Note: Descriptions are shown in the official language in which they were submitted.
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BIOASSAY FOR IDENTIFYING ESTROGEN RECEPTOR-~i/oc SELECTIVE
MODULATORS
Technical Field
The present invention relates to hormone receptors, and, more
particularly to methods for identifying compounds that selectively activate
estrogen receptors (ER) of the alpha or beta subtype, as well as a test kit
for use in the methods.
Background of the Invention
Proteins which regulate gene expression are essential for cell
function. A well-studied family of gene regulatory proteins is the steroid
hormone receptor superfamily. These receptor proteins enable cells to
respond to various hormones by activating or repressing specific genes.
One member of this family is the ER. Estrogens are classically known as
important hormones in sexual development and reproductive function. It
is well known that estrogens affect cell proliferation and differentiation in
target tissues by binding to ERs in target cells. Estrogen replacement
therapy is a well established treatment for prevention and/or amelioration
of osteoporosis in postmenopausal women (Sagraves, 1995,; Lobo, 1995)
because these compounds have been demonstrated to prevent bone
loss and fractures in women. Additionally, estrogen replacement therapy
has been associated with a decreased mortality from cardiovascular
disease. Finally, ongoing studies suggest that estrogens may provide
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2
benefit to the central nervous system with regard to cognitive
improvement and a decrease in Alzheimer's disease.
The classic ER, now designated ER-a, exhibits a modular structure
with distinct domains involved in ligand binding, receptor dimerization,
DNA binding, and transcriptional activation (Green et al, 19861. The
nucleotide sequence of the DNA binding domain is conserved among
steroid hormone receptors. Kuiper et al ( 1996) exploited this similarity in
an attempt to identify additional members of this family. The new
member they discovered was recognized to be an ER because the amino
acids in the DNA binding domain were almost identical to those of ER-a, ,
the in vitro translated protein specifically bound [3H]-estradiol with
nanomolar affinity and was able to regulate transcription from a simple
estrogen response element. This protein has been designated ER-(3 to
distinguish it from the previously known form Inow called ER-a). Human
and mouse ER-(3 have also been cloned Bhat et.al. 1998; Mosselman,
1996; Pettersson et al, 1997; Tremblay et al, 1997). Following ER-(3's
discovery, most work has focused on mapping the distribution of its
mRNA in normal and neopfastic tissues, characterizing its binding affinity
for a wide variety of ligands, and assessing its interaction with ER-a.
ER-~3 mRNA is detectable by RT-PCR and in situ hybridization in a
wide variety of tissues. While its distribution overlaps that of ER-a, some
tissues express only one receptor type. For example both receptors are
found in the uterus, ovary and pituitary, whereas ER-~3 appears more
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3
prominent in the prostate, lung and bladder (Kuiper et al, 1996). When
examined by in situ hybridization, further distinctions between receptor
distribution can be made. ER-~i mRNA is expressed in the epithelial cells
of the rat prostate; ER-a is found in the stromal compartment (Kuiper et
al, 1996). In the rat ovary, ER-(3 appears in the granulosa cells; ER-a in
the stroma (Shughrue et al, 1996; personal communication). In the rat
hypothalamus, ER-~3 but not ER-a, message is expressed in the
paraventricular region, whereas both messages are seen in the preoptic
area (Shughrue et al, 19961. Interestingly, significant species differences
may exist in the relative levels of ER-(3 and ER-a mRNA in certain organs.
For example, ER-(3 mRNA is highly expressed in the rat prostate, whereas
more modest levels are detected in the human prostate. ER-/3
predominates over ER-a in the rat prostate (Kuiper et al, 1996; Lau et al,
1998; Enmark et al, 1997), but the levels are more equal in the mouse
prostate (Couse et al, 19971.
Several groups have characterized ER expression in tumor samples
and cancer cell lines. Most work has been done in the breast where one
study reports ER-~i mRNA using RT-PCR in 70% of forty breast biopsy
samples (Dotzlaw et al, 1996). No correlation was noted between ER-a
and ER-(3 expression. Another study also reports heterogeneity of ER-a/~i
mRNA expression in breast tumor samples, with some tumors having only
ER-a mRNA and others expressing mRNA for both receptor subtypes
(Enmark et al, 1997). Among breast cancer cell lines, MDA MB 231 has
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4
only ER-~3 mRNA, and conflicting reports exist about the ER status of
MCF-7 cells (Enmark, 1997; Kuiper et al, 1997; Vladusic et al, 1998; R.
