before our discovery
of the partial agonist activities of Dex-Mes and Dex-Ox because neither
of the existing antiprogestins, RU 486 or RTI-020, displays appreciable
agonist activity (Figs. 2
, 3
, and 7
).
When TIF2 is cotransfected with PR in 1470.2 cells, the total
transactivation increases about 25%, as expected for a coactivator. At
the same time, a weak but statistically significant shift to lower
steroid concentrations is seen in the dose-response curve [Fig. 8A
: 1.95 ± 0.53 fold
(SD), n = 4, P = 0.038]. At the same
time, added TIF2 enhances the partial agonist activity of Dex-Mes (Fig. 8B
). Interestingly, while the effect of added TIF2 is not as great as
added PR on the positioning of the dose-response curve for R5020 (Fig. 8A
), TIF2 is equally effective in increasing the partial agonist
activity of Dex-Mes (Fig. 8B
). This difference in sensitivity to TIF2,
relative to increased PR, suggests that the mechanisms involved in TIF2
modulation of agonist and antagonist induction properties are not the
same.

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Figure 8. Modification of Induction Properties of PR-Agonist
and Antagonist Complexes in 1470.2 Cells by Added Coactivator TIF2
Triplicate cultures were transfected as in Fig. 3A with 1.5 ng PR
± 0.4 µg TIF2, or 30 ng PR, plus the indicated concentrations of
R5020 or 1 µM Dex-Mes. The data for R5020 (A) and Dex-Mes
(B) are normalized for cotransfected Renilla and plotted as percent of
the maximal values for R5020, as in Fig. 3D . The treatments in panel B
are 1.5 ng PR (open bar), 1.5 ng PR + 0.4 µg TIF2
(patterned bar), and 30 ng PR (solid
bar). The SD of triplicate values is indicated by
the error bars. Similar results were obtained in three
additional experiments.
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It should be noted that TIF2 had no effect on the induction properties
of either PR or GR in T47D cells under conditions where increased GR
did have an effect on the Dex EC50 (data not
shown). Thus, it appears that the ability of TIF2 to modulate PR, or
GR, induction properties can be cell specific.
SRC-1 (steroid receptor coactivator 1) is another coactivator
that is closely related to TIF2 (45, 46) and thus might be expected to
have similar effects as TIF2. To examine the effects of a range of
SRC-1 concentrations, we did not perform an entire dose-response curve
but rather assayed the percent of maximal activity of just a single
subsaturating concentration of R5020. As shown in Fig. 5
(dashed
lines), this abbreviated assay is a valid test for changes in the
R5020 dose-response curve or EC50. Surprisingly,
additions of up to 1.8 µg of SRC-1 have no significant effect on
either the EC50 for R5020, as indicated by the
activity of a subsaturating concentration of R5020 (Fig. 9
), or the total amount of
transactivation (data not shown). Similarly, neither CREB-binding
protein (CBP) nor 0.45 µg SRC-1 plus 0.2 µg CBP had any effect on
the EC50 for R5020 or the total amount of
transactivation (data not shown). This absence of effect was not due to
a total inactivity of the SRC-1 because 1.8 µg of SRC-1 did increase
the partial agonist activity of Dex-Mes with 3 ng of PR to about 65%
of that seen with 30 ng of PR (n = 3; P = 0.048;
see also Fig. 9
). Also, the same SRC-1 and CBP plasmid preparations
elevated the percent of maximal activities of 1
nM Dex and 1 µM Dex-Mes
with GR in CV-1 cells (data not shown and Ref. 26). Therefore, it
appears that not all coactivators have the same effects on the
activities of PR complexes in 1470.2 cells.

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Figure 9. Influence of Coactivator SRC-1 on Induction
Properties of PR-Agonist and Antagonist Complexes in 1470.2 Cells
Triplicate cultures were transfected as in Fig. 3A with PR ±
varying amounts of SRC-1, as indicated, and then treated with
vehicle ± 60 pM R5020, 30 nM R5020, or 1
µM Dex-Mes. The induction by 60 pM R5020, or
1 µM Dex-Mes, was normalized for cotransfected Renilla
and plotted as percent of the maximal activity with 30 nM
R5020, as in Fig. 3D . The SD of triplicate values is
indicated by the error bars (*, P =
0.04; **, P = 0.001). Similar results were obtained
in two additional experiments.
|
|
In contrast to coactivators, corepressors, such as SMRT (19) and NCoR
(18), typically produce the opposite effects of coactivators. They can
decrease the total transactivation of receptors (8, 18, 19, 26) and
reduce the partial agonist activity of antisteroids (8, 9, 15, 17, 25, 26). It was therefore of interest to determine what effect increased
concentrations of corepressor would have on PR-agonist and partial
agonist complexes. Cotransfection of the corepressor SMRT causes a
reproducible 2.6 ± 0.4-fold (n = 2) right shift in the
dose-response curve (Fig. 10A
), and
decreased the partial agonist activity of Dex-Mes from 11.1 ±
1.6% to 0.4 ± 1.6% (SD, n = 4;
P < 0.0001, Fig. 10B
), while simultaneously acting as
a corepressor to decrease the levels of total transactivation by
13 ± 6% (SEM, n = 5,
P = 0.09).

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Figure 10. Effect of Added Corepressor SMRT on Induction
Properties of PR-Agonist and Antagonist Complexes in 1470.2 Cells
Triplicate cultures were transfected as in Fig. 3A with 30 ng PR
± 1.8 µg SMRT plus the indicated concentrations of R5020 or 1
µM Dex-Mes. The data are normalized for cotransfected
Renilla and plotted as percent of the maximal values for
R5020 for (A) one of two experiments with R5020 and for (B) the average
(±SD) of four experiments with Dex-Mes, as in Fig. 3D .
|
|
The addition of NCoR, another corepressor (18), also suppresses the
total transactivation, this time by 28 ± 9% (SEM,
n = 9, P = 0.01). However, NCoR causes an increase
in the activity of subsaturating concentrations of R5020 to shift the
dose-response curve to lower EC50s (Fig. 11A
). At the same time, added NCoR
increased the partial agonist activity of saturating concentrations of
Dex-Mes (Fig. 11B
; averages values = 36 ± 17% without NCoR
vs. 69 ± 16% with NCoR, n = 6,
SD, P = 0.0064). Therefore, both
SMRT and NCoR appear to act as corepressors by decreasing the total
transactivation, but they elicit almost exactly opposite behaviors in
the induction properties of PR complexes.

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Figure 11. Modulation of Induction Properties of PR-Agonist
and Antagonist Complexes in 1470.2 Cells by Added Corepressor NCoR
Triplicate cultures were transfected as in Fig. 3A with 30 ng PR
± 1.2 µg NCoR plus the indicated concentrations of R5020 or 1
µM Dex-Mes. The data for R5020 (A) and Dex-Mes (B) are
normalized for cotransfected Renilla and plotted as percent of the
maximal values for R5020, as in Fig. 3D . The SD of
triplicate values is indicated by the error bars.
Similar results were obtained in two (A) or five (B) additional
experiments.
|
|
 |
DISCUSSION
|
---|
This study describes evidence that Dex-Mes and Dex-Ox, two C-17
position derivatives of the GR-selective ligand Dex, are new
antiprogestins with partial agonist activity. These steroids are active
with PR-A and -B isoforms, different reporters [CAT (chloramphenicol
acetyltransferase) and LUC (luciferase)], and different
promoters and enhancers (GREtk and MMTV) in a variety of cells (CV-1,
1470.2, and T47D). To the best of our knowledge, Dex-Mes is the first
compound to exhibit partial agonist activity with PR-A (Fig. 2A
) (14, 47, 48, 49). The high level of agonist activity of Dex-Ox with the
endogenous PR of T47D cells, which is predominantly PR-A (50), suggests
that Dex-Ox, like Dex-Mes, also possesses agonist activity with PR-A
receptors. The data with T47D cells further demonstrate that the
activities of Dex-Ox in CV-1 and 1470.2 cells are not an artifact of
transient transfection. This, in turn, indicates that the partial
agonist activity of Dex-Mes with PR is also not limited to transfected
PR.
With any new steroid, an obvious question is whether the observed
biological activities result from the formation of receptor-steroid
complexes. Five lines of evidence indicate that the effects of both
Dex-Mes and Dex-Ox are mediated by direct interactions with PR. First,
the partial agonist activity of both steroids in 1470.2 (Fig. 3
, B and
C) and CV-1 (data not shown) cells is dependent upon transfected PR.
Second, cell-free competitive binding studies show that Dex-Ox binds
PR-B with an affinity that is 1% of the potent progestin R5020 and
suggest that the affinity of Dex-Mes is 0.1% of R5020 (Fig. 4A
).
Third, the relative concentrations of R5020, Dex-Ox, and Dex-Mes
required for 50% inhibition in the cell-free binding studies
(1:100:1,000, respectively) correspond closely to the biological
potencies of each steroid as an agonist, as determined from the
dose-response curves with 3 ng of PR-B cDNA in Figs. 5
and 6
(EC50s: R5020, 0.35 nM; Dex-Ox, 30
nM; and Dex-Mes, 350 nM). Fourth, the 10-fold
greater potency of Dex-Ox vs. Dex-Mes as an antiprogestin
(Fig. 4B
) correlates with the 10-fold higher apparent affinity of
Dex-Ox for PR. Fifth, the low activity of Dex-Mes in intact cells can
be partially reversed by added R5020 (Fig. 4B
), which argues that at
least some of the effects of Dex-Mes are mediated by specific
interactions with PR. Collectively, we feel these data strongly support
the conclusion that Dex-Mes and Dex-Ox are two new antiprogestins that
display significant amounts of partial agonist activity under a variety
of conditions due to their binding to PR.
Very few partial agonists for PR exist in the literature. The most
active such compound to be reported is RTI-020 and some closely related
derivatives (16), which are structurally similar to the almost pure
antiprogestin RU 486 (Fig. 1
). Interestingly, RTI-020 is inactive in
CV-1 (16) and 1470.2 cells, even under conditions where Dex-Mes and
Dex-Ox are active (Figs. 2A
and 3
). These data suggest that D-ring
derivatives at the C-17 position of Dex (Fig. 1
) may be a rich new
source of future antiprogestins with partial agonist activity. As the
oxetanone modification of Dex (Fig. 1
) increased the apparent affinity
for PR by about a factor of 10 (Fig. 4A
), it is reasonable to expect
that other derivatives with even higher affinity for PR might be found
to retain appreciable amounts of partial PR agonist activity.
The discovery of two new antiprogestins that possess partial agonist
activity (Dex-Mes and Dex-Ox) facilitated our subsequent studies of
whether the induction properties of PR can be modified by changing the
receptor concentration, as has recently been described for GR (26, 27, 28).
Thus, in addition to looking at the ability of variations in PR level
to alter the position of the dose-response curve of agonists (or
EC50), we could now determine whether this is
accompanied by coordinated changes in the partial agonist activity of
antiprogestins. The present results demonstrate for the first time that
both properties of PR gene induction are influenced by PR
concentration. The fact that PR concentration is not constant among
PR-responsive tissues leads to the additional prediction that the
partial agonist activity of Dex-Mes and Dex-Ox will also not be
constant in all tissues. Furthermore, the level of PR in a given cell
can be modified, as seen in breast cells where estrogens usually induce
PR levels (51). Therefore, even in the same cell, the partial agonist
activity of Dex-Mes or Dex-Ox may change.
Another modulator of the partial agonist activity of antiprogestins is
shown here to be coregulators. Elevated levels of the coactivators TIF2
and SRC-1, along with the corepressor NCoR, can each augment the
partial agonist activity of Dex-Mes. As the levels of these
coregulators are not uniform among cells (30, 31, 32), or even in a given
cell (25, 30), it is likely that partial agonist activity of Dex-Mes
will vary from cell to cell. Furthermore, the capacity of PR to respond
to added TIF2 is cell-dependent, as seen here by the effects on PR in
1470.2 cells but not in T47D cells. This suggests that the influence of
PR and coregulator concentrations on the partial agonist activity of
Dex-Mes, or Dex-Ox, will be additionally modified by other
cell-specific factors. This combinatorial diversity makes Dex-Mes and
Dex-Ox promising candidates as new SPRMs that could exhibit varying
amounts of partial agonist activity for a given gene among diverse
cells and potentially even different genes within a given cell. It
should also be noted that these same variables have been found to
influence the dose-response curve (or EC50) of
PR-agonist complexes (Figs. 1
and 5
) and thus could afford differential
control of transcription of responsive genes in different cells by
modifying the sensitivity to subsaturating concentrations of progestins
and possibly of different genes within the same cell.
The absence of any effect of SRC-1 on the EC50 or
total transactivation of PR-agonist complexes in 1470.2 cells was
unexpected as SRC-1 was first discovered for its ability to increase
the transcriptional activity of PR (43). Similarly, we did not observe
the synergistic increase in the levels of PR-mediated transactivation
reported for the combination of SRC-1 and CBP (52). It is important to
realize that SRC-1 was not totally inactive in our system but did
augment the partial agonist activity of the antiprogestin Dex-Mes.
Therefore, the simplest explanation for these different responses of PR
to SRC-1 is that they reflect tissue-specific variations. Almost all
reports of SRC-1 activity with PR have been in HeLa cells (43, 44, 53)
and none have appeared for 1470.2 cells. Similar examples of
cell-specific activities of cofactors have been described (26, 45), and
seen here, with TIF2. Whether this reflects different levels of
endogenous coactivators (25, 26, 30, 32, 33), or a more basic
selectivity in the effects of various coactivators on PR induction
parameters, remains to be established.
TIF2/GRIP1 is reported not to interact with PR complexed with RU 486
(45, 46). However, with the new antiprogestin Dex-Mes, there is a clear
effect of added TIF2 on the PR complexes (Fig. 8
), consistent with a
functional interaction. This suggests that the conformation of the
Dex-Mes complex is different from that with RU 486 and permits
associations with both coactivators and corepressors. It is tempting to
speculate that the smaller size of Dex-Mes vs. RU 486 (54)
permits an equilibrium mixture of a wider variety of tertiary
structures of the ligand-bound PR, with different structures having
modified affinities for coactivators or corepressors. The magnitude of
conformational changes need not be dramatic because coactivators
and corepressors can both bind to the same general hydrophobic pocket
of steroid/nuclear receptors (20, 21, 24).
The observed effects of added SMRT and NCoR (Figs. 10
and 11
) indicate
a functional interaction of corepressors with both PR-agonist and
PR-antagonist complexes. SMRT and NCoR both act as corepressors in
1470.2 cells in that they suppress the total levels of PR
transactivation but have exactly opposite effects on other induction
parameters (Figs. 10
and 11
). SMRT causes a right shift of the R5020
dose-response curve to higher EC50s while NCoR
induces a left shift. The partial agonist activity of the antisteroid
Dex-Mes is decreased by SMRT but increased by NCoR. In many respects,
NCoR acts more like the coactivator TIF2. While part of the explanation
of these divergent results may be yet another example of cell-specific
differences, they complicate a clear-cut distinction between
coactivators and corepressors. These differences between NCoR and SMRT
are reminiscent of the reports that basal transcription by TR is
increased by added SMRT but not NCoR (55) and that DAX-1 (56) and
RevErb (57) display differential interactions for SMRT and NCoR.
We have recently reported analogous effects of receptor and coregulator
concentrations on GR induction properties (26). These results led to a
model in which the EC50, and partial agonist
activity, of transcriptionally active GR complexes is influenced by a
dynamic equilibrium of receptor and coregulator proteins (26). More
recent studies (27) indicate that the effects of varying concentrations
of both GR and coactivators are manifested via a shared mechanistic
step (X in Fig. 12
). We suspect that a
similar common intermediate is involved in the modulation of PR complex
activities with changing PR and coregulator concentrations. However,
this intermediate is unlikely to be the same as that for GR
(i.e. different from X in Fig. 12
) because overexpressed PR
does not alter the GR dose-response curve or partial agonist activity
of antiglucocorticoids (28). Therefore, the proposed common
intermediate for PR action would be either downstream of X or in a
different pathway (Fig. 12
). The identity of these proposed
intermediates is clearly of interest. Unfortunately, very little is
known about the role of individual cofactors at any stage of
receptor-mediated transactivation.

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Figure 12. Proposed Model by Which Added PR, and Increasing
Concentrations of Coregulators, Modify the Dose-Response Curve of
PR-Agonist Complexes and the Partial Agonist Activity of PR-Antagonist
Complexes
Both GR and PR are capable of inducing the same reporter gene, but the
components that can alter the induction properties of PR (PR and
coregulator concentrations) are speculated to affect a different step
or pathway than for GR, where several components (glucocorticoid
modulatory element, GR concentration, and coregulator concentration)
have been found to competitively interact at a step labeled X (27 ). See
text for further details.
|
|
The effects of added PR on the EC50 are
consistently much more pronounced for agonists than for antagonists
(Fig. 6
, A and B). Conversely, the effects of TIF2 and SRC-1 on the
partial agonist activity of Dex-Mes are more than on the
EC50 for R5020 (Figs. 8
and 9
). This suggests
that the mechanistic details for the modification of PR induction
parameters are not the same for agonist and partial agonist complexes.
This is not surprising, given the different x-ray structures of
receptor ligand-binding domains with agonist vs. antagonist
steroids (58, 59). Whether this structural diversity is translated into
altered affinities for common cofactors or very different affinities
for completely separate cofactors remains to be determined.
Dex-Mes and Dex-Ox are also antiglucocorticoids (34, 35). While they
evoke negligible agonist activity through the endogenous GR of 1470.2
cells (Figs. 3A
and 4A
), they display significant amounts of
glucocorticoid activity in other cells (26, 28, 34, 35, 39). This is in
contrast to the most commonly used antiprogestin, RU 486, which is a
pure antiglucocorticoid in a wide variety of cells. Therefore, the use
of Dex-Mes and Dex-Ox as antiprogestins should be accompanied by
reduced side effects since fewer actions of GRs will also be blocked.
Dex-Mes is an electrophilic affinity label but does not covalently
label PR (60, 61). While the chemical reactivity of Dex-Mes, and
reaction with a variety of proteins, is not a problem when used to
examine PR- or GR-mediated responses in tissue culture cells, it
probably limits the applications in intact animals and humans (62).
Dex-Ox, however, is not subject to any such restrictions and may find
more widespread applications.
In conclusion, alterations in the intracellular levels of PR,
coactivators, and corepressors can affect two physiologically relevant
induction properties of PR: the partial agonist activity of some
antiprogestins and the EC50 of PR agonists.
Cellular variations in the level of coactivators and corepressors (25, 29, 30, 31, 32, 33) are common. When this effect is superimposed on the alterations
in PR levels, especially in response to induction by estrogen receptors
(51), the differences in the transactivation of target gene in various
cells with both subsaturating concentrations of agonist and saturating
concentrations of antagonist may be substantial. Given the many
mechanistic similarities of all of the steroid receptors, it will be
interesting to test the hypothesis that the induction properties of
androgen, estrogen, and mineralocorticoid receptors can be similarly
modified.
 |
MATERIALS AND METHODS
|
---|
Unless otherwise indicated, all operations were performed
at 0 C.
Chemicals, Buffers, and Plasmids
[17
-methyl-3H]Promegestone (R5020, 85Ci/mmol)
and nonradioactive R5020 were obtained from NEN Life Science Products (Boston, MA) and dexamethasone (Dex) from
Sigma (St. Louis, MO). Dex-Ox (63) and Dex-Mes (64) were
prepared as described, and RTI-3021020 (RTI-020) was a gift from C.
Edgar Cook (Research Triangle Institute, Research Triangle Park,
NC). Restriction enzymes and digestions were performed according to the
manufacturers specifications (New England Biolabs, Inc.,
Beverly, MA).
MMTVLuc (pLTRLuc) was provided by Gordon Hager (NIH, Bethesda, MD). The
Renilla null luciferase reporter was purchased from
Promega Corp. (Madison, WI). The cDNA plasmids of SRC-1a
(Bert OMalley, Baylor College of Medicine, Houston, TX), TIF2 and A
and B forms of human PR (Hinrich Gronemeyer, IGBMC, Strasbourg,
France), CBP (Richard Goodman, Vollum Institute, Portland, OR), NCoR
(Michael Rosenfeld, University of California, San Diego), and SMRT (Ron
Evans, Salk Institute, La Jolla, CA) were each received as gifts.
Cell Culture and Transfection
Monolayer cultures of COS-7 and 1470.2 cells were grown at 37 C
with 5% CO2 in DMEM (Life Technologies, Inc., Gaithersburg, MD) and DMEM with 4.5 g glucose/liter
(Quality Biologicals, Inc., Gaithersburg, MD), respectively,
supplemented with 5% and 10% of FBS, respectively. CV-1 (28) and T47D
(50) cells were grown as described. Coregulator plasmids were
transiently cotransfected into 1470.2 cells using calcium phosphate
with the human PR-B (hPR-B) or hPR-A construct, 1 µg of MMTVLuc (or 2
µg of GREtkCAT or GREtkLuc), and 50 ng Renilla null
luciferase, with the total transfected DNA brought up to 3 µg/60-mm
dish with pBSK+ DNA (28). In experiments with
coregulator cDNA plasmid, all cells not transfected with coregulator
were cotransfected with an equimolar amount of the same plasmid vector
to control for artifacts of the vector DNA. The cells were treated for
24 h with steroids in media containing 10% charcoal-stripped FBS
(HyClone Laboratories, Inc. Logan, UT) (regular FBS for
other cells) and harvested in 1x Passive Lysis Buffer (0.5 ml/dish,
Promega Corp.). Twenty microliters of the cell lysates
were used to assay for luciferase activity using the Dual-Luciferase
Assay System from Promega Corp. according to instructions
of the supplier. The data were then normalized either for total protein
or cotransfected Renilla activity.
Assay for Steroid Competition
Transient transfection of COS-7 cells with 10 µg/10-cm plate
of human PR-B cDNA was performed as described previously (65). Cytosols
of transfected cells containing the steroid-free receptors were
obtained by the lysis of cells at 80 C and centrifugation at
15,000 x g (66). Thirty percent cytosol with 20
mM sodium molybdate was adjusted to 2.5
nM [3H]R5020 ±
varying amounts of nonradioactive competitor and incubated at 0 C for
18 h. Unbound [3H]R5020 was removed by
dextran-coated charcoal. The specific binding, calculated by
subtracting the background disintegrations/min (2,000-fold R5020) from
the total [3H]R5020 binding in the
presence or absence of cold competitor, was divided by the
specific disintegrations/min of the uncompeted
[3H]R5020 and expressed as the percent of
uncompeted binding.
Analysis of Data
The activity for subsaturating concentrations of agonist, or
saturating concentrations of antagonist, was expressed as percent of
maximal activity with saturating concentrations of agonist (30
nM R5020 unless otherwise noted). The fold induction with
30 nM R5020 was calculated as the luciferase activity
(relative firefly light units/relative Renilla light units)
activity with 30 nM R5020 divided by the basal
activity obtained with ethanol. Individual values were generally
within ± 20% of the average, which was plotted. Unless otherwise
noted, all statistical analyses were by two-tailed Students
t test using the program InStat 2.03 for Macintosh
(GraphPad Software, Inc., San Diego, CA).
 |
ACKNOWLEDGMENTS
|
---|
We thank Edgar Cook, Ron Evans, Richard Goodman, Hinrich
Gronemeyer, Gordon Hager, Bert OMalley, and Michael Rosenfeld for the
gift of research materials and George Chrousos (NIH) for critical
review of the paper.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. S. Stoney Simons, Jr., Building 8, Room B2A-07, NIDDK/LMCB, NIH, Bethesda, Maryland 20892-0805.
1 Current address: Sequenom, 142-F North Road, Suite 150, Sudbury,
Massachusetts 01776. 
2 Current address: 306 Henderson Street, Chapel Hill, North Carolina
27514. 
Received for publication August 22, 2000.
Revision received October 12, 2000.
Accepted for publication October 30, 2000.
 |
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