ABSTRACT
Retinoids are important physiological agents that regulate
epithelial cell differentiation and proliferation. The importance of
these agents in regulating growth, development, and differentiation has
led to a search for new retinoid agonists and antagonists. In the
present manuscript we show that AGN193109, a retinoid analog, is an
efficient antagonist of retinoid action in human cervical epithelial
cells. Treatment of ECE16-1 cells with natural or synthetic retinoids
reduces cytokeratin K5, K6, K14, K16, and K17 levels, increases
cytokeratin K7, K8, and K19 levels, increases retinoic acid
receptor-
(RAR
) mRNA levels, suppresses proliferation, and
alters cell morphology. Co-treatment with AGN193109 prevents these
responses. Half-maximal and maximal antagonism is observed at a molar
ratio of AGN193109:retinoid agonist of 1:1 and 10:1, respectively. When
administered alone AGN193109 has no agonist activity. Thus, AGN193109,
which binds to RAR
, RAR
, and RAR
with K
values = 2, 2, and 3
nM, respectively, but is unable to bind to the retinoid X
receptors, is a highly active antagonist of retinoid action in ECE16-1
cells.
There is currently a great deal of interest in developing new
retinoids for therapeutic
application(1, 14, 36, 43) . This
interest has been fueled by the finding that retinoids are natural
cancer preventive agents and that administration of retinoid can be
used to treat existing cancer(2, 3, 15) .
Retinoids interact with the retinoid receptors that are
ligand-activated, trans-acting transcription factors (4, 16, 17) . The retinoid receptor family
includes six major forms. Three of these, RAR
, (
)RAR
, and RAR
, display a preference for binding
to all-trans-retinoic acid
(tRA)(5, 6, 7, 8, 9, 10, 11, 17) .
The other three forms, RXR
, RXR
, and RXR
, bind to
9-cis-retinoic acid (9-cRA)(18, 19) . Various
retinoids have been described that interact selectively or specifically
with the RAR or RXR receptors or with specific subtypes (
,
,
or
) within each
class(1, 37, 38, 43) . However, few
antagonists of retinoid action have been described and they are mostly
low affinity antagonists that have been poorly characterized at the
receptor level(12, 20, 21, 39) .
In the present study we describe the effects of a new retinoid,
AGN193109, a specific and highly effective antagonist of RAR function (40) , on the differentiation and proliferation of the ECE16-1
human cervical cancer cell line. ECE16-1 cells are an human
papillomavirus type 16-immortalized cell line that was generated by
transfection of normal human cervical cells with the molecularly cloned
human papillomavirus type 16 virus(22) . They are a model for
study of the early stages in cervical dysplasia and retinoid effects on
cervical cell differentiation. These cells respond to retinoid
stimulation with characteristic changes in proliferation rate,
morphology, and marker gene
expression(13, 22, 23, 24, 25) .
The predominant receptor forms expressed in ECE16-1 cells are RAR
,
RXR
, and RAR
; however, RAR
is induced by retinoid
treatment(37) .
In the present study we demonstrate that
AGN193109 is a highly active antagonist of retinoid action in ECE16-1
cells. We show that it is an efficient antagonist of retinoid-dependent
changes in ECE16-1 cell morphology, gene expression, and proliferation,
but has no intrinsic agonist activity.
MATERIALS AND METHODS
Cell Culture
ECE16-1 cells were routinely
cultured in complete medium containing Dulbecco's modified
Eagle's medium:F-12 (3:1), nonessential amino acids, 5% fetal
calf serum, 5 µg/ml transferrin, 2 nM triiodothyronine,
0.1 nM cholera toxin, 2 mML-glutamine, 1.8
10
M adenine, and 10 ng/ml
epidermal growth factor (EGF). For proliferation experiments, the cells
were shifted to defined medium (DM) containing Dulbecco's
modified Eagle's medium:F-12 (3:1), 2 mML-glutamine, nonessential amino acids, 0.1% bovine serum
albumin, 1.8
10
M adenine, 5
µg/ml transferrin, 2 nM triiodothyronine, 50 µg/ml
ascorbic acid, 100 µg/ml streptomycin, 100 units/ml penicillin, and
50 µg/ml gentamicin(26) . DM was supplemented with EGF
and/or retinoids as indicated.
Retinoids
tRA and 13-cis-retinoic acid
(13-cRA) were obtained from Sigma, and 9-cRA,
AGN193109, and TTNPB (27, 41) were synthesized in the
Department of Chemistry, Allergan, Inc. Retinoid stocks were dissolved
in dimethyl sulfoxide (Me
SO) as 1000
concentrates
and the DMSO concentration in the medium did not exceed
0.1%(28, 29) .
Cell Proliferation Studies
ECE16-1 cell
proliferation studies were performed exactly as described
previously(23, 25, 26) . Cells
(10,000/cm
) were seeded in complete medium and allowed to
attach overnight. The cells were then shifted to DM, allowed to
equilibrate for 24 h, and treatment was initiated by addition of fresh
DM or DM containing EGF or retinoid as indicated. After 3 days of daily
treatment with retinoid, the cells were harvested with 0.025% trypsin,
1 mM EDTA, fixed in isotonic buffer containing 4%
formaldehyde, and counted using a Coulter counter (26) .
Protein and Nucleic Acid
Methods
Poly(A)
RNA was prepared,
electrophoresed, transferred to nylon membrane, and hybridized with
P-labeled cDNAs encoding glyceraldehyde-3-phosphate
dehydrogenase(30) , cytokeratin K6(31) , or RAR
(9) as described
previously(29, 32, 42) . For detection of
cytokeratins, ECE16-1 cells were labeled with
[
S]methionine and cytokeratins were prepared and
electrophoresed in two dimensions exactly as described previously (22) .
RESULTS
Retinoid Effects on Cell Morphology
Fig. 1shows the morphology of untreated ECE16-1 cells (A) or cells treated with 10 nM TTNPB (B),
10 nM TTNPB + 100 nM AGN193109 (C), or
100 nM AGN193109 alone (D). TTNPB, a potent
RAR-specific agonist, produces significant changes in cell morphology
which are completely reversed by addition of a 10-fold molar excess of
AGN193109. Although it efficiently antagonizes TTNPB effects, AGN193109
alone produces no change in ECE16-1 cell morphology. Dose-response
curves indicate that the AGN193109 inhibits the TTNPB-dependent
morphological change in a concentration-dependent manner.
Figure 1:
TTNPB and AGN193109 regulation
of cell morphology. ECE16-1 cells were grown for 3 days in DM
supplemented with 10 ng/ml EGF (DM/EGF) (A) or DM/EGF
containing 10 nM TTNPB (B), 10 nM TTNPB
+ 100 nM AGN193109 (C), or 100 nM AGN193109 (D). The cells were photographed using a Nikon
Optiphot phase contrast microscope at a magnification of
40.
AGN193109 Antagonizes Retinoid-dependent Growth
Suppression
ECE16-1 cells remain quiescent in defined medium (Fig. 2A, open circle)(25, 26) .
However, they proliferate rapidly in the presence of epidermal growth
factor (filled square) (Fig. 2A) (25, 26, 37) and addition of 10 nM TTNPB suppresses this proliferation. Addition of increasing
concentrations of AGN193109 in the presence of 10 nM TTNPB
prevents the TTNPB-dependent suppression of proliferation (filled
circles). The growth suppression is half-reversed by 10 nM and completely reversed by 100 nM AGN193109. Although it
can antagonize the effects of TTNPB, 1 uM AGN193109 given
alone has no effect on cell proliferation (filled triangle).
Figure 2:
AGN193109 regulation of ECE16-1 cell
proliferation. Cell proliferation experiments were performed exactly as
outlined under ``Materials and
Methods''(23, 25, 26) . All treatment
groups were grown in DM containing 10 ng/ml EGF, except for the DM
group, which lacked EGF treatment (single open circle in each
panel). In each panel, the single solid square indicates
proliferation in the presence of 10 ng/ml EGF alone without added
retinoid. The single solid upward triangle indicates
proliferation in the presence of EGF-containing medium and 1000 nM AGN193109. For the dose-response curves, 10 nM TTNPB (A) or 10 nM tRA, 9-cRA, or 13-cRA (B) were
added to EGF-containing medium in the presence of increasing quantities
of AGN193109. The values indicate the number of cells present at the
end of 3 days of treatment with the indicated agent or combination of
agents. Identical responses were observed in each of three
experiments.
The ability of AGN193109 to antagonize the effects of TTNPB could be
related to the fact that TTNPB is an RAR-specific ligand that does not
interact with RXR receptors (Table 1). We therefore determined
whether AGN193109 could reverse the effects of tRA, 13-cRA, and 9-cRA.
These retinoids interact with RAR or both RAR and RXR receptors (Table 1). As with TTNPB, AGN193109, when present at a 10-fold
molar excess, completely reverses the growth suppressive effects of
tRA, 13-cRA, and 9-cRA (Fig. 2B).
AGN193109 Effects on Biochemical Markers of Retinoid
Action
We next determined whether AGN193109 could antagonize
retinoid effects on expression of retinoid-responsive marker genes.
Cytokeratin proteins form the intermediate filaments in epithelial
cells and are important markers of retinoid action in cervical
epithelial cells(22, 33, 34, 35) .
As shown in Fig. 3A, ECE16-1 cells grown in
retinoid-free medium express cytokeratins K5, K6, K7, K8, K13, K14,
K16, K17, and K19 (A)(22) . Treatment with 10 nM TTNPB suppresses K5, K6, K14, K16, and K17 levels and increases
K7, K8, and K19 levels (Fig. 3B). Simultaneous
treatment with 100 nM AGN193109 eliminates the effects of 10
nM TTNPB (Fig. 3C). No changes in keratin
expression are observed when cells were incubated with 0.01-1000
nM AGN193109 alone (not shown). We have shown previously that
changes in keratin mRNA level mediate changes in keratin protein
level(13, 22, 35) . As a representative
keratin, we show the effects of each treatment on the level of mRNA
encoding K6 (Fig. 3B). AGN193109 (100 nM) has
no effect and 10 nM TTNPB suppresses K6 mRNA levels. The
TTNPB-dependent suppression is completely inhibited when cells are
incubated with 10 nM TTNPB plus a 10-fold molar excess of
AGN193109. AGN193109 also inhibits the retinoid-dependent change in the
level of mRNA encoding the other keratins (not shown).
Figure 3:
AGN193109 regulation of
retinoid-responsive marker gene expression. In A, ECE16-1
cells were grown in DM containing 10 ng/ml EGF (DM/EGF) (A) or
DM/EGF supplemented with 10 nM TTNPB (B) or 10 nM TTNPB + 100 nM AGN193109 (C) for 3 days.
The cells were labeled with [
S]methionine for 24
h prior to harvest for preparation of cytokeratins. Equal numbers of
radioactive counts from each sample were electrophoresed in
nonequilibrium pH gradient electrophoresis gels as described previously (22) . The second dimension was a 6% acrylamide gel. In B, poly(A)
RNA was prepared from cells
treated for 3 days with the indicated agent under the same conditions
as in A. The RNA was transferred to Biodyne A membrane and
hybridized with
P-labeled cDNAs encoding cytokeratin K6,
RAR
, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Glyceraldehyde-3-phosphate dehydrogenase is included as a control, as
its level is not regulated by retinoids.
Regulation of RAR
mRNA Levels
We have shown
previously that RAR
mRNA levels are increased by
retinoids(37) . The results in Fig. 3B show
that TTNPB increases RAR
mRNA levels, that the increase is
completely antagonized by AGN193109 and that AGN193109 has no intrinsic
retinoid activity in regulating this marker.
DISCUSSION
AGN193109 Antagonizes a Range of Responses in ECE16-1
Cells
Retinoids produce multiple effects on tissues and cells
and different classes of retinoids can produce differing effects within
and among cell lines(29, 37) . In ECE16-1 cells,
RAR-specific ligands are more active retinoids than RXR-specific
ligands(37) . In the present study, we examined the effects of
a novel synthetic retinoid, AGN193109, on a variety of
retinoid-dependent responses in ECE16-1 cells. We found that AGN193109
is able to eliminate retinoid-dependent changes in cell morphology and
prevent retinoid-dependent suppression of proliferation. Moreover, at
the biochemical level, AGN193109 prevents retinoid-dependent changes in
marker gene expression. We examined genes that are increased (K7, K8,
K19, RAR
), as well as those that are suppressed (K5, K6, K14, K16,
K17) by retinoid treatment(22) . In each case, the
retinoid-dependent response was antagonized by AGN193109. Thus,
AGN193109 prevents both stimulatory and suppressive responses. Taken
together these results suggest that AGN193109 may be a global
antagonist of retinoid action in ECE16-1 cells (i.e. it is
likely to antagonize most, and perhaps all, retinoid-dependent
responses).
AGN193109 Is an Efficient Antagonist with No Agonist
Activity
Several problems can be encountered with receptor
antagonists. First, they may have partial agonist activity. This can
complicate interpretation of their effects in in vivo systems.
To determine whether AGN193109 has intrinsic agonist or antagonist
activity, we evaluated its effects on ECE16-1 cell function in the
absence of added retinoid agonist. Concentrations of AGN193109 ranging
from 0.01 to 1000 nM produced no detectable changes in cell
morphology, cell proliferation, or retinoid-responsive marker gene
expression. Based on these results we conclude (i) that AGN193109 is a
highly effective antagonist of retinoid action in these cells and (ii)
that it has not intrinsic agonist activity.Second, antagonists may
have a low affinity for the target receptor. Such ligands must be used
at extremely high levels to accomplish the antagonism. AGN193109,
however, has an affinity for the RARs of 2-3 nM.
Considering that the receptor affinities of tRA, 13-cRA, and 9-cRA
range from 7 to 141 nM and TTNPB has affinities ranging from
20 to 51 nM, AGN193109 is a high affinity ligand. This is
reflected in its potency at preventing biological end responses. The
compound is maximally effective at inhibiting retinoid agonist activity
when present at a 10-fold molar excess and is half-maximally effective
when present in equal molar amounts. Thus, AGN193109 is a high affinity
antagonist.
Natural Ligands That Interact with RAR and RXR
Receptors
Our initial studies showed that the synthetic
retinoid, AGN193109, which interacts solely with RAR receptors, is
effective at antagonizing the effects of the RAR-specific retinoid
agonist, TTNPB. However, it was unclear whether the activity of natural
ligands would be inhibited by AGN193109. We therefore tested the
ability of AGN193109 to antagonize the effects of tRA, 13-cRA, and
9-cRA. 13-cRA, 9-cRA, and tRA interact with RARs, while 9-cRA also has
a high affinity for RXRs. The results show that AGN193109 is an
effective antagonist of the activity of each of these ligands. Thus,
although AGN193109 only interacts with the RAR forms, it can also
antagonize the effects of an agent (9-cRA) that interacts with
relatively high affinity with both RARs and RXRs. Since AGN193109 does
not interact with the RXRs, it is not likely that it directly prevents
9-cRA from interacting with RXR receptor. Moreover, RXR-specific
ligands are relatively inactive in ECE16-1 cells(37) . Since
9-cRA can efficiently bind to RARs, it is likely that AGN193109 is
inhibiting 9-cRA effects mediated via RARs.Receptor antagonists can
be extremely useful ligands, both for understanding mechanism of action
and for designing new therapies. In the present manuscript, we show in
ECE16-1 cells, a human ectocervical epithelial cell line, that
AGN193109 is an extremely active high affinity antagonist of retinoid
action that appears to have no intrinsic retinoid agonist activity.