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DIFFERENTIAL LIGAND ACTIVATION OF ESTROGEN
RECEPTORS ERa AND ER(i AT AP1 STTES
CROSS-REFERENCE TO RELATED INVENTIONS
[ Not Applicable ]
This invention was made with the Government support under Grant No.
GM 50872, awarded by the National Institutes of Health. The Government of the
United
States of America may have certain rights in this invention.
BACKGROUND OF THE I1WENTION
Estrogens, antiestrogens, and other nuclear transcription factor ligands are
used in a wide variety of therapeutic contexts. Thus, for example, estrogens
are used in
the treatment of osteoporosis and other aspects {e.g., vasomotor instability)
of
menopause, in the treatment of hypoestrogenism, and in the regulation of
fertility.
Antiestrogens are used in the treatment of cancer. Tamoxifen, for example, is
an
antiestrogen that is used in breast cancer chemotherapy and is believed to
function as an
antitumor agent by inhibiting the action of the estrogen receptor (ER) in
breast tissue (see,
e.g., {Sutherland et al. {1987) Cancer Treat. Revs, 15: 183-194).
Glucocorticoids are
used in the treatment of pure red cell anemia, acute renal failure due to
acute
glomerulonephritis or ~ lymphocytic leukemias, lymphomas, and other
conditions.
Progestins or progestational agents such as medroxyprogesterone or megestrol
acetate
are used in the treatment of endometrial carcinoma and breast carcinoma, and
are used in
the regulation of fertility.
It has long been known that nuclear transcription factor ligands may have
profound and contradictory effects upon patients depending on physiological
context. For
example, estrogen and estrogen agonists may have beneficial effects, such as
preveming
osteoporosis and reducing serum cholesterol (hove, et al. ( 1992) New Eng. J.
Med. 326:
852-856; Love, etal. (1990)J. Natl. Cancerlnst. 82:1327-1332). Conversely,
agonistic
activity may also be harmful. Tamoxifen for example sometimes increases
endometrial
tumor incidence (Tmo et al. (1991) Cancer Tread &Res. 53: 228-237) or switches
from
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0 inhibition to stimulation of estrogen dependent growth in breast tumor
progression
(Parker ( 1992), Cancer Surveys 14: Growth Regulation byNuclear Hormo~re
Receptors.
Cold Spring Harbor Laboratory Press).
The related benzothiophene analog raloxifene (Figure 1 A) has been
reported to retain the amiestrogen properties of tamoxifen in breast tissue
and to show
minimal estrogen effects in the uterus; in addition, it has potentially
beneficial estrogen-
like effects (in nonreproductive tissue such as bone and cardiovascular tissue
(Jones et al.
(1984) J. Med Chem., 27: 1057-1066; Black et al. (1994) J. Clip. Irrvest., 93:
63-69;
Sato et al. (1996) FASEB J., 10: 905-912; Yang et al. (1996) Endocrinol., 137:
2075-
2084; Yang et al., (1996) Science, 273: 1222-1225).
One explanation for these tissue-specific actions of antiestrogens is that the
ligand-bound ER has different transactivation properties when bound to
different types
of DNA enhancer elements. The estrogen receptor (ER) has been shown to mediate
gene
transcription both from the classical estrogen response element (ERE) and from
an AP 1
enhancer element that requires ligand and the AP1 transcription factors Fos
and Jun for
transcriptional activation (Fig.1B). In transactivation experiments, tamoxifen
inhibits the
transcription of genes that are regulated by a classical ERE, but like the
natural estrogen
hormone 17/3-estradiol [F.,~ (Fig. 1 A)], tamoxifen activates the
transcription of genes that
are under the control of an AP 1 element (Webb, et al ( 1995) Mol. Endo., 9:
443-456).
At the end of 1995, a second ER (ER~i) was cloned from a rat prostate
cDNA library (Kuiper et al. (1996) Proc. Natl. Acac~ Sci., USA, 93: 5925-
5930). The
human (Mosselman et al. (1996) FEES Lett., 392: 49-53) and mouse (Tremblay et
al.
(1997) Mol. E~rdocrinol., 11: 353-365) homologs have also been cloned. The
first
identified ER has been renamed ERa (Kuiper et al. ( 1996) supra. ). The
existence of two
ERs was postulated to present a potential new mechanism tissue-specific
estrogen
regulation.
From the foregoing, it is clear that the activity and regulation of nuclear
transcription fiictor ligands, especially estrogens, is complex and the use of
various
transcription factor ligands can lead to contradictory and often adverse
consequences.
Thus, when electing to use a nuclear transcription factor ligand in a
therapeutic context,
it is desirable to eluadate as precisely as possible the various modes of
action (biological
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0 activities) of the agents) under consideration. Similarly, it has long been
known that
various environmental compounds have estrogenic and possl'bly antiestrogenic
activity.
When evaluating the impact of such environmernal estrogens and/or
antiestrogens, it is
desirable to evaluate their effect on all metabolic pathways in which they
might prove
active.
SUMMARY OF THE INVENTION
The present invention provides methods to rapidly and effectively screen
compounds for their ability to activate or inactivate gene transcription in a
previously
unknown regulatory pathway: an estrogen receptor beta (ER~i)-mediated AP 1
pathway.
This invemion is premised, in part, on the surprising discovery that ER~i is
capable of
intersecting with AP 1 to induce transcription of a gene under AP 1 control.
Even more
surprising was the discovery that ER~i-mediated AP 1 interactions can produce
results
significantly different than ERac-mediated AP 1 interactions. For example,
estradiol, which
activates gene expression through an ERai-mediated AP1 interaction, actually
inhibits
gene activation through an ERa-mediated AP 1 interaction.
In one embodiment, this invention provides methods of screening test
compounds for differential ERac-mediated and ERA-mediated activation at an APl
site.
The methods typically involve providing a first cell comprising an estrogen
receptor (3
(ER~i), an AP 1 protein, and a conshuct comprising a promoter comprising an AP
1 site
which regulates expression of a first reporter gene. The first cell is
contacted with the test
compound and the expression of the first reporter gene is compared with ERa-
mediated
expression of a gene at an AP 1 site in response to the same test compound.
The cell can
contain a heterologous estrogen receptor beta (ER~i) and preferred ER(3s
comprise an
amino acid seqeunce of SEQ ID NO: 3 or SEQ ID NO: 5. The cell can also contain
a
heterologous AP1 protein. Preferred reporter genes used in this assay include
chloramphenicol acetyl transferees (CAT), luciferase, (3 -galactosidase (~i-
gal), alkaline
phosphatase, horse radish peroxidase (HIZP), growth hormone (GH), and green
fluorescent protein (GFP) with a luciferase gene or a green fluorescent
protein (gene)
being preferred. The test compound can be a compound known or suspected to
have anti-
estrogenic activity. The method can be one in which the ERa-mediated
expression of a
gene at an AP1 site is determined by providing a second cell comprising an
estrogen
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0 receptor a (ERac), AP1 proteins, and a construct comprising a promoter
comprising an
AP1 site which regulates expression of a second reporter gene. The second cell
is
contacted with the test compound; and expression of the second reporter gene
is detected.
One preferred standard estrogen response element is from the Xenopus
vitellogenin AZ
gene. The second reporter gene and the first reporter gene can be the same
species of
reporter gene. The cell and the second cell are the same cell.
In one embodiment, this invention provides methods screening a test
compound for the ability to activate or inhibit estrogen receptor beta (ER(3)
mediated gene
activation at an AP 1 site. The methods typically involve providing a first
cell comprising
an estrogen receptor ji {ERA), AP 1 proteins, and a construct comprising a
promoter
comprising an AP1 site which regulates expression of a first reporter gene.
The cell is
contacted with a test compound and expression ofthe first reporter gene is
detected. The
cell can contain a native or heterologous estrogen receptor beta (ER~i). In a
preferred
embodiment, the ER j3 the amino acid sequence of Sequence ID No: 3 or Sequence
ID No:
5. The first cell can also contain a heterologous AP1 protein (e.g,. jun
and/or fos).
Virtually any reporter gene may be used. Preferred reporter genes include, but
are not
limited to chloramphenicol acetyl transferase (CAT), luciferase, (3 -
galactosidase (~i-gal),
alkaline phosphatase, horse radish peroxidase (HItP), or Been fluorescent
protein (GFP)
with a luciferase or a green fluorescent protein (GFP) being most preferred.
Virtually any
compound can be screened according to the methods of this invention. However,
preferred test compounds are compounds known to have anti-estrogenic activity.
In another embodiment, the above method can fiuther involve providing
a second cell comprising an estrogen receptor a (ERa), AP 1 proteins, and a
construct
comprising a promoter comprising an AP1 site which regulates expression of a
second
reporter gene. The second cell is contacted with the test compound and the
expression
of the second reporter gene is then detected. In addition, or alternatively,
the above
method can involve providing a third cell comprising an estrogen receptor a
(ERa), and
a construct comprising a promoter comprising a standard estrogen response
element
(ERE) which regulates expression of a third reporter gene. The third cell is
contacted
with the test compound; and expression of the third reporter gene is then
detected. One
standard estrogen response element can be from the Xenopus vitellogenin A2
gene.
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fourth cell
comprising an estrogen receptor ~ (ER(i), and a construct comprising a
promoter
comprising a standard estrogen response element (ERE) which regulates
expression of a
fourth reporter gene. The fourth cell is contacted with the test compound and
expression
of the fourth reporter gene is detected. Again the standard estrogen response
element can
5 be from the Xenopus vitellogenin A2 gene. In one embodiment, the first cell
and said third
cell are the same cell, while in another embodiment, the first cell and said
fourth cell are
the same cell.
Any of the above-described assays can be nm to detect or identify
inhibitors that block compounds that activate ER/i-mediated AP 1 gene
transcription. This
typically involves performing the assays as described above, but, in addition,
contacting
the first cell with a second compound, in addition to the test compound,
wherein said
second compound is known to activate transcription through estrogen receptor
(3 (ER(3)
mediated gene activation at an AP 1 site. Detecting then comprises detecting
test
compound mediated decrease in said estrogen receptor [i (ERA) mediated gene
activation
at an AP1 site. In a particularly preferred embodiment, the detecting can
involve
comparing the expression of the first reporter gene in the presence of the
test compound
and the second compound with the expression of the reporter gene in the
presence of the
second compound without the test compound.
In one embodiment, the second compound known to activate transcription
through estrogen receptor (3 (ER(i) mediated gene activation at an AP1 site is
identified
by a method involving providing a second cell comprising an estrogen receptor
~ (ER~i),
and AP 1 protein, and a construct comprising a promoter comprising an AP 1
site that
regulates expression of a second reporter gene. The second cell is contacted
with the
s~;ond compound and the expression of the second reporter gene is detected
where an
increase in expression of the second reporter gene produced by the compound
indicates
that said second compound activates transcription through ERA at an AP1 site.
The assays ofthis invention can also be used to detect or identify inhibitors
that block compounds that inhibit ER/3-mediated AP 1 gene transcription. These
methods
involve performing the assays as described above, while additionally
contacting the first
cell with a second compound, in addition to the test compound, where the
second
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0 compound is known to inhibit transcription through estrogen receptor ~ (ERA)
mediated
activity at an AP1 site. Expression of the reporter gene is detected where the
detection
comprises detecting test compound mediated increase in estrogen receptor (3
(ERA)
mediated gene activation at an AP 1 site. The detecting can involve comparing
expression
of the first reporter gene in the presence of both the second compound and the
test
compound with expression of the first reporter gene in the presence of the
second
compound without the test compound.
The second compound known to inhibit transcription through estrogen
receptor ø (ERA) mediated gene activation at an AP 1 site can be identified by
providing
a second cell comprising an estrogen receptor (3 (ER~i), and AP 1 protein, and
a construct
comprising a promoter comprising an AP1 site that regulates expression of a
second
reporter gene. The second cell is contacted with the second compound; and
expression
of the second reporter gene is detected. A decrease in expression of said
second reporter
gene produced by the second compound indicates that the second compound
inhibits
transcription through ERA at the AP 1 site.
This invention also provides for any ofthe cells described above or herein.
In one embodiment the cell comprises an estrogen receptor (3 (ER~i), an AP 1
protein (e.g.,
jun or fos), and a construct comprising a promoter comprising an AP 1 site
which
regulates expression of a first reporter gene. The cell can additionally
include a receptor
for a nuclear transcription factor ligand preferably for a nuclear
transcription factor ligand
other than estrogen. The cell preferably contains a heterologous ER(i, more
preferably
an ER(i comprising an amino acid sequence of Sequence 1D No: 3 or Sequence m
No:
5. The AP 1 protein can be a native AP 1 protein or a heterologous AP 1
protein. The
reporter gene can be one selected from the group consisting of chloramphenicol
acetyl
transferees (CAT), luciferase, (i -galactosidase ([i-gal), alkaline
phosphatase, horse radish
peroxidase (HRP), and green fluorescent protein (GFP), but in particulary
preferred
embodiment, the reporter gene encodes a luciferase or a green fluorescent
protein (GFP).
The cell can additionaly include a standard estrogen response element (FRE)
which
regulates expression of a second reporter gene. One preferred standard
estrogen response
element is from the Xenopus viteUogenin A2 gene. Preferred cells of this
invention are
mammalian cells and particularly preferred cells are derived from breast
tissue or from
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described assays can
be run to detect or identify inhibitors that block compounds that activate ERø-
mediated
AP 1 gene transcription.
In still another embodiment, this invention provides methods of screening
a nuclear tr~aascription factor ligand for the ability to modulate estrogen
receptor (3
S mediated activation or inactivation of transcription at an AP 1 site. The
methods involve
providing a first cell containing an estrogen receptor ~ (ER~i), an AP 1
protein, a receptor
for the nuclear transcription factor ligand, and a construct comprising a
promoter
comprising an AP1 site which regulates expression of a first reporter gene.
The cell is
contacted with the transcription fi~ctor ligand and with a compound having ERA
mediated
activity at the AP 1 site. Expression of the first reporter gene is then
detected.
The method can fiuther involve providing a second cell containing an
estrogen receptor (3 (ER~i), a receptor for the nuclear transcription fi~ctor
ligand, and a
construct comprising a promoter comprising an estrogen response element (ERE)
that
regulates expression of a second reporter gene. The second cell is contacted
with the
transcription factor ligand and with the compound having AP-1 mediated
estrogenic
activity and expression of the second reporter gene is detected. The first and
second cells
can be the same or different.
Alternatively, or in addition, the method can further involve providing a
second cell comaining a cognate receptor of the transcription factor ligand,
and a
promoter comprising a response element for the cognate receptor that regulates
expression of a second reporter gene. The second cell is contacted with the
transcription
factor ligand and with the compound having compound having ER(i mediated
activity at
said AP1 site expression of the second reporter gene is detected. Again, the
first and
second cells can be the same or different cells.
In any of the above-described methods the nuclear transcription factor
ligand can be selected from the group consisting of a glucocorticoid, a
progestin, vitamin
D, retinoic acid, a an androgen, a mineralcorticoid, and a prostaglandin.
Similarly, the
cognate receptor can be selected from the group consisting of an estrogen
receptor, a
glucocorticoid receptor, a progestin PR A receptor, and progestin PR B
receptor,
androgen receptor, a mineralcorticoid receptor, and a prostaglandin receptor.
In a
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0 particularly preferred ~nbodiment, the ERS comprises an amino acid sequence
of Figure
or Figure 6A. The ERA can be a heterologous ER~i. Similarly, the receptor for
the
nuclear transcription factor ligand can be heterologous to the cell. The cell
can express
an AP 1 protein (e.g., jun or fos) from a heterologous DNA. In one
particularly preferred
embodiment, the nuclear transcription factor is a progestin; and said receptor
for the
5 nuclear transcription factor ligand is a progestin receptor. In another
preferred
embodiment, the nuclear transcription factor is a glucocorticoid and said
receptor for said
nuclear transcription factor ligand is a GR receptor.
This invention also provides methods of screening an agent for the ability
to alter modulation of estrogen receptor [3 (ER~i) activation or inactivation
of
transcription at an AP 1 site by a nuclear transcription factor ligand. The
methods involve
providing a first cell containing an estrogen receptor ji (ER/3), an AP 1
protein, a receptor
for the nuclear transcription factor ligand, and a promoter comprising an API
site which
regulates expression of a first reporter gene. The first cell is contacted
with the
transcription factor ligand, with a compound having ERA mediated activity at
an AP 1 site,
and with the agent and expression of the first reporter gene is detected.
This method can further involve providing a second cell containing an
estrogen receptor (3 (ER~i), a receptor for the nuclear transcription factor
ligand, and a
promoter comprising an estrogen response element (ERE) that regulates
expression of a
second reporter gene. The second cell is contacted with the transcription
factor ligand
and with the compound having AP-1 mediated estrogenic activity and expression
of the
reporter gene is detected. The first and second cell can be the same cell or
different cells.
The nuclear transcription factor can be one selected from the group consisting
of a
glucocorticoid, a progestin, vitamin D, retinoic acid, an androgen, a
mineralcorticoid, a
2S prostaglandin. Similarly, the nuclear transcription factor ligand is
selected from the group
consisting of an estrogen receptor, a glucocorticoid receptor, a progestin PR
A receptor,
progestin PR-B receptor, an androgen receptor, a mineralcorticoid receptor,
and a
prostaglandin receptor. Again, in any of the assays described herein, the ERj3
can be a
heterologous ER(i and in a preferred embodiment, the ER~i comprises an amino
acid
sequence of Sequence m No: 3 or S~uence ID No: 5 or is encoded by a nucleic
acid
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0 sequence of Sequence ID No: 3 or Sequence ID No: 6. The AP 1 proteins)
and/or the
receptor for the nuclear transcription factor ligand can also be native to the
cell or
heterologous. In one particularly preferred embodimem, the nuclear
transcription factor
is a progestin; and the receptor for said nuclear transcription factor ligand
is a progestin
receptor, while in another preferred embodiment, the nuclear transcription
factor is a
glucocorticoid and the receptor for said nuclear transcription factor ligand
is a GR
receptor.
This invention also provides kits for screening a compound for the ability
to activate or inhibit estrogen receptor (3 (ER(3) mediated gene activation at
an AP1 site.
The kits can include a container containing a cell comprising an estrogen
receptor ~3
(ER~i), an AP 1 protein (e.g., jun and/or fos), and a construct comprising a
promoter
comprising an AP 1 site which regulates expression of a first reporter gene.
The cell of the
kits can further a receptor for a nuclear transcription factor ligand,
preferably a nuclear
transcription factor ligand other than estrogen. The kits can also further
include
instructional materials containing protocols for the practice of any of the
assay methods
described herein.
DEFINITIONS
The terms "activate transcription" or "inhibit transcription" as used herein
refer to the upregulation of transcription of a gene or the downregulation of
transcription
of a gene. It will be appreciation that either complete, or partial, "turning
on" or "turning
off' are is regaxded herein as activation or inhibition, respectively.
Activation and
inhibition of transcription are typically measured with respect to a control
or controls
where the comrol or controls involve a similar treatment lacking the compound
or agent
in question and/or contain a standard agent (e.g., E2 or tamoxifen). It will
also be
appreciated that there may exist a baseline level of transcription (e.g, of a
particular
reporter gene) even where an assay cell of this imrention is '~nstimulated"
(e.g. the
receptor in question is unliganded), i. e., without exogenously supplied
ligand). In this
case, it may be possible to see inhibition without necessarily applying
exogenous activator
see, e.g., Example 1).
As used herein an antiestrogen is a compound that substantially inhibits
estrogen activity as measured in an assay for estrogenic activity, for
example, cellular
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generally,
a "transcription factor antagonist" is a compound that substamially inhibits
transcription
factor activity as meas~ued in a standard assay for that transcription factor
activity.
A "nuclear transcription factor" as used herein refers to members of the
nuclear transcription factor supetfiunily. This is a family of receptors that
are capable of
5 entering the nucleus of a cell and once there, effecting the up-regulation
or down-
regulation of one or more genes. A "nuclear transcription factor ligand" is a
compound
that binds to a nuclear transcription factor. Preferred nuclear transcription
factors are
typically steroid receptors, however, the group is not so limited. Nuclear
transcription
factor ligands include, but are not limited to estrogen, progestins,
androgens,
10 mineralcorticoids, glucocorticoids, retinoic acid, vitamin D, and
prostaglandins.
Transcription factor ligands also include analogues of naturally occurring
factors~and
blocking agents (antagonists) of such factors. Transcription factors also
include, as they
are identified, the ligands that bind orphan receptors (those nuclear
transcription factors
which have been identified by sequence homology, but whose ligand is yet
unidentified).
It will be appreciated that when used in the context of a modulator of
estrogen activity,
the nuclear transcription factor ligand is typically one other than estrogen
(or other than
the estrogen or estrogen agonist whose activity is being modulated). Nuclear
transcription factors typically mediate their activity through binding of a
cognate receptor
in the cell nucleus. The term cognate receptor" refers to a receptor of the
type that is
typically bound by the transcription ligand in question. Thus; the cognate
receptor for an
estrogen is an estrogen receptor, the cognate receptor for a glucocorticoid is
a
glucocorticoid receptor, the receptor for a progestin is a progestin receptor,
and so forth.
The cognate receptor includes the native (naturally occurring) form as well as
modified
receptors.
The phrase estrogen receptor beta (ER~irmediated activation or
inactivation of gene transcription at an AP 1 site refers to the activation or
inactivation of
a gene (e.g., a reporter gene) under control of an AP 1 site by the
interaction of that AP 1
site with a liganded ER(i receptor. Similarly ERa-mediated activation or
inactivation
refers to gene regulation mediated by the interaction of ERa. Inactivation or
inactivation
at an ERE refers to activation or inactivation of a gene under control of an
ERE.
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0 The phrase "digsl ERa-mediated and ERA-mediatod activation at an
AP 1 site" refers to differences between ERa- and ER(3-mediated gene
activation at an
AP1 site in response to the same ligand. Differential activation can be
reflected in
significant differences in levels of gene activation or inactivation by the
same ligand
depending on whether it interacts with ERa or ERA. Differential activation can
also
reflect differences in the "sign" of gene activation. Thus differential
activation can refer
to ER[3-mediated activation oftranscription at an AP 1 site and ERa-mediated
inactivation
of gene transcription at an AP1 site in response to the same ligand.
Conversely,
differential activation can refer to ERj3-mediated inactivation
oftranscription at an AP1
site and ERa-mediated activation of gene transcription at an AP 1 site in
response to the
same ligand.
API-mediated estrogenic/agonist activity, as used herein, refers to
activation of a gene under the control of an AP 1 site (also referred to as an
AP 1 response
element) mediated by the interaction of a nuclear transcription factor with
the AP1 site.
When used in reference to ER mediated activation of a gene controlled by the
AP1 site,
the pathway is referred to as the indirect estrogen response (in contrast to
the classical
estrogen response which is mediated through an ERE). A general description of
the AP 1
site is found in Angel & Kann, Biochem. Bioplrys Acta:, 1072: 129-157 (1991)
and
Angel, et al., Cell, 49: 729-739 (1987).
A "compound having AP 1 mediated estrogenic activity" refers to a
compound that, when present in a cell containing a gene under control of an AP
1 site and
AP 1 proteins, activates transcription of the gene under comrol of the AP 1
site.
A "compound having the ability to inactivate or inhibit estrogen receptor
beta (ER~i) mediated gene activation at an AP1 site refers to a compound that
is capable
of upregulating or downregulating transcription of a gene under the control of
an AP 1 site
through its imeraetion (e.g., binding) of an ERA.
The phrases "modulate estrogen activation" or "modulation of estrogen
activation" refer to alteration of the estrogen induced expression of a
particular gene.
Where the phrase additionally recites "at an AP 1 site or at an ERE" the
phrase refers to
alteration of the level of estrogen induced expression of one or more genes
under control
of the AP1 site or ERE site respectively. The phrase "detecting expression"
when used
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0 with reference to a reports gene refers to detection of presence or absence
of expression
of the reporter gene or to quantification of expression level of the reporter
gene. The
quantification can be either an absolute measurement or a relative measurement
(e.g., in
comparison to another expressed gale).
The term "operably linked" refers to functional linkage between a nucleic
acid expression control sequence (such as a promoter, signal sequence, or
transcription
factor binding site) and a second nucleic acid sequence, wherein the
expression control
sequence affects transcription and/or translation of the nucleic acid
corresponding to the
second sequence.
The term "recombinant" when used with reference to a cell indicates that
the cell replicates a heterologous nucleic acid, or expresses a peptide or
protein encoded
by a heterologous nucleic acid. Recombinant cells can express genes that are
not found
within the native (non-recombinant) form of the cell. Recombinant cells can
also express
genes found in the native form of the cell wherein the genes are modified and
re-
introduced into the cell by artificial means. Recombinant expression refers to
the
expression of the heterologous nucleic acid by such a recombinant cell.
A "heterologous nucleic acid", as used herein, is one that originates from
a foreign source (or species) or, if from the same source, is modified from
its original
form. Thus, a heterologous nucleic acid operably linked to a promoter is from
a source
different from that from which the promoter was derived, or, if from the same
source, is
modified from its original form. Modification of the heterologous sequence may
occur,
e.g., by treating the DNA with a restriction enzyme to generate a DNA fragment
that is
capable of being operably linked to the promoter. Techniques such as
sit~directed
mutagenesis are also useful for modifying a heterologous sequence. Similarly,
a
"heterologous protein" refers to a protein 'that originates from a foreign
source (e.g.,
different cell or species) or, if from the same source, is modified from its
original form,
or is expressed fibm a heterologous nucleic acid.
A "recombinant expression cassette" or simply an "expression cassette"
is a nucleic acid construct, generated recombinantly or synthetically, with
nucleic acid
elements that are capable of effecting expression of a structural gene in
hosts compatible
with such sequences. Expression cassettes include at least promoters and
optionally,
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0 transcription termination signals. Typically, the recombinant expression
cassette includes
a nucleic acid to be transcribed (e.g., a nucleic acid encoding a desired
polypeptide), and
a promoter. Additional factors necessary or helpful in effecting expression
may also be
used as described herein. For example, an expression cassette can also include
nucleotide
sequences that encode a signal sequence that directs secretion of an expressed
protein
from the host cell.
Xenogens are defined here to include any compound having
estrogenic activity in the assays described herein, which is derived from a
source outside
the human body. Environmental compounds as used herein can be derived from a
wide
variety of sources including plants, soil, water, foods. They also include
synthetic
compounds such as chlorinated organics, polycyclic aromatic hydrocarbons,
herbicides,
pesticides, pharmaceuticals and the like.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 A illustrates the structure of five estrogen receptor (ER) ligands:
Estradiol (E~, diethylastilbestrol (DES), ICI 184,384, raloxifene (Ral), and
tamoxifen
(Tam).
Figure iB illu~rates two estrogen receptor (ER) response elements: a
simple (classical) estrogen response element (ERE) and an ER dependent AP 1
element
described also in USSN 08/410,807, in USSN 60/051,309, and by Webb etal (
1995)Mol.
Endo., 9: 443-456.
Figure 2 illustrates ER~i action at an estrogen response element (ERE).
HeLa cells were transfected with an ERE-regulated luciferase reporter plasmid
and an
expression vector for rat ER(3 as described herein. Transfected cells were
treated with the
ligands (E2, 0.1 pM; DES, 1 pM, Ral, l pM, tamoxifen 5 pM; and ICI,1 ~ or an
ethyl
alcohol (EtOH) vehicle control. All assays were done with at least triplicate
transfections.
Error bars show deviations between wells from a single representative
transfection.
Figure 3 illustrates ERa action at an AP 1 element. HeLa cells were
traasfected with an AP 1 reporter plasmid and an ERa expression plasmid and
treated with
the five Iigands (see, e.g., Figure 2). Ligand concentrations were E2, 0.1 pM;
DES, 1
~M,; Ral, 1 pM; Tam, 5 p,M, and ICI, 1 pM. Error bars are as in Figure 2.
CA 02301143 2000-02-17
wo ~nm6o rcrnJS9snso3o
14
0 Figure 4 illustrates ER~3 activation and inhibition at AP1. (A) ERA action
at an AP 1 response element. HeLa cells were fed with an AP 1 reporter plasmid
and a rat ER[3 expression plasmid as described herein. Transfected cells were
treated with
the following ligand concentrations: E2, 0.1 pM; DES,1 pM; Ral, l pM, Tam, 5
pM; and
ICI, 1 E,~M. (B) Dose response of raloxifene induction with ERS at an AP1
element.
HeLa cells transfected as described for A were treated with the indicated
range of
raloxifene concentrations. (D) Comparative inhibition of raloxifene induction
by E= and
DES. HeLa cells were transfected as described for (A) and treated with
ligands. The left
panel shows transactivation induction by raloxifene ( 1 pM), the lack of
induction by E2
(0.1 uM) and induction to the amount observed with the control (no ligand
added). The
right panel shows the dose dependence of inhibition of raloxifene (1 ~
induction by
DES (solid line) and EZ (Dashed line). (D) Raloxifene overriding EZ
inhibition. HeLa cells
were transfected as described for (A) and treated with Iigands. The left panel
shows the
transcription induction resulting from the vehicle control (EtOH), Ral ( 1 pM)
plus EZ ( 10
nM), and EZ (10 nM) alone. The right panel shows the dose dependence of
raloxifene
induction in the presence of E~ (10 nMJ.
Figure 5 illustrates ligand-dependent ER~i activity in three cell types;
Ishikawa cells, MCF7 cells and MDA453 cells. (A) Ligand-dependent ER~3 action
at an
AP 1 element in Ishikawa cells. Ishikawa cells were transfected with an AP 1-
regulated
luciferase reporter plasmid and an ER(3 expression plasmid. Transfected cells
were treated
with one or two ligands as indicated (Ez, 0.1 pM; DES, 1 ~M; Ral, 1 pM, Tam, 5
pM;
and ICI, 1 pM; or an EtOH vehicle (co~rol)). (B) Ligand dependent ERø action
at an
AP 1 eleme~ in MCF7 cells. MCF7 cells were treated and analyzed as described
for (A).
Ligand dependent ERA action at an AP1 element in MDA453 cells. MDA453 cells
were
treated and analyzed as described for (A).
DETAILED DESCRIPTION
Antiestrogens are therapeutic agents for the treatment and possible
prevention of breast cancer. Tamoxifen (Figure 1 A), for example, is an
antiestrogen that
is used in breast cancer chemotherapy and is believed to function as an
antitumor agent
by inhibiting the action of the estrogen receptor (ER) in breast tissue
(Grainger et al.
(1996) Nature Med, 2: 381-385). Paradoxically, tamoxifen appears to function
as an
CA 02301143 2000-02-17
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0 estrogen-like ligand in uterine tissue, and this tissue-specific iatrogenic
effect may explain
the increased risk of uterine cancer that is observed with prolonged tamoxifen
therapy
(Kedar et al. (1994) Lancet, 343: 1318-1321).
The related benzothiophene analog raloxifene (Fig. l A) has been reported
to retain the antiestrogen properties of tamoxifen in breast tissue and to
show minimal
5 estrogen effects in the uterus; in addition, it has potentially beneficial
estrogen-like effects
(in nonreproductive tissue such as bone and cardiovascular tissue (Jones et
al. (1984) J.
Med Chem., 27: 1057-1066; Black et al. (1994) J. Clip. Imest., 93: 63-69; Sato
et al.
(1996) FASEB J., 10: 905-912; Yang et al. (1996) F.~docrinol., 137: 2075-2084;
Yang
et al., ( 1996) Science, 273 :1222-1225)). One explanation for these tissue-
specific actions
10 of antiestrogens is that the Ggand-bound ER may have different
transactivation properties
when bound to different types of DNA enhancer elements.
The classical estrogen response element (ERE) is composed of two
inverted hexanucleotide repeats, and ligand-bound ER binds to the ERE as a
homodimer
(Fig. 1B). The ER also mediates gene transcription from an AP1 ecihancer
element that
15 requires ligand and the AP 1 transcription factors Fos and Jun for
t<ansaiptional activation
(Fig. l B) (Umayahara et al. ( 1994) J. Biol. Chem., 269:16433-16442). In
transactivation
experiments, tamoxifen inhibits the transcription of genes that are regulated
by a classical
ERE, but like the natural estrogen hormone 17b-estradiol jE2 (Fig. 1 A)],
tamoxifen
activates the transcription of genes that are under the control of an AP 1
element (Webb
et al. ( 1995) Mol. Endocrinol., 9: 443-456).
At the end of 1995, a second ER (ERA) was cloned firm a rat prostate
cDNA library (Kuiper et al. (1996) Proc. Natl. Acad Sci.USA, 93: 5925-5930).
The
human (Mosselman et al. (1996) FEES Lett., 392: 49=53) and mouse (Tremblay et
al.
{1997)Mol. E»dOCrinol., 11: 353-365) homologs were also cloned. The first
idemified
ER has been renamed ERa (Kuiper et al. (1996) supra). It was a discovery of
this
invention that ERA presents another source of tissue-specific estrogen
regulation,
particularly as mediaxed through the AP 1 site. In particular, it was a
discovery of this
invention that ERa and ER[i respond differently to certain Ligands at an AP 1
element.
The results described herein suggest different regulatory fixnctions for the
two ER
subtypes. This invention thus provides materials and methods for screening for
CA 02301143 2000-02-17
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16
0 compounds that exhibit differential activity depending on whether their
activity is
mediated through ERa or ER~i. In addition, this invention provides materials
and
methods for determining whether a compound is capable of activate or inhibit
estrogen
receptor (3 (ER~i) mediated gene activation (transcription) at an AP1 site.
I. Screening Methods and Compositions.
It was a discovery of this ERA can interact with a AP 1 site to activate or
inactivate expression (e.g.transcription) of a gene under the control of the
AP1 site.
Moreover, it was a particularly surprising discovery that putative estrogens
can actually
demonstrate "antiestrogenic" activity in an ER~/AP 1 pathway (where
antiestrogenic
activity in this context is as compared to the activity of an estrogen in the
classical
ERaJERE pathway). Thus, where an estrogen would activate transcription in an
ERa/ERE pathway the estrogen inactivates transcription in an ERaIAP l pathway.
Conversely, putative antiestrogens can demonstrate estrogenic activity in an
ER/3/AP 1
pathway. This invention thus provides methods for detecting antiestrogenic
activity of
putative estrogens, or for detecting estrogenic activity of putative
antiestrogens. More
generally, as explained below, this invention provides methods of screening
compounds
for the ability to activate or inhibit estrogen receptor [3 (ER(3) mediated
gene activation
at an AP 1 site. This allows identification of previously unsuspected
environmental
estrogens or antiestrogens or for screening of compounds for those that have
desirable
estrogenic or antiestrogenic properties. Such compounds are expected to be
useful for
the treatment or the prevention of various cancers (e.g.breast cancer, ovarian
cancer,
endometrial cancer) and other diseases (e.g. endometriosis) mediated by
estrogen.
A) Screening for ER[3 mediated AP 1 activation or inhibition.
This invention provides efficient ways to screen large numbers of test
compounds for the ability to activate or inhibit estrogen receptor a (ERj3)
mediated gene
activation at an AP1 site. In one embodiment, the methods utilize a cell
containing an
estrogen receptor beta (ER~i), an AP 1 protein, and a construct comprising a
promoter and
reporter gene under the contro! of an AP 1 site such that ER~i irneraction
with the AP 1
site, can increase or inhibit expression (e.g., transcription) ofthe reporter
gene. The cell
is contacted with one or more compounds whose ER(3 activity at AP1 it is
desired to
evaluate. In a preferred embodiment, the expression level of the reporter gene
in the cell
CA 02301143 2000-02-17
wo ~nt~6o rcrms9mso3o
17
0 contacted with the compound is compared to the expression level of a cell
contacted by
a control (e.g., identical culture conditions lacking the test compound and/or
with a
reference compound e.g., estradiol or tamoxifen). A decrease in expression
level of the
reporter gene indicates that the test compound inhibits ER(3-mediated
expression
(transcription) at an AP1, site, while an increase in expression level of the
reporter gene
indicates that the test compound activates ER[3-mediated expression
(transcription) at an
AP 1 site.
The criteria used to evaluate a change in expression level of the reporter
gene in this assay, and the other assays described herein, are those standard
in the art.
Thus, for example, a statistically significant difference in expression level
between the test
and control experiments are scored as a valid change. In a preferred
embodiment, the
expression level may change by a factor 1.5 or more, preferably by factor of 2
or more,
more preferably by a factor of 4 or more, and most preferably by a factor of 5
or even 10
or more.
Screening for differential ERac and ER/i mediated activity.
it will be appreciated that using the methods of this invention, the ability
of compounds to activate or inhibit ERA-mediated transcription at an AP 1 site
can be
compared to the ability of those compounds to activate or inhibit ER(~-
mediated activity
at an ERE site or to the ability of those compounds to activate or inhibit ERa-
mediated
activity at an APlor ERE. In this manner, compounds having a highly specific
mode of
activity across a wide tissue distribution, or alternatively compounds having
a highly
variable mode of activity can be identified.
Four preferred estrogen receptor based assays are illustrated in Table 1.
These correspond to ERa-mediated ERE activity, ERa-mediated AP1 activity, ER(3-
mediated ERE activity, and ER~i-mediated AP 1 activity. It was a discovery
ofthe present
invention that various compounds exhibit differential activity in these
various assays.
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18
0
Table 1. Iuush~ation of estrogen receptor based assays.
ER ER
a
ERElreporter Cla (3
exre ssical pathwaclassical
S AP1/reporter Ind a
ene irect ath indirect athwa
This is illustrated in Table 2, where it can be seen that estrogen activates
transcription in both the classical response (at an ERE) and in the indirect
response (at an
AP 1 ) when the interaction is mediated by ERa. In contrast, estrogen acts as
an inhibitor
of transcription at AP 1 when the interaction is mediated by ER~i. In
contrast, the estrogen
antagonist tamoxifen appears to always act as an inhibitor at an ERE, but an
activator of
transcription at an AP 1 site. Moreover, the activity of ERA does not appear
to be tissue
restricted.
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19
0 Tsble 2. Illustration of the activity of estradiol (E~ and an estrogen
antagonist (tamoxifen) in each of the ER assays.
ER ER
a
ERFJreporter
gene Ac Ac
Estradiol tivates tivates
Inh Inh
Tamoxifen ibits ibits
AP 1/reporter
gene Ac Inh
Estradiol tivates ibits
Ac Ac
Tamoxifen tivates tivates
The assay for ERA-mediated AP 1 activity is described above. The
remaining assays are performed in an analogous meaner. Thus, the ERa-mediated
activity
assays simply involve substituting ERa for ER(3, and the ERE activity assays
simply
involve substituting the EREJreporter gene construct for the AP 1 /reporter
gene construct.
The ERa assays (both for ERE and AP1 activity) are described in detail in USSN
08/410,807, in USSN 60/051,309, and by Webb et al (1995) Mol. Enafo., 9: 443-
456).
The assay for ERA-mediated ERE activity utilizes a cell comaining an
estrogen receptor beta (ER(3), and a construct comprising a promoter and
reporter gene
under the control of an ERE site such that ERA imeraction with the ERE site,
can increase
or inhibit expression (e.g., transcription) of the reporter gene. The cell is
contacted with
one or more compounds whose ERj3 activity at an ERE it is desired to evaluate.
In a
preferred embodiment, the expression level of the reporter gene in the cell
contacted with
the compound is compared to the expression level of a cell contacted by a
comrol (e.g.,
identical culture conditions lacking the test compound and/or with a reference
compound
eg., estradiok or tamoxifen). A decrease in expression level of the reporter
gene indicates
that the test compound inhibits ERA-mediated expression (transcription) at an
ERE, while
CA 02301143 2000-02-17
wo ~nm6o pc~rivsmso3o
0 an increase in acpression level of the reporter gene indicates that the test
compound
activates ERA-mediated expression (transcription) at an ERE site.
While, in a preferred embodiment, each assay is performed in a separate
cell, it will be appreciated that AP1 and ERE assays can be combined and
performed in
a single cell. In this case, the APllreporter gene construct preferably
utilizes a different
5 reporter gene than the ERE/reporter gene construct so that AP 1 activation
or inactivation
can be distinguished from ERE activation or inactivation.
Screening for inhibitor activity.
The above-describe assays can also be used to identify (screen for)
compounds that inhibit other compounds which have ERa-mediated or ERA-mediated
10 activity an ERE or at an AP-1 site. These assays are performed in the same
manner as the
assays described above. In this instance, however, the cell is contacted with
two
compounds, a test compound that is being screened for inhibitory activity and
a second
compound for which an inhibitor (or alternatively an agonist) is sought.
Thus, for example, where it is desired to identify a test compound having
15 ER(3-mediated estrogen inhibitory activity at an AP 1 site, the cell
containing ERA, an AP 1
protein, and a reporter gene under control of an AP1 site is contacted with
estrogen and
the test compound. If the compound inhibits the characteristic ER/i-mediated
estrogen
activity at API, the compound is an inlu'bitor. It should be noted that in
this case, ERJ3-
mediated estrogen activity at AP 1 inhibits transcription, thus an estrogen
inhibitor in this
20 context actually increases ER(i-mediated transcription at AP1. This is
illustrated in
Example 1, where it is shown that tamoxifen is one such inhibitor.
Inhibitors, or agonists, of ERø-mediated or ERa-mediated estrogenic or
antiestrogenic activity at ERE and at AP 1 can be screened in an analogous
manner.
D) Screening for environmental estrogens or antiestrogens.
As indicated above, this invention allows for screening of test compounds
for estrogenic or antiestrogenic activity mediated through ERA or ERa at an
ERE or at
an AP1 site. The assays are particularly useful for screening environmental
compounds
for estrogenic or antiestrogenic activity. Environmental compounds having
estrogenic
activity are referred to here as xenoestrogens. Xenoestrogens include any
compound
derived from a source outside the human body, having estrogenic activity in
the assays
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21
0 described herein. Environmental compounds as used herein can be derive from
a wide
variety of sources including plants, soil, water, foods. They also include
synthetic
compounds such as chlorinated organics, polycyclic aromatic hydrocarbons,
herbicides,
pesticides, pharmaceuticals and the like.
It will be appreciated that environmental estrogens often are only weakly
active. Consequently, particularly when testing an environmental compound for
estrogenic
or antiestrogenic activity, it is often desirably to maximize sensitivity of
the assay. This
may be accomplished by using cells that produce the methods typically comprise
cultured
cells that produce high levels of the human estrogen receptor (ERa or ER~i).
Such cells
include, but are not limited to MCF-7 cells (ATCC No. I-iTB 22), MDA453 cells
(ATCC
No. HTB 131), ZR-75-1 cells (ATCC No. CRL 1500) or ERC1 cells described in
Kushner et al. (1990) Mol. F~idocri»ol., 4:1465-1473, and ERC2 and ERC3 cells
as
described by Webb et al. (1993) Mol. F.»docrirrol., 6:157-167.
It is also known that environmental estrogens may show synergistic activity
in combination. Thus, in one embodiment, two or more suspected environmental
estrogens are assayed according to the above methods in combination. It will
be
recogNZe~, however, that such combined testing is not limited simply to
environmental
estrogens but rather, amr combination of agents can be screened
simultaneously.
Screening for transcription factor modulation of ER(3 activity at AP 1.
It has been demonstrated that various nuclear transcription factors (e.g.,
progesterone, gtucocorticoids, etc. ) interact with the ERa-mediated
estrogenic activity
at the AP1 site (see, e.g., USSN 60/051,309). It is believed that ER(3 is also
capable of
such interactions at AP1. Thus, in another embodiment, this invention provides
assays
(methods of screening) nuclear transcription factor ligands, and putative or
known
transcription factor ligand agonists or antagonists for the ability to
modulate ER(3-
mediated activation or inactivation of transcription at an AP 1 site.
These assays are performed in the same manner as the assays described
above, however the assay cell additionally contains a receptor for a second
nuclear
transcription ligand (preferably a ligand other than estrogen). Thus, the cell
contains an
estrogen receptor beta (ER~i), an AP 1 protein, a receptor for a second
nuclear
transcription factor ligand, and a construct comprising a promoter comprising
an AP 1 site
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22
0 which regulates expression of a reporter gene. The cell is contacted with
both a
transcription factor ligand that is to be wed and with a compound having ER[3
mediated activity at an AP i site.
Alteration of the typical activity (level of AP1 regulated reporter gene
expression) ofthe compound having ER[3-mediated activity at an AP 1 site by
the presence
of the compound being scxeened (the test transcription factor iigand)
indicates that the
screened compound is capable of modulating an ER~i-mediated AP 1 response of
the
compound having ER(i-mediated activity at an AP 1 site. Preferred second
nuclear
transcription factor ligands include, but are not limited to glucocorticoids,
progestins,
vitamin D, retinoic acid, androgens, mineralcorticoids, and prostaglandins.
Similarly, inhibitors, or agonists, of the test compound can be screened by
running the same assay in the presence of the inhibitor that is to be
screened.
II. Cell Types
The assay methods of this invention provide methods for evaluating the
ability of a test, or control, compound to activate or inhibit transcription
through
interaction with a transcription factor receptor (e.g., estrogen receptor).
Thus, in a
preferred embodiment, the cells used in the assays of this invention
preferably contain at
least one transcription factor receptor.
For example, where it is desired to screen for activity of a compound
mediated by the estrogen receptor a (ERa) cells are preferably provided that
contain ERa
and where it is desired to screen for activity of a compound mediated by
estrogen receptor
~3 (ER[3) cells are preferably provided that contain ER~i.
Where it is desired to screen for the ability of a nuclear tt~nscxiption
factor
ligand modulate estrogen receptor (a or (i) mediated activation or
inactivation of
transcription at an AP 1 site, the cell preferably include, in addition to the
particular ERa
or ER[3 at least a second nuclear transcription. factor receptor (e.g.,
glucocorticoid
receptor (GR)). Cells that naturally express one or more of the desired
receptor types can
be used in the assays of this imemion. Alternatively, cells can be modified
(e.g., through
recombinant DNA techniques) to express ERa and/or ER.(~ and/or the
transcription factor
receptor of choice.
CA 02301143 2000-02-17
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23
0 Suitable cells for practicing the methods of this invention include, but are
not limited to cells derived from a uterine cervical adenocarcinoma (HeZ,a) ,
a
hypothalamic cel! line (GT1-1 (Melton et al. (1990) Neuron, 5: 1-10), MCF-7
cells
(ATCC No. HTB 22), MDA453 cells (ATCC No. HTB 131), ZR 75-1 cells (ATCC No.
CRL 1500) or ERC1 cells described in Kushnex et al., Mol. Faralocrinol.,
4:1465-1473
(1990). ERC2 and ERC3 calls as described by Webb, et al. Mol. F,ndocrinol.,
6:157-167
(1993). It will be appreciated that the invention is not limited to practice
in mammalian
cells and may be practiced, for example in yeast and insect cells, transfected
with the
appropriate genes and recombinant constructs.
A) Cells naturally expressing two or more receptor types.
_ Many cells that express a second transcription factor receptor in addition
to the estrogen receptor (ER) are well known to those of skill in the art.
Thus, for
example, in the uterus there is evidence that ER and glucocorticoid receptors
(GR) co-
exist in the endometrium (Prodi et al. (1979) Tumor. 65: 241-253). In the
brain, maps of
ER and GR immunoreactivity and mRNA localization suggest co-localization in
certain
cerebral nuclei such as the paraventricular nucleus of the hypothalamus, the
hypothalamic
arcuate nucleus, and the central nucleus of the amygdala (Fuxe et al. ( 1985)
Endocrinol.,
118: 1803-1812; Simerly et al. (1990) J. Comp. Neurol. 294: 76-95). In bone,
ER and
have been found in cultured osteoblast-like cells (Liesegang et al. (1994) J.
Andrology,
14: 194-199). ER has also been demonstrated in osteoclasts {Oursler et al.
(1994) Proc.
Natl. Acad Sci., USA, 91: 5227-5231) and data suggest that the glucocorticoid
dexamethasone (Dex) regulates metaboli~n in these cells (along (1979) J. Biol.
Chem.,
254: 6337-b340) raising the possibility that osteoclasts contain functional GR
as well. In
addition, numerous tumor cell lines have been demonstrated to have both ER and
GR
(Swing et al. (1989) Int. J. Cancer., 44: 744-752.
B) Cells recombinamly modified to express two or more receptor types.
Cel~s normally lacking the ERa or ER~i or other transcription factor
cognate receptors can be recombinantly modified to express one or more of the
desired
receptors. Typically this involves transfecting the cell with an expression
cassette
comprising a nucleic acid encoding the receptor of interest and culturing the
cell under
conditions where the receptor is expressed (e.g., in the presence of an
appropriate inducer
CA 02301143 2000-02-17
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24
0 if the promoter regulating exp~ion of the receptor is inducible). Typically,
the cassette
is selected to provide constitutive expression of the receptor.
A cell that naturally expresses one receptor need only be modified to
express the second receptor. However, if the cell expresses neither receptor,
it may be
transfected with expression cassettes expressing both receptors. Even where a
cell
S naturally expresses one or both receptors, it may be recombinantly modified
to express
those receptors at a higher level (e.g., by introducing expression cassettes
encoding the
receptors) whose expression level it is desired to increase).
The cells need not contain "native" receptors, but may be modified to
provide truncated or chimeric receptors to provide increased affnity and/or
sensitivity of
the assay. Thus, for example, Berry, et al.(1990), F.~I~BD J., 9: 2811-2818,
describe the
production of cells containing truncated or chimeric ER receptors.
Methods of modifying cells to express particular receptors are well known
to those of skill in the art. Thus, for example, cells modified to express
high levels of
estrogen receptor are described by Kushner et al. ( 1990), Mol. Er~iocrinol. ,
4:1465-1473.
See also ITlrst et al. (1990) Mol. Endocrinol., 4: 162-170). Transfection of
cells to
express ERac is described below, in the Examples, and in US SN 08/410, 807.
Transfection
of cells to express ER~i is described herein, and transfection of cells to
express
glucocorticoid receptors (GR), progestin receptors (PR), and other receptors
is described
in copending USSN 60/043,059.
C) Cells Containing AP1 proteins.
In assays that involve screening for transcription factor receptor mediated
activation or inactivation oftranscription at APl, the cells preferably
contain one or more
AP 1 proteins (the Jun or Fos proteins or other members of that protein
family, see
Bohmaan, et al. (1987) Science, 238: 1386-1392) in addition to the
transcription factor
receptor(s).
The cells can naturally express the AP 1 proteins) or they can be modified
(e.g., by transfection with a suitable expression cassette) to express a
heterologous AP1
protein. Methods of expressing AP 1 proteins are well known to those of skill
in the art
(see, e.g., Turner et al. (1989) Science, 243:1689-1694 and Cohen et al.
(1989) Genes
CA 02301143 2000-02-17
wo ~nm6o rrrnJSmso3o
0 & Dev., 3 : 173-184, and Example 1 ). Cells that naturally express one or
more AP 1
proteins may still be so modified to increase intracellular jun and/or fos
levels.
IlT) Expression of Nuclear Transcription Factor Receptors.
As explained above the assays of this invention utilize cells containing one
or more nuclear transcription factor receptors (e.g., ERa, ER~3, GR, PR, etc.
) an estrogen
5 receptor and a receptor for a nuclear transcription factor (typically a
transcription factor
other than estrogen). The factor can be one that is expressed endogenously by
the cell or,
alternatively, the cell can be modified (e.g., a recombinant cell) so that it
expresses the
receptor.
A) Estrogen Receptor Alpha (ERa)
10 An estrogen receptor, as used herein, includes an estrogen receptor alpha
(ERa) in its native (naturally occurring) form as well as modified estrogen
receptors.
Numerous modifications of estrogen receptors are known to those of skill in
the art.
These include, but are not limited to VP 16-ER, V-ER, a chimeric receptor
comprising the
strong VP 16 transcriptional activation domain linked to the amino terminus of
the ER, V-
15 ER in which the ER DNA binding domain (DBD) is deleted, Hl 1 an ER lacking
the DNA
binding domain, and the like (see e.g., Kumar et al., Cell, 51: 941-951 (1987)
and Elliston
et al. ( 1990) J Biol Chem 265:11517-21 ).
Means of recombinantly expressing the estrogen receptor alpha (ERa) are
well known to those of skill in the art (see, e.g., USSN 08/410,807 and Webb
et al ( 1995)
20 Mol. Fardocrinol., 9: 443-456).
B) Estrogen Receptor Beta (ER(3).
Estrogen receptor beta (F.R(3) is a second estrogen receptor (ER) cloned
from a rat prostate cDNA library (Kuiper et al. ( 1996) Proc. Natl. Acad Sci.
USA, 93
5925-5930). Subsequently the human (Mosselman et al. (1996) FEBSLett., 392: 49-
53)
25 and mouse (Tremblay et al. (1997) Mol. Errdocrinol., 11: 353-365) homologs
were
cloned. Accordingly, the original estrogen receptor (ER) has been renamed ERa
(Kuiper
et al. ( 1996) supra. ).
Using the known sequence information one of skill in the art can routinely
construct vectors that express an ER~i when transfected into a suitable host
cell. Detailed
CA 02301143 2000-02-17
WO 99/11760 PGTNS98/18030
26
0 protocols for the preparation of an ER(3 vector can be found in Kuiper et
al. ( 1996) Proc.
Natl. Acad Sci. USA, 93: 5925-5930 and in WO 97/09348.
It will be appreciated that exist s number of different estrogen beta
receptors comprising various splice variants, mutations, and so forth. It will
be
appreciated that ER~i as used herein is intended to include all FR~3 variants.
However, in
a preferred embodiment, the ER(3 variants used in this invention correspond to
the so
called "intermediate length" ER~i variants such as those described in WO
97/09348.
Particularly preferred ERA variants are shown in sequence listings 3, 4, and 5
herein which
correspond to figures 1 and 13A and 13B of WO 97/09348,
C) Nuclear transcription factor ligand and cognate receptor
As indicated above, the in addition to the estrogen receptor (ERa and/or
FR~i), the cells can contain a cognate receptor for a nuclear transcription
factor ligand
whose interaction (preferably a cognate receptor other than an estrogen
receptor). As
used herein, the term "cognate receptor" refers to a receptor of the type that
is typically
bound by the transcription factor ligand in question. Thus, the cognate
receptor for an
estrogen is an estrogen receptor, the cognate receptor for a glucocorticoid is
a
glucocorticoid receptor, the receptor for a progestin is a progestin receptor,
and so forth.
As with the estrogen receptor, the cognate receptor includes the native
(naturally
occurring ) form as well as modified receptors.
Natural and modified cognate receptors for nuclear transcription factor
ligands, particularly for steroid nuclear transcription factors, are well
known to those of
skill in the art. These include, but are not limited to the glucocorticoid
receptors, the
progestin receptors (e.g., PR A, PR B (see, e.g., Law et al. ( 1987) Proc.
Natl. AcaaL Sci.
USA 84: 2877-2881; Wei et al. (1988) Mol. Fr~do. 2: 62-72; and Kushner et al.
(1990)
Mol. Endocrinol, 4:1465-1473), vitamin D receptors, mineralcorticoid
receptors,
androgen receptors, and thyroid hormone receptors (see, Mangelsdorf (1995)
Cell, 83:
835-839).
IV. ERE and AP1 Reporter Constructs
The cells of this invention preferably contain (e.g., are transfected with)
nucleic acid constzucts comprising one or more reporter genes under the
control of a
response element (either the AP1 site or estrogen response element (ER,E)).
Where two
CA 02301143 2000-02-17
WO 99/11760 PGT/US98/18030
27
0 different response elements are monitored in a single cell, two differ~t
reporter genes are
used. Thus, for example, one gene can reports transcription induced by the
classical
estrogen response system (ERE), while the other gene reports transcription
induced by
the indirect (AP 1 ) estrogen response. The two reporter genes and response
elements are
typically placed in separate cells, but the methods can also be used with both
constructs
in the same cell.
A) AP1/Reporter construct.
In one embodiment the methods of this invention imrolve providing a cell
containing an estrogen receptor (ERa or ER(3), and a promoter comprising an
AP1 site
that regulates expression of a reporter gene (also referred to herein as the
reporter gene
for the indirect estrogen response pathway (see, e.g., USSN 08/410,807 and
Webb et al
(1995) Mol. Endocrinol., 9: 443-456).
The reporter gene for the indirect estrogen response pathway comains an
AP1 site preferably upstream of the target promoter and capable of regulating
(i.e.,
operably linked to) that promoter. AP 1 site are sites that are bound by AP 1
(the Jun and
Fos proteins) or other members of that protein family. In a preferred
embodiment, the
consensus AP1 site (or AP1 response element) is TGA(C/G)TCA (SEQ ID NO: 1).
One of skill would recognize that the particular AP1 site used is not a
critical aspect of the imrention. Any sequence capable of being bound by AP 1
or members
of that family and regulating a promoter is suitable. This would include
promoters which
encompass a naturally occurring AP 1 site. Typical promoters include, but are
not
restricted to meralloprotease genes such as stromelysin, gelatinase,
matrilysin, and the
human collagenase gene.
Alternatively promoters may be construcxed which contain a non-naturally
occurring AP1, or related, binding site. This facilitates the creation of
reporter gene
systems that are not typically found under the control of AP 1. In addition,
promoters may
be constructed which contain multiple copies of the AP1 site thereby
increasing the
sensitivity or possibly modulating the response the reporter gene system.
B) ERE/Reporter Construct
The methods of this invention can also involve providing a cell containing
a promoter comprising an estrogen response element that regulates expression
of a
CA 02301143 2000-02-17
WO 99/11760 PCTNS98/18030
28
0 reporter gene (also referred to herein as the reporter gene for the direct
or classical
estrogen response pathway (see, e.g., U.S.S.N. 08/410,807 and Webb, etal.
(1995)Mol.
E~alo., 9: 443-456). This permits detection of the "direct" (classical)
estrogen response
and evaluation of the interaction or modulation of the classical response by
the nuclear
transcription factor ligand.
Typically, the estrogen response element (ERE) is upstream of the target
promoter and capable of regulating that promoter. In a preferred embodiment
the FRF
may be the consensus estrogen response element AGGTCACAGTGACCT (SEQ ID NO:
2) from the Xenopus vitellogenin A2 gene. The particular ERE used in the cell
is not a
critical aspect of the invention and the present invention is not limited to
the use of any
one particular ERE. Suitable EREs are well known to those of skill. For
instance, other
sources of naturally occx~iTing EREs include the vitellogenin B2 gene, the
chicken
ovalbumin gene, and the PS2 gene. Alternatively, non-naturally occurring EREs
may be
inserted into particular promoters. The consensus ERE from theXenopus
vitellogenin A2
gene is widely used for this purpose, but other EREs may be used as well
C) Reporter Genes)
The present invention is not limited to a particular reporter gene. Any
gene that expresses an easily assayable product will provide a suitable
indicator for the
present assay. Suitable reporter genes are weU known to those of skill in the
art.
Examples of reporter genes include, but are not limited to CAT
(chloramphenicol acetyl
transferees) (Alton and Vapnek ( 1979) Nature 282: 864-869), luciferase, and
other
enzyme detection systems, such as beta-galactosidase; firefly luciferase
(deWet et al.
( 1987) Mol. Cell. Biol. 7:725-737); bacterial luciferase (Engebrecht et al. (
1984) Proc.
Natl. Aced Sci., USA,1: 4154-4158; Baldwin et al. ( 1984) Biochemistry 23
:3663-3667);
alkaline phosphatase (Toh et al. (1989) Eur. J. Biochem.182: 231-238; Hall et
al. (1983)
J. Mol. Appl. Gen. 2: 1 O 1 ), and green fluorescent protein.
One of skill will recognize that various recombinant constructs comprising
the AP-1 site can be used in combination with any promoter and reporter gene
compatible
with the cell being used. The promoter will preferably be one susceptible to
regulation
by the AP 1 site.
D) Construction of the Promoter/Reporter Expression Cassette.
CA 02301143 2000-02-17
wo ~nm6o rcr~s9snso3o
29
0 The promoter/reporter ion cassettes and, other expression cassettes
(constructs) described herein, can be constructed according to ordinary
methods well
known to those of skill in the art. Construction of these cassettes is
variously exemplified
in Example 1, in USSN 08/410,807, in Webb et al. (1995)Mol. Endo. 9: 443-456,
and
in other references cited herein.
The constructs can all be created using standard amplification and cloning
methodologies well known to those of skill in the art. Examples of these
techniques and
instructions sufficient to direct persons of skill through many cloning
exercises are found
in Berger and Kimmel, Guide toMolecular Cloning Techniques: Methods in
Fnzymology,
152 Academic Press, Inc., San Diego, CA; Sambrook et al. ( 1989) Molecular
Cloning -
A LaboratoryManual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold
Spring
Harbor Press, NY,; Current Protocols in Molecular Biology, Ausubel et al.,
eds., Current
Protocols, a joint venture between Greene Publishing Associates, Inc. and John
Wiley &
Sons, Inc., (1994 Supplement) (Ausubel); Cashionetal., U.S. Patent No:
5,017,478; and
Carr, European Patent No. 0,246, 864. Examples of techniques sufficient to
direct persons
of skill through in vitro amplification methods are found in Berger supra.,
Sambrook
supra., and Ausubel supra., as well as Mullis et al., (1987) U.S. Patent No.
4,683,202;
Inr>is et al. ( 1990) PCR Protocols A Guide to Methods and Applications,
Academic Press
Inc. San Diego, CA; Arnheim & Levinson (October 1, 1990) C&EN 36-47; The
Journal
Of MH Research ( 1991 ) 3 : 81-94; Kwoh et al. ( 1989) Proc. Natl. Acac~ Sci.
USA 86:
1173; Guatelli et al. ( 1990) Proc. Natl. Acad Sci. USA 87, 1874; Lomell et
al. ( 1989) J.
Clin. Chem., 35: 1826; Landegren et al., (1988) Science, 241: 1077-1080; Van
Brunt
(1990) Biotechnology, 8: 291-294; Wu and Wallace, (1989} Gene, 4: 560; and
Barringer
et al. (1990) Gene, 89: 117.
V. ER(3-mediated Activation through tethered coativactors.
In still another embodiment, ERA can mediate gene activation through
virtually any response element using a tethered transcription factor
coactivator strategy.
The methods involve contacting a nucleic acid that includes the gene of
interest operably
linked to a response element with a tethered coactivator. The tethered
coactivator is
composed of a polypeptide that comprises an activation fimction derived from a
transcriptional coactivator, and a DNA binding moiety that is capable of
specifically
CA 02301143 2000-02-17
WO 99/11760 PGT/US98J18030
0 binding to the response element. The tethered coactivator is cornacted with
an activated
transcription factor polypeptide (e.g., ER.~) that includes an activation
function derive
from a tt~ansaiption factor. The contacting of the tethered coactivator with
the activated
transcription factor polypeptide stimulates expression of the gene. The
transcription
factor can be, for example, a nuclear hormone receptor such as the estrogen
receptor or
5 the estrogen receptor beta, or an AP 1 transcription factor, however, in a
preferred
embodiment, the transcription factor is ER~i. Detailed protocols for the
tethered
transcription factor activation strategy are provided in copending USSN
60/043,059.
VI. Detection of the reporter genes.
Detection of the reporter genes of this imrention is by standard methods
10 well known to those of skill in the art. Where the reporter gene is
detected through its
enzymatic acxivity this typically involves providing the enzyme with its
appropriate
substrate and detecting the reaction product (e.g., light produced by
luciferase). The
detection may involve simply detecting presence or absence of reporter gene
produce, or
alternatively, detection may involve quantification of the level of expression
of reporter
15 gene products. The quantification can be absolute quantification, or
alternatively, can be
comparative e.g., with respect to the expression levels of one or more
"housekeeping"
genes. Methods of quantifying the expression levels of particular reporter
genes are well
known to those of skill in the art. It will be appreciated that such detection
can be
performed "manually" or may be automated e.g., as in a high-throughput
screening
20 system.
I~gh throughput assays for the presence, absence, or quantification ofgene
expression (e.g., via the detection ofthe transcribed nucleic acid (mRNA) or
the detection
of gene expression (protein product)) are well known to those of skill in the
art. Thus, for
example, U. S. Patent 5,559,410 discloses highthroughput screening methods for
proteins,
25 U.S. Patent 5,585,639 discloses high throughput screening methods for
nucleic acid
binding (i.e., in arrays), while U.S. Patents 5,576,220 and 5,541,061 disclose
high
throughput methods of screening for ligand/antibody binding.
In addition, high throughput screening systems are commercially available
(see, e.g., Zymark Corp., Hopkinton, MA; Air Technical Industries, Mentor, OH;
30 Beckman Instruments, Inc. Fullerton, CA; Precision Systems, Inc., Natick,
MA, etc.).
CA 02301143 2000-02-17
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31
0 These systems typically automate entire procedures including all sample and
reagent
pipetting, liquid dispensing, timed incubations, and final readings of the
microplate in
detectors) appropriate for the assay. These configurable systems provide high
throughput and rapid start up as well as a high degree of flexibility and
customization.
Compounds to be Screened.
It will be appreciated that virtually any compound can be screened by the
methods of this invention. Such compounds include, but are not limited to
known or
suspected estrogens or antiestrogens including environmernal estrogens or
environmental
antiestrogens as described above.
It will be appreaated that compounds are expected to be show the most
estrogenic or antiestrogenic activity if they are capable of penetrating to
the nucleus of a
cell and binding to a transcription factor receptor (e.g., ERa or ER(i). Such
compounds
are often lipophilic or capable of entering cells passively through pores or
gates, through
active transport, or through endocytosis. Particularly preferred compounds
include, but
are not limited to, steroid compounds or steroid analogs.
VIII. Assay Kits
In another embodiment, this invention provides kits for the practice of the
methods of this imrention. The kits preferably include one or more cornainers
containing
the cells described herein for the practice of the assays of this invention.
Thus, for
example, the cells may include, but are not limited to, cells containing an
estrogen
receptor ~i (ERA), AP 1 protein(s), and a construct comprising a promoter
comprising an
AP 1 site which regulates expression of a first reporter gene, or such cells
additionally
containing a receptor for a nuclear transcription factor ligand other than
estrogen. The
AP1/recporter gene and the ERE/reporter gene constructs can be in separate
cells or
together in the same cell. The cells may additionally express high levels of
AP 1 proteins
such as fos and/or jun. Alternatively, or in addition, the kits can contain
the AP 1 /reporter
gene and/or the ERFJreporter gene constructs described herein and/or the ERa,
ER[i, or
other nuclear transcription factor receptor vectors. The kits may optionally
contain any
of the buffers, reagents, culture media, culture plates, reporter gene
detection reagents,
and so forth that are useful for the practice of the methods of this
invernion.
CA 02301143 2000-02-17
WO 99/il~b0 PCT/US98/18030
32
0 In .addition, the kits may include instructional materials containing
directions (i. e., protocols) for the pracxiice of the assay methods of this
invention. While
the instructional materials typically comprise written or printed materials
they are not
limited to such. Arly medium capable of storing such instructions and
communicating
them to an end user is contemplated by this invention. Such media include, but
are not
limited to electronic storage media (e.g., magnetic discs, tapes, cartridges,
chips), optical
media (e.g., CD ROM), and the like. Such media may include addresses to
Internet sites
that provide such instructional materials.
EXAMPLES
The following examples are offered to illustrate, but not to limit the present
invention.
Example 1
Comparison of the Transac~ivation Properties of ERa and ERA
This example describes the investigation of the transactivation properties
of ERa and ER(i with a panel of five ER ligands with the use of a reporter
gene under the
control of either a classical ERE or an AP 1 element. The results presented
herein show
that ERa and ERA respond differently to certain ligands at an AP 1 element
suggesting
different regulatory fimctions for the two ER subtypes.
Screeni»gMethais
The transactivation properties of ERa and ER(i were compared with a
panel of five estrogen receptor (ER) ligands using a reporter gene under the
control of
either a classical estrogen response element {ERE) or an AP 1 element. The ERE
and AP 1
driven luciferase reporter plasmids (EREII-LucG145 and Ocoll78, respectively)
and the
ERa expression plasmid (pSGS-HEO) were used as described in Webb et al. (
1995) Mol.
Endocrinol., 9: 443-456, and in USSN 08/410,807 now issued as U. S. Patent
The rat ER(i expression vector has been previously described (Kuiper et
al. (1996) Proc. Natl. Acad Sci.USA, 93: 5925-5930). The foil-length human
ER~i
cDNA which was isolated from an ovarian cDNA library and found to be identical
to the
previously reported partial cDNA clone (Mossehnan et al. (1996) FEBSLett.,
392: 49-
CA 02301143 2000-02-17
wo ~n mho rcTnrs9srt solo
33
0 53) was cloned into the pCMVS eukaryotic expression vector and the resulting
ERj3
expression vector was used for these experiments (see, Kuiper et al. ( 1996)
Proc. Natl.
Accu~ Sct. USA, 93: 5925-5930). The ligands used to compare ERa and ER(~
transactivation properties included the estrogens ~i-estradiol (E~ and
diethylstilbestrol
(DES) and the antiestrogens Imperial Chemical Industries (ICI) 164384,
tamoxifen, and
raloxifene. Raloxifene was synthesized according to published procedure (Jones
et al.
( I 984) J. Med Chem., 27: 1057). Structure and purity were verified by 'H
nuclear
magnetic resonance (NMit), "C NMR, ultraviolet thin layer chromatography, and
high
resolution mass spectrometry. ICI 164384 was obtained from a private source
and the
other compounds were obtained from commercial sources.
The experiments were conducted by transfecting HeLa cells with either an
ERa or ER~i expression plasmid along with a reporter plasmid that contained a
luciferase
gene under the transcriptional control of an estrogen response elerilent
(ERE).
Cells were gown in Nunc Delta Surface tissue culture plates to a density
of not more than 5 x 104 per cm2. Cells were grown in 0.1 pm sterile filtered
DME-F-12
Coon's Modified Medium (Sigma Cell Culture) with 15 mM Hepes, 0.438 g/L L-
glutamine, 1.338 g/L NaHC03, 10% Seru-Max 4 (an iron supplemented , formula
fed
newborn calf serum, Sigma Cell culture; from a lot tested for low estrogenic
activity),
0.05 mg/mL Gentamycin, 100 mg/ml Streptomycin SO,, and 100 units/ml penicillin
"G".
Ishikawa cells were gown in a medium containing 100 nM tamoxifen and
MCF-7 cells were grown in medium containing 10 nM estradiol.
For the transfection assays, cells were suspended 0./5 ml ofelectroporation
buffer in 0.4 cm gap electroporation cuvettes (BioRad) at 106 to 2 x 106 cells
per cuvette.
The electroporation buffer was prepared as a solution of 500 ml phosphate
buffered saline
(PBS), 5 ml of 10% glucose, and 50 pL of Biobrene. Five pg of reporter plasmid
and 6
pg ofER expression plasmid were added and the cuvette was agitated to
facilitate mixing
of the solution and homogeneous cell distribution in the cuvette. Cells were
then
immediately transfected by electroporation with a BioRad GenePulser
electroporation
apparatus at a potential of 0.25 kV and a capacitance of 960 uF. To the
electroporation
cuvettes was added 1 ML growth medium (described above).
CA 02301143 2000-02-17
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34
0 The transfected cells for one experiment were pooled and carefully
resuspended in growth medium at a density of 8 x 10'- 1.6 x 10' cells/mL.
After a
homogenous cell distribution was obtained by thorough mixing cells were plated
on Nunc
6-well dishes at 2 mL per well. After 2 h of incubation hormones were added
and the
medium was mixed by gentle swirling. Cells were then incubated in the presence
of
hormone for 40-48 hours.
Growth medium was removed from the wells, and the cells were washed
with Mg2+ and Ca2+ free PBS, and then they were lysed chemically with 0.2 mL
of 100
mM potassium phosphate buffer (pH 7. S) containing 0.2% Triton X-100 and 1 mM
DTT).
The plates were then frozen to -80'C, thawed and scraped with a rubber
policeman to
loosen and break up cell Fragments. The lysate was centrifuged in a microfuge
for 2 min,
0.1 mL of the supernatant was combined with 0.3 mL luciferase assay solution,
and the
chemiluminescence was measured immediately for a period of 10 s.
The luciferase assay solution consisted of 25 nM gtycylglycine, 15 mM
MgS04, 4 mM EGTA, 15 mM potassium phosphate at pH 7.8, with the addition of
DTT
to a f>rtal concentration of 1 mM, ATP to a final concentration of 2 mM and
luciferin
(Analytical Luminescence Laboratories) to a final concentration of200 uM
shortly before
commencing the assay. Luminescence measurements were performed on a MonoGght
1500 (Analytical Luminescence Laboratories). The relative light units reported
here were
adjusted to a scale of 100 for uniformity.
The data were collected using the HEO ER variant. HEO shows reduced
transactivation response from the unliganded receptor compared with the wild-
type ER
resulting in clearer ligand-induced transactivation data Each experiment with
ERac was
also checked with the wild-type ER (HEGO), and the general ligand induction
trends were
found to the same as those obtained with HEO. The only difference was that the
ligand-
induced ttansactivation responses were lower with HEGO than with the control
(no ligand
added).
Transacdvation experiments were performed with both rat and human ER~i
and identical trends in ligand behavior and similar induction levels were seen
with both
ER~is in HeLa cells. The data shown in Figure 2B and Figure 4 were obtained
with the
rat ERA expression plasmid.
CA 02301143 2000-02-17
WO 99/11760 PCT/US98/1$030
0
Fxperime»ts and Results
The transactivation properties of ERa and ER(3 at a classical ERE in
response to the estrogens EZ and diethylstilbestrol (DES) and the
antiestrogens Imperial
Chemical Industries (ICn 164384, tamoxifen, and raloxifene were first
investigated. Both
5 ERa (18) and ER~i (Fig. 2) showed the same transactivation profiles with the
panel of
ligands. E2 and DES stimulated luciferase production 10-fold over ICI 164384;
raloxifene, tamoxifen, and the control (no ligand added). The antiestrogens
blocked EZ
stimulation in ligand competition experiments.
Next, the ligand-induced transactivation behavior of ERa and ERA at an
10 AP1 site was examined. With ERa, all five ligands stimulated luciferase
transcription,
including the antiestrogens ICI 164384, tamoxifen, and raloxifene (Fig. 3).
This
stimulation was dependent on h~ansfected ER, as cells transfected with only
the reporter
plasmid showed no induction of reporter transcription. Of the five ligands,
raloxifene
induced transcription the least, showing twofold induction compared with the
sixfold
15 inductions typically seen with EZ and tamoxifen. The raloxifene-induced
transactivation
was dose dependent with a concentration value required for one-half maximal
activation
(ECM) of about 1 nM. In addition, raloxifene reduced the activation caused by
EZ in a
dose-dependent manner to the amount observed with raloxifene alone,
demonstrating that
raloxifene induction is weaker than induction by EZ and that raloxifene-
induced
20 transactivation results from binding to ERa. If EZ is classified as a full
activator of ERa
at an AP 1 element (ERac-AP 1 ), then raloxifene fimctions as a partial
activator and
tamoxifen functions as a fill activator.
In contrast to the results seen with ERa-AP1, a difference in the ligand
activation profile of ER[3 at an AP1 element (ER~i-AP1) was observed. In cells
25 transfected with ER[i, treatment with the estrogens EZ and DES did not
increase luciferase
transcription over the control (no ligand added), whereas treatment with the
antiestrogens
ICI 164384, raloxifene, and tamoxifen increased luciferase transcription (Fig.
4A). This
transcription activation required transfected ER(I, as wells that were
transfected with only
the reporter plasmid did not show transcriptional activation by the
antiestrogens. The
30 transcriptional activation caused by raloxifene was dose dependent with an
ECM value of
CA 02301143 2000-02-17
WO 99/11760 PCT/US98I18030
36
0 about 50 nM (Fig. 4B). In ligand competition experiments, both E2 and DES
were able
to block the raloxifene induction, and both estrogen ligands were able to
reduce raloxifene
induction to the basal level of transcription in a dose-dependent manner with
concentration values required for one-half maximal inhibition of 1 to 10 nM
(Fig. 4C).
In a different ligand competition experiment, the inln'bitory effect on
transcription resulting from EZ treatment could be overcome by higher
concentrations of
raloxifene in a dose-dependent manner (Fig. 4D). Thus, it appears that the
pharmacology
of ER ligands is reversed at an AP 1 element with ER(3; with F.R~i-AP 1, the
antiestrogens
act as transcription activators, and the estrogens act as transcription
inhibitors.
It was next investigated whether the action ofER[3-AP 1 could be observed
in cell lines derived from estrogen target tissues such as the uterus and
breast.
Transactivation assays for ERj3-AP1 were performed in Ishikawa cells (a human
uterine
cell line) (Fig. SA) and in MCF7 (Fig. 5B) and IVIDA453 (Fig. Sc) human breast
cancer
cells. (The human ERA was used for transactivation in these cells.) In each of
these cell
lines, the ligands acted the same as they did in the HeLa cells; the thr~
antiestrogens
activated and the estrogens inhibited ER(3-dependent transcription from an APl
site (Fig.
5). No induction was seen with cells that were not transfected with the ER.~
expression
plasmid, indicating that the antiestrogen induction required ER(3.
Antiestrogen induction
in the breast cell lines was higher than that observed in HeLa cells.
Transfected MCF7
cells treated with raloxifene gave a 20- to 80-fold transactivation response
over the
control (no ligand added). In addition, raloxifene and ICI 164384 induced
transcription
more than tamoxifen in the breast cell lines(Fig. 5, B and C).
MCF7 cells did not appear to contain high concentrations of endogenous
ER(i mRNA (Kuiper et al. (1997) Endocri»ol., 138: 553); however, the results
suggest
that the additional transactivation machinery required for ERA-AP 1 function
is present in
these cells. With two of these target tissue cell lines, E~ treatment reduced
the amount of
transcription to less than that seen with the control (no ligand added). In
MDA453 (Fig.
SC) and Ishikawa cells (Fig. SA), E~ treatment resulted in a consistent 40 to
75%
reduction of reporter transcription levels compared with the control. This
effect was also
observed in ligand competition experiments (Fig. 5, A and C); EZ and DES
blocked
raloxifene induction and reduced the amount of transcription to less than that
seen for the
CA 02301143 2000-02-17
WO 99/11760 PCT1US98/18030
37
0 control. Thus, when ERA is bound by the estrogen hormone E2 or the synthetic
estrogen
DES, it functions as a negative regulator of genes controlled by an ER
dependent AP1
element.
The ER is the only known member of the steroidal subfamily of nuclear
receptors that has different subtypes (Mangeldorf et al. (1996) Cell, 83: 835-
839).
Nuclear receptors that respond to nonsteroidal hormones that have different
known
subtypes include the thyroid receptor {TRa and TR~i), the retinoic acid
receptor (RARa,
RARE, and RARY), and the retinoid X receptor (RXRa, RXR[3, and RXRy)
(Mangelsdorf
et al. (1996) Cell, 83: 841-850). The results presented herein demonstrate
that two
nuclear receptor subtypes can respond in opposite regulatory modes to the
natural
hormone from the same DNA response element. Moreover, the ligand-induced
responses
with ER(3 at an AP 1 site provide an example of negative transcriptional
regulation by the
natural hormone and strong positive regulation by synthetic antiestrogens.
(The genes for
transforming growth factor and quinone reductase are ER regulated genes
controlled by
promoters containing nonclassical EREs that are activated by antiestrogens.
However,
the action of ERA at either of these promoters has not been reported. The
action of ERa
on the quinone reductase gene shows a similar ligand profile to that of ERA at
an AP1
site; antiestrogens are transcription activators, and E= is a transcription
inhibitor.
If signaling from ER dependent AP 1 elements occurs in estrogen target
tissues, the finding herein that ERa and ER[3 respond differently to ligands
at AP1 sites
reveals a potential control mechanism for transcriptional regulation
ofestrogen-responsive
genes and adds a layer of complexity in analyzing the pharmacology of
antiestrogen
therapeutics. The role of E2 completed to ER(3 would be to turn off the
transcription of
these genes, whereas the antiestrogens raloxifene, tamoxifen, and ICI 164384
could
override this blockade and activate gene transcription. It wil! be T helpful
to search for
genes in estrogen target tissues that are transcriptionally regulated by ER[3
at an AP 1 site
and to characterize the phenotype of cells in which these genes are activated.
It is understood that the examples and embodiments described herein are
for illustrative purposes only and that various modifications or changes in
light thereof will
be suggested to persons skilled in the art and are to be included within the
spirit and
CA 02301143 2000-02-17
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38
0 purview of this application and scope of the appended claims. All
publications, patents,
and patent applications cited herein are hereby incorporated by reference.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: THE REGENTS OF THE UN111ERSiTY OF CALIFORNIA
(ii) TITLE OF INVENTION: DIFFERENTIAL LIGAND ACTIVATION OF
ESTROGEN RECEPTORS ERalpha AND ERbeta AT AP1 SITES
(Iii) NUMBER OF SEQUENCES: 6
(iv) OORRESPOIiDENCE ADDRESS:
(A) ADDRESSEE: Fulbri9ht i ,lerarski L.L.P.
(8) STREET: 865 S. FiQueroa Street, 29'" Floor
(C) CITY: Los A~pelee
(D) STATE: Cslifornia
(E) COWITRY: USA
(F) ZIP: 90017-2576
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC caa~pstible
(C) OPERATING SYSTQI: PC-DOS/MS-DOS
(D) SOFTWRE: Patentln Release d1.0, Version 51.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION N<18:ER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: BERLINER, Robert
(B) REGISTRATI011 NtlIIBER: 20,121
(C) REFERENCE/DOCKET NUMBER: 5555-497
(ix) TELECOIIIUNICATION INFORMATIO11:
(A) TELEPIIOIIE: 213-892-9200
(B) TELEFAIf: 213-680-4518
(2) INFORMATION FOR SE0 tD N0:1:
(i) SEQUEtICE CHARACTERISTICS:
(A) LENGTH: 7 base pain
(B) TYPE: nucleic said
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (oenoinic)
(ix) FEATURE:
(A) NAIIE/KEY: -
(B) LOCATI011: 1..7
(D) OTHER INFORMATION: /note~ «AP1 response elaiaent"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
TGASTCA 7
(2) INFORMATI011 F0lt SE0 ID 110:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
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39
(C) STRANDEDNESS: sin4le
(D) TOPOLOGY: linear
(ii) lbLECULE TYPE: DNA (~noaiie)
(ix) FEATINtE:
(A) NIWE/KET: -
(8) LOCUTION: 1..15
(D) OTHER INFORMATION: /note" "ERE fraa~the Xenopta vitellopenin A2 Gene"
(xi) SEqJENt~ DESCRIPTIOIIs SE0 ID 110:2:
AGGTCAGGT GACCT 15
(2) INFORMATION FOR SEG ID N0:3:
(i) 5E4VENCE CHARACTERISTICS:
(A) LENGTH: 2568 base pairs
(B) TYPE: nucleic acid
(C) STRAIDEDNESS: side
(D) TOPOLOGY: linear
(ii) MO<.ECULE TYPE: DNA (9eno~ie)
(ix) FEATURE:
(A) NAI~/KEY: CDS
(B) LOGTION: 424..1878
(D) OTHER INFORMATION: /note" "Il~ino acid sequence of a
rat ERbeta"
(xi) SEGIH:NCE DESCRIPTION: SE0 ID N0:3:
GGMTTTCGG GGCIIGCTCGC CCAGGGGGAG CGGCtGGTGC60
TGCGCTGGC ATCCCTAGGC
ACCGGGTCT GCMTAAAGT CTGGCAGCG CTGGTGGCT 120
GAGCGACAJIC GGTGaCTGG
GAGTCCGGCT CTGTGfiCTGA GGMAGCACC TGTCTGGTT180
TAG11GMTGC AMATIIGAGII
ATGTTTACCT GCGGTGTT AGTCTGAGT CCGTGAGTC 240
tCTGIIGAAG TMTGTCGT
CTGTACCTCT TCTGCMGG AGTTTTCTG GCTGCGACCC 300
TCTGAAGIIG TGGAGATCM
AMCTGCCG TCGIIGt'.CTTA GTtCCCTGCT TCCTATMCT360
GTAGCGGTC GTCCTACCC
CTGGAGCACG GCCCGTCTA GTCCCTTCC TCCTACGTAGi20
ACMCCGCG TGAGTATTG
GCT ATG AG TTC TAC AGT CCT GCT GTG ATG <68
MC TAC AGT GTt CCC GGC
Met Thr Phe Tyr Ser Pro Ala Val Net Asn
tyr Ser Val Pro Ely
1 5 10 15
AGC ACC AGT MC CTG GAC GGt GGG CCT GTC 516
CGA CTG AGC AG AGC CG
Ser Thr Ser Asn Leu Asp Gly Gly Pro Vat
Arp Leu Ser Thr Ser Pro
25 30
MT GTG CTA TGG CG ACT TCT GGG CAC CTG 5b4
TCT CCT TTA GCG ACC GT
Asn Val Leu Trp Pro Thr Ser Gly His Leu
Ser Pro Leu Ala Thr His
35 40 45
TCC CM TG TCG CTC CTC TAT GG GM CCT CM 612
MG AGT CCt TCG TGT
Cys Gtn Ser Ser Lnu Leu Tyr Ala Glu Pro
Gln Lys Ser Pro Trp Cys
50 55 60
GM GCA AGA TG CTA GAG GC ACC TTA CCT GTA 660
MC AGA GA6 AG CTG
Glu Ala Arp Ser Leu Glu His Thr Leu Pro
Yal Asn ArS Glu Thr Leu
65 70 75
MG AGG AAG CTT AtiT GGG AGC AGT TGT GCC 708
AGC CCT GTT ACT AGT CG
Lys Arp Lys Leu Ser Gly Ser Ser Cys Ala
Ser Pro Vat Thr Ser Pro
80 85 9p g5
CA 02301143 2000-02-17
wo ~nm6o rcrnrsmeo3o
MC GG M6 AGG GAT GCT CAC TTC TGC CCC 756
GTC TGC AGC GAT TAt 6G
Asn Als Lys Arg Asp Ala Nis Phe Cys pro
Val Cys Ser Asp Tyr Als
110
TCT GGG TAT GT TAC GGC GTT TGG TG TGT 804
GM GGf1 TGT MG GCC tTT
Ser Gly Tyr Nis Tyr Gly Val Trp Ser Cys
Glu Gly Cys Lys Als Phe
115 120 125
TTT AM AGA AGC ATT GA GGA GT MT GAT TAT 852
ATC TGT CG GCC ACG
Phe Lys Arg Ser Ile Gln Gly Nis Asn Asp
Tyr Ila Cys Pro Ala Thr
130 135 140
MT CAG TGT ACC ATA GAC MG MC CGG CGT 900
AM AGC TGC GG GCC TGC
Asn Gln Cys Thr ile Asp Lys Asn Arg Arg
Lys Ser Cys Gln Als Cys
145 150 155
CGA CTT CGC MG TGT TAT GM GTA GGA ATG 948
GTC MG TGT GGi1 TCC AGG
Arg Leu Arg Lys Cys Tyr Gtu Vat Gly Net
Val Lys Cys Gly Ser Arg
160 165 170 175
AGA GM CGG TGT GCG TAC CGT ATA GtG CGG 996
AGG GG AGA AGT TCT AGC
Arg Glu Arg Cys Gly Tyr Arg Ile Val Arg
Arg Gln Arg Ser Ser Ser
180 185 190
GAG G6 GTA GC TGC CTG AGC AM GCC MG AGA 1044
MC GGT GGG GT GG
Glu Gln Yal His Cys Leu Ser Lys Ala Lys
Arg Asn Gly Gly His Ala
195 200 205
CCC CGG GTG AAG GAG CTA CTG CTG AGC ACC 1092
TTG AGT CG GAG ClU1 CTG
Pro Arg Val Lys Glu Leu Leu Lsu Ser Thr
Leu Ser Pro Glu Gln Leu
210 215 220
GTG CTC ACC CTC CTG GM GCT GM CG CCC 1146
Mt GTG CTG GTG AGC CGT
Vel Leu Thr Leu Leu Glu Ala Glu Pro Pro
Asn Val Leu Val Ser Arg
225 ~0 ~5
CCC AGC ATG CCC TTC ACC GAG GCC TCC ATG 1188
ATG ATG tCC CTC ACT MG
Pro Ser Net Pro Phe Thr Glu Ala Ser Met
Net Net Ser Leu Thr Lys
240 245 250 ~5
CTG GCG GAC MG GM CTG GTG GC ATG ATT 1236
GGC TGG GCC MG AM ATC
Leu Als Asp Lys Glu Lw Vat Nis Net Ile
Qty Trp Als Lys Lys Ile
2~ 265 270
CCT GGC TTT GTG GAG CTC AGC CTG TTG GAC 1284
CM GTC CGG CTC TTA GM
Pro Gly Phe Val Glu Lau Ser Leu Leu Mp
Gln Val Arg Leu Leu Glu
275 280 285
AGC TGC TCG ATG GAG GTG CTA ATG GTG GGA 1332
CTG ATG TGG CGC TCC ATC
Ser Cys ~ Net Glu Val Leu ~t Val Gly
Leu Net
Arg Ser Ile
3~
GAC GC txC GGC MG CTC ATT TTC GCT CCC 1380
GAC CTC GTT CTG GAC AGG
Asp His Pro Gly Lys Leu lle Phe Ala Pro
Asp Leu Val Leu Asp Arg
3~ 310 315
GAT GAG GGG MG TGC GTA GM GGG ATT CTG 1428
GM ATC TTT GI1C ATG CTC
Asp Glu Gly Lys Cys Val Gtu Gly Ile Leu
Glu Ile Phe Asp Net Leu
325 330 335
CTG GCG ACG ACG TG AGG TTC CGT GAG TTA 1476
AM CTC GG GC MG GAG
Leu Ala Thr Thr Ser Arg Phe Arg Glu Leu
Lys Leu Gln His Lys Glu
345 350
TAT CTC TGT GTG MG GCC ATG ATC CTC CTC 1524
MC TCC AGT ATG TAC CCC
Tyr Leu Cys Val Lys Ala Net Ile Leu Leu
Asn Ser Ser Net Tyr Pro
355 ~0 ~5
TTG GCT TCT GG MC CAG GAG GG GM AGT AGC 1572
CGG MG CTG AG GC
Leu Ala Ser Ala Asn Gln Glu Ata Glu Ser
Ser Arg Lys Leu Thr Nis
370 375 380
CTA CTG MC GCG GTG AG GAT GCC CTG GTC TGG GTG ATT GCG MG AGT 1620
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Leu ~ Asn Ala Vat Thr A~ Ale Leu Yal
Trp 3~ Ile Als Lya Ser
GGT ATC TCC tCC GG CAG GG TG GTC CGA 1668
CTG GCC MC CTC CtG ATG
Gly Ile Ser Ser Gln Gln Gln Ser Yal Arg
Leu Ala Asn Leu Lau !!et
400 405 410 415
CTT CTT TCT CAC GTC AGG CAC ATC AGT MC 1716
MG GGC ATG GM GT CTG
Leu Leu Ser Nis Yal Arg Nis Ile Ser Asn
Lys Gty Met Glu His Leu
420 425 430
CTC AGC ATG MG TGC AM MT GTG GTC CCG 1764
GtG TAT GAC CTG CTG CTG
Leu Ser Met Lys Cys Lys Mn Vat Vat Pro
Vel Tyr Asp Leu Leu Leu
435 440 445
GAG ATG CTG MT GCT GC ACG CTT CGA GGG 1812
TAC AAG TCC TG ATC TCG
Glu lief Leu Asn Ala Nis Thr Leu Arg
Gly Tyr lys Ser Ser Ile Ser
450 455 460
GGG TCT GAG TGC AGC TG AG GAG GAC AGT 1860
MG MC AM GAG AGC TCC
Gly Ser Glu Cys Ser Ser thr Glu Asp Ser
Lys Asn Lys Glu Ser Ser
465 470 475
GG MC CTA CAG TCT GG TG11TGGCGG GCCTGAGGCG1908
GAGGACTAC
Gln Asn Leu Gln Ser Gln
480 485
AGAGATGGTC MMGTGGM GTGtACCCT AGGTCTGGG 1968
GGTTCCTCTT AGGGCTGCCT
TGGTTACGG CCCCTTACCC AGCTGGCT TCCGGGAGT 2028
GGGGTGGTT GTGTGGCGGT
GTTCCTGTA CGGGATGTA CGCCWATG CGAGTTCTA 2088
ACTTGTATAG CCTTGAI1GGC
TCTCGGTGTA CTTACTTTCT GTCTCCTTGC CGCTTGGAII2148
AGTCTGAM GGTTCTGGM
CTAMGGTG MGTCTGATT TGGAACGlITT GTCCTTAGTC2208
AGGJ1AMGGA ATATGGGTG
TGAGGGCT ATMGMATG GACTGTAGGA CTGTGTGGCC 2268
ATAMATCAA CCTTTGG11TG
GCGTCTTCTA GACGCTTGA TTGTAGGATT GMMCGG 2328
TTGAGATG GCTGTTTCG
GTTCCTGCC TCACGGGTCT GTGAGGACTC ATTMTGTG2388
TGGGTTATTC TATGMGAC
CAGMlIGATA GTGCAAGCTT AGATGTACCT TGTTCCTCCT2448
CCGGACCCT TGGGTTAGT
CCTTAGIIGCC TGCTTATTTG GTCTGTCTGA ATGTGGTGT2508
TGTGTGGGT TMGATTTM
ATCTCTTTGT MTATTGGCT TCCTTGAAGC TATGTGTCT2568
TTCTCTCTCT CCCGGMTTT
(2) INFORMATION FOR SEQ ID N0:4:
<i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 485 amino acids
tB) TTPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(ix) FEATtNtE:
(A)
(B) -
(D) OTHER INFORMATION: /note= "Amino acid seyue:xe of a
rat ERbeta"
(xi ) SEQUENCE DESCRIPTt011: SEQ 10 110:4:
Met Thr Phe Tyr Ser Pro Ala Val Met Asn Tyr Ser Val Pro Gly Ser
1 5 10 15
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Thr Ser Asn Lau Asp Gly Gly Pro Val Arg Leu Ser Thr Ser Pro Asn
20 25 30
Yat teu Trp Pro Thr Ser Gly His Leu Ser Pro Leu Ala Thr His Cys
35 40 45
Gln Ser Ser Lau Leu Tyr Ala Glu Pro Gln tys Ssr Pro Trp Cys Glu
5o s5 bo
Ala Arg Ser Leu Glu His Thr Leu Pro Vat Asn Arg Gtu Thr Leu Lys
65 70 75 gp
Arg Lys Lau Ser Gly Ser Ser Cya Ala Ser Pro Vsl Thr Ser Pro Asn
85 90 95
Ala Lys Arg Asp Ata Nis Phe Cys Pro Val Cys Ser Asp Tyr Ata Ser
105 110
G!y Tyr His Tyr Gly Val Trp Ser Cys Glu Gly Cys Lys Ata Phe Phe
115 120 125
Lys Arg Ser Ile Gtn Gly His Asn Asp Tyr Ile Cys Pro Ala Thr Asn
130 135 140
Gln Cys Thr ile Asp Lys Asn Arg Arg Lys Ser Cys Gln Ala Cys Arg
145 150 155 160
Leu Arg Lys Cys Tyr Glu Val Gly list Val Lys Cys Gly Ssr Arg Arg
165 170 175
Glu Arg Cys Gly Tyr Arg Ile Val Arg Arg Gln Arg Ser Ser Ser Glu
180 185 190
Gln Val His Cys Leu Ser Lys Ala Lys Arg Asn Gly Gly His Ala Pro
195 200 205
Arg Vat Lys Glu Leu Leu Lsu Ser Thr Leu Ser Pro Glu Gtn Leu Val
210 215 220
Lau Thr Lsu Leu Glu Ale Glu Pro Pro Asn Vat Leu Val Ser Arg Pro
2Z5 230 Z35 240
Ser list Pro Phe Thr Glu Als Ser Het Met list Ser Leu Thr Lys Leu
245 250 255
Ala Asp lys Glu Leu yal His Het Ile Gly Trp Ala Lys Lys Ile Pro
265 270
Gly Phe Vsl Glu Leu Ser Leu Lau Asp Gln Val Arg Leu Leu Glu Ser
275 280 285
Cys ~ filet Glu.Val Lsu ~t Val Gly Leu Net Trip Arg Ser Ile Asp
His Pro Gly tys Leu Its Phe Ale Pro Asp Leu Val Leu Asp Arg Asp
305 310 315 320
Glu Gly Lys Cys Val Glu Gly Its Leu Glu Ile Phe Asp Net Leu Leu
325 330 335
Ata Thr Thr Ser Arg Phe Arg Glu Leu Lys Lsu Gln Nis Lys Glu Tyr
340 345 350
Leu Cys Vat Lys Ala Net file Leu Leu Asn Ssr Ser Net Tyr Pro Leu
355 360 365
Ala Ssr Ala Asn Gln Gtu Ale Glu Ser Ser Arg Lys Lau Thr His Leu
370 375
Leu Asn Ala Val Thr Asp Ala Leu Val Trp Val Ile Ala Lys Ser Gly
3~ 395 400
Ile Ser Ser Gln Gln Gln Ser Vat Arg Leu Ala Asn Leu Leu Net Leu
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4~ 410 415
Leu Ser His Val Arg His Ile Ser Asn Lys Gly Met Glu His Leu Leu
4~ 425 430
Ser llet Lys Cys Lys Asn Val Val Pro Vat Tyr Asp Leu Leu Leu Gtu
435 440 445
Ilet Leu Asn Ala His Thr Leu Arg Gly Tyr Lys Ser Ser Ile Ser Gly
450 455 460
Ser Glu Cys Ser Ser Thr Glu Asp Ser Lys Asn Lys Glu Ser Ser Gln
465 4T0 475 480
Asn Leu Gln Ser 6ln
485
(2) INFORNATIOM FOR SE0 1D 110:5:
(i) SEOUEMCE CHARACTERISTICS:
(A) IEMGTN: 485 amino acids
(8) TYPE: amino acid
(C) STRANDEDI~SS: uNuwxn
(D) TOPOLOGr: ur~nown
(ii) MOLECULE TYPE: protein
(ix) FEATURE:
(A)
(8) LOCAT1011: 1..485
(D) OTHER IMFORMATI011: /note. ~Mino acid sequence of
huasn ERbeta~
(xi) SEQUENCE DESCRIPTION: SE0 ID N0:5:
Met Thr Phe Tyr Ser Pro Ala Val Met Asn Tyr Ser Ile Pro Ser Asn
1 5 10 15
Vat Thr Asn Lau Gtu Gly Gly Pro Gly Arg Gln Thr Thr Ser Pro Asn
20 25 30
Val Leu Trp Pro Thr Pro Gly Nis Leu Ser Pro Leu Val Val His Arg
35 40 45
Gln Leu Ser Nis Lau Tyr Ala Glu Pro Gln Lys Ser Pro Trp Cys Glu
50 55 60
Ala Arg Ser Leu Glu His Thr Le~u Pro Val Asn Arg Glu Thr Leu Lys
65 70 75 80
Arg Lys Val Ser G~ly Asn Arg Cys Ala ~r Pro Val Thr Gly ~ Gly
Ser Lys Arg Asp Ala Nis Phe Cys Ala Vsl Cys Ser Asp Tyr Ala Ser
100 105 110
Gly Tyr Nis Tyr Gly Vsl Trp Ser Cys Glu Gly Cys Lys Ala Phe Phe
115 120 125
Lys Arg Ser Ile Gln Gly His Asn Asp Tyr Ile Cys Pro Ala Thr Asn
130 135 140
Gln Cys Thr ile Asp Lys Asn Arg Arg Lys Ser Cys Gln Ala Cys Arg
145 150 155 160
Leu Arg Lys Cys Tyr Glu Val Gly Ilet Val Lys Cys Gly Ser Arg Arg
165 170 175
Glu Arg Cys Gty Tyr Arg Leu Vsl Arg Arg Gln Arg Ser Ala Asp Glu
180 185 190
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Gln Leu ;ids, Cys Ala Gly Lys A2l~a Lys Arg Ser Gly Gly Nis Ala Pro
205
Arg Val Arg Glu Leu Leu Leu Asp Ala Leu Ser Pro Glu Gln Leu Yal
210 215 220
Leu Thr Leu Leu Glu Ala Glu Pro Pro Nis Vsl leu ile Ser Arg Pro
225 230 235 240
Ser Ala Pro Phe Thr Glu Ala Ser Ilet Met Met Lau Ser Thr Lys Leu
265 250 255
Ala Asp Lys Glu leu Val His Met Ile Ser Trp Ala Lys Lys 1le Pro
2~ 265 270
Gly Phe Val Glu Leu Ser Leu Phe Asp Gln Val Arg Lou Leu Glu Ser
275 280 285
Cys Trp Met Glu Val Leu Met ltet Gly Leu Met Trp Arg Ser Ile Asp
2~ 295 300
His Pro Gly Lys Leu Ile Phe Ala Pro Asp Lau Val Leu Asp Arg Asp
305 310 315 320
Glu Gly Lys.Cys Vel Glu Gly Ile Leu Glu Ile Phe Asp Met Leu Leu
325 330 335
Ala Thr Thr Ser Arg Phe Arg Glu Leu Lys Leu Gln His Lys Glu Tyr
340 345 350
Leu Cys Val Lys Ala Met Ile Leu Leu Asn Ser Ser Net Tyr Pro Leu
355 360 365
Val Thr Ala Thr Gln Asp Ala Asp Ser Ser Arg Lys Leu Ale Nis Leu
370 375 380
Leu Asn Ala Val Thr Asp Ala Leu Vat Trp Vat Ile Ala Lys Ser Gly
385 390 395 400
Ile Ser Ser Gln Gln Gln Ser Met Arg Lau Ale Asn Leu Leu Met Leu
405 410 415
Leu Ser Nis Val Arg His Ala Ser Asn Lys Gly Ilet Glu Nis Leu Leu
420 425 430
Asn Met Lys Cys Lys Asn Vsl Val Pro Vat Tyr Asp Leu Leu Leu Glu
435 440 445
Met Leu Asn Ala His Val Leu Arg Gly Cys Lys Ser Ser Ile Thr Gly
450 455 460
Ser Glu Cys Ser Pro Ala Glu Asp Ser Lys Ser Lys Glu Gty Ser Gln
465 470 475 480
Asn Leu Gln Ser Gln
485
(2) 1HFORMATI011 FOR SEQ ID 110:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1460 bees pairs
(B) TYPE: nucleic scid
(C) STRANDEDHESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genoaic)
(fx) FEATURE:
(A) NAME/ICEY: -
(B) LOCATIOM: 1..1460
(D) OTHER IMFORMATION: /notes "DNA sequence of huaen
ERbeta"
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WO 99/11760 PCT/US98/18030
(xi) SEDIIENCE DEixRIPTI0fl:, SEa tD
N0:6:
CTATGAGTT CTAGGTCCT GCTGTGATGA ATTACAGGT60
TCCGGCMT GTCACTAACT
TGG~AGGTGG GCCTGGTCGG GGIICCACAA GCCCAAIITGT120
GTTGTGGCG ACACCTGGGC
ACCTTTCTCC TTTAGTGGTC GTCGCGGT TATGGTCT 180
GTATGCGGA11 CCTGAAAGA
GTCCCTGGTG TGAAGCMGA TCGCTAGAJ1C AGCCTTACC240
TGTAAACAGA GAGAGCTGA
AAAGGMGGT TAGTGGGMC CGTTGCGCG GCCCTGTTAC300
TGGTCGGGT TCAAAGI1GGG
ATGCTGCTT CTGCGCTGTC TGCJIGCGATT ACGGTCGGG360
ATATGCTAT GGAGTCTGGT
CGTGTGAAGG ATGTAAGGCC TTTTTTAAM GAAGGGGG420
AGGAGTAAT GATTATATTT
GTCCAGCTAC AMTCAGTGT ACMTCGATA AAAACCGGCG480
CAAGAGCTGC GGGCCTGCC
GACTTCGGM GTGTTACGM GTGGGAATGG TGAAGTGTGG540
CTCCCGGAGA GAGAGIITGTG
GGTACCGCCT TGTGCGGAGA GGAGMGTG CCGACGAGG600
GCTGGCTGT GCCGGGAGG
CGAGAGMG TGGCGGCGC GCGCCCCGAG TGCGGGAGCT660
GCTGCTGGAC GCCCTG11GCC
CCGAGGGCT AGTGCTGCC CTCCTGGJ1GG CTGAGCCGCC720
CGTGTGCTG ATGGCCGCC
CGGTGCGCC CTTGCCGAG GCCTCGTGA TGATGTCCCT780
GACCAAGTTG GCCGACAAGG
AGTTGGTAG GTGATGGC TGGGCCA11GA AGATTCCCGG840
CTTTGTGGAG CTGGCCTGT
TCGACCMGT GCGGCTCTTG GAGAGCTGTT GGATGGAGGT900
GTTAATGATG GGGCTGI1TGT
GGCGCTGAT TGACGCCCC GGGAGCTG TCTTTGCTCC 960
AGATCTTGTT CTGGAGGGG
ATGAGGGGAA ATGCGTAGAA GGAATTCTGG AAATCTTTGA1020
GTGCTCCTG GCA11CTACTT
GAGGTTTCG AGIIGTTAAAA CTCCMGG AAGMTATCT 1080
CTGTGTCAAG GCGTGATCC
TGCTCMTTC GGTATGTAC CCTCTGGTCA GGCGACCG 1140
GGI1TGCTGAC AGGGCCGGA
AGCTGGCTG CTTGCTGMC GCCGTGACCG ATGCTTTGGT1200
TTGGGTGATT GCCAAGAGCG
GCATCTCCTC CGGGGCM TCGTGCGCC TGGCTAACCT 1260
CCTGATGCTC CTGTCCGCG
TGGGGTGC GAGTAAGAG GGGTGGAAC ATCTGCTCAA 1320
GTGAAGTGC AAAAATGTGG
TCCGGTGTA TGACCTGCTG CTGGAGATGC TGMTGCCG1380
CGTGCTTCGC GGGTGCAAGT
CCTCGTGC GGGGTCCGAG TGCAGCCCGG GGAGGAGG 1440
TAAMGCAAA GAGGGCTCCC
AGAACCTAG GTCTGGTGA