Henderson, unpublished observationsl.
Although the figand binding domain of human ER-a and ER-~i are
59% identical at the amino acid level (Enmark et al, 1997), the binding
affinity of 17(3-estradiol is quite similar. Some compounds show marked
selectivity for either ER-a or ER-~3. Genistein is a phytoestrogen which
binds with approximately 10-25 fold higher affinity for ER-(3 (Kuiper 1996,
H. Harris unpublished observations). On the other hand raloxifene binds
about 20 fold better to ER-a than ER-(3 (H. Harris, unpublished
observations).
When ER-a and ER-~i are coexpressed , interaction can be
measured by several methods including a mammalian two-hybrid system,
glutathione S transferase pulldown and gel shift/supershift assays
(Cowley et al, 1997; Pettersson et al, 1997; Ogawa et al, 19981. Since
these receptors can heterodimerize, estrogens' effect on tissues
containing both receptors may be mediated by a complex interaction
between ER-a and ER-Vii.
One valuable tool in determining the function of ER-(3 is the
estrogen receptor-a knockout (ERKO) mouse (Lubahn et al, 1993).
Because these mice lack functional ER-a, they can help define the
physiological roles of both ER-a and ER-Vii. One study has compared the
effectiveness of 17-~i estradiol treatment in ameliorating consequences of
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artificially induced vascular injury to carotid arteries in wild type and
ERKO mice (lafrati et ai, 19971. In both types of mice, pharmacological
doses of 17-(3 estradiol suppressed the increase in medial area and
smooth muscle cell proliferation seen in the vehicle treated d animals. It
5 is thought that these responses to endothelial denudation may narrow the
lumen of the vessel, thus restricting blood flow. Because 17-(3 estradiol
was equally effective in ERKO as wild type mice, one interpretation is that
ER-a is not necessary for this response. Because ER-(3 mRNA is also
expressed in these vessels, perhaps it mediates 17-(3 estradiol's action.
However, direct evidence supporting this hypothesis is lacking. Another
example of ERKO mice responding to 17-(3 estradiol replacement was
described in a recent poster and abstract (Pan et al, 1997, and personal
communication). This study reports a loss of femoral bone mineral
density and trabecular bone volume in ERKO mice after ovariectomy and
an increase in these parameters upon treatment with 17-~3 estradiol (but
not dihydrotestosteronel. Again, based on indirect evidence, a
suggestion is made that ER-/3 has a physiological role.
Thus, while certain aspects of ER-~i have been characterized, the
art fails to provide methods for identifying ligands which are functionally
selective for ER-a or ER-j3. Such an assay would be greatly advantageous
to the pharmacological industry to uncover the possible therapeutic
applications of the ER subtypes.
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6
Summary of the Invention
The present invention provides method for screening a test
compound that binds to an ER in a receptor binding assay, wherein said
method detects ER-(3 polypeptide-mediated transcription, said method
comprising the steps of:
(a) providing a cell which comprises at least one estrogen-induced
DNA sequence encoding metallothionein IMT-II) and at least one DNA
sequence encoding ER-p polypeptide, wherein said receptor is
transcriptionally active;
(b) contacting said cell with either said test compound which binds
ER or a control; and
(c) detecting the expression of said MT-II, wherein enhanced
expression of said MT-II relative to a control indicates that said test
compound has estrogen agonist activity.
This invention further provides a method for screening a test
compound that binds to the ER in a receptor binding assay, wherein said
method detects inhibition of ER-(3 )- polypeptide-mediated transcription,
said methad comprising the steps of:
(a) providing a cell which comprises at least one estrogen-induced
DNA sequence encoding metallothionein (MT-II) and at least one DNA
sequence encoding ER-~i polypeptide, wherein said receptor is
transcriptionally active;
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(b) contacting said cell with one or more estrogen(s) in the
presence of the test compound known to bind ER; and
(c) and detecting the expression of said MT-II, wherein decreased
expression of said MT-II relative to the addition of one or more estrogenls)
alone indicates that said test compound has estrogen antagonist activity
A method of screening test compounds to identify drug candidates
which mimic estrogens' effect on ER-Vii- or ER-a-mediated transcription is
further provided wherein said method comprises the steps of:
(a) contacting said test compound with a plurality of:
(i1 first cells comprising at least one DNA sequence
encoding metallothionein (MT-II) and at least one DNA
sequence encoding an ER-~3 polypeptide, wherein said
receptor is transcriptionally active and
(ii) second cells comprising an ERE reporter gene construct,
wherein said cells express ER-a polypeptide;
(b) identifying compounds which increase expression of MT-II in
said first cells relative to control but have minimal effect on
expression of said reporter gene in said second cells, wherein said
compounds are considered ER-p selective; or
(c) identifying compounds which increase expression of the
reporter gene in said second cells relative to control but have
minimal effect on expression of MT-II in said first cells, wherein
said compounds are considered ER-a selective.
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8
Lastly, the present invention provides a method of screening test
compounds to identify drug candidates which inhibit estrogen's effect on
ER-(3- or ER-a-mediated transcription, said method comprising the steps
of:
(a) contacting said test compound in the presence and absence of
one or more estrogen(s) with a plurality of:
(i) first cells comprising at least one endogenous DNA
encoding a metallothionein (MT-II) gene and DNA encoding
a ER-~i polypeptide, wherein said receptor is transcriptionally
active and
(ii) second cells comprising an ERE reporter gene construct,
wherein said cells express ER-a polypeptide; and
(b) identifying compounds which decrease expression of MT-il in
said first cells relative to treatment with one or more estrogen(s)
alone but have minimal effect on expression of said reporter gene
in said second cells, wherein said compounds are considered ER-(3
selective; or
(c) identifying compounds which decrease expression of the
reporter gene in said second cells relative to treatment with one or
more estrogen(s) alone but have minimal effect on expression of
MT-II in said first cells, wherein said compounds are considered
ER-a selective.
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9
Brief Descriytion of the Fi4ures
Figure 1: RT-PCR amplification of ER-~3 and ER-a a from Saos -2 and
LNCaPLN3 cells.
Figure 2: Sequencing gel separating amplified cDNAs and 17-~i estradiol
upregulation of fragment 6a.
Figure 3: (A) Nucleotide sequence ISEQ ID No. 12) of the regulated
fragment and its alignment with human MT-II, SEQ ID No. 13. (B)
Translation of fragment sequence from first methionine to stop codon ('),
SEQ ID No. 14
Figure 4: Regulation of MT-II in two cell lines. To determine fold change
in mRNA, MT-II signal was normali2ed to that of GAPDH, and compared
to the control cells.
Figure 5: Dose response of 17-~3 estradiol regulation of MT-II in Saos-2
cells. Result are shown for four different experiments and the ECso
shown for each.
AMENDED SHEET
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26-08-2000 US 009929856
Figure 6: Time course of metallothionein-II regulation in Saos-2 cells.
Data is normalized with GAPDH and fold change calculated from the
vehicle control at time =Ohr.
5 Figure 7: Receptor specificity of MT-II regulation in Saos-2 cells.
Figure 8: Regulation of MT-II in Saos-2 cells by various compounds.
Figure 9: Metallothionein-II regulation in cycloheximide-treated Saos-2
10 cells. (A) Measurement of protein synthesis during treatment with
cycloheximide. IB) Whole cell ER binding assay after 8 hours of
cycloheximide treatment. (C,DI Metallothionein-II regulation after 8 and
24 hours of treatment respectively.
Figure 10: MT is regulated by estrogens in the rat prostate.
Figure 1 1: Induction of MT-II in the rat prostate requires at least two
days of dosing.
Figure 12: Screening strategy for ligands which selectively activate ER-~i
and/or ER-a.
Detailed Description of the Invention
AMENDED SHEET
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The present invention provides an efficient way to screen large
numbers of test compounds which selectively activate ERs of the a or (3
subtype. These compounds may have desirable properties for either the
treatment or the prevention of various diseases mediated by estrogens,
including but not limited to cancers (e.g. breast, ovarian, endometrial,
prostate), endometriosis, osteoporosis and cardiovascular and central
nervous system diseases.
Definitions
(n the present description and claims, the following terms shall be
defined as indicated below.
"hER(3L" is defined as the human form of ER-(3 described in
Example 1 (the cDNA or its translated protein product).
"Estrogen" is defined as any ligand that can function as an
estrogen agonist.
"Estrogen agonist" is defined as a compound that substantially
mimics 17-(3 estradiol as measured in a standard assay for estrogenic
activity, for example, cellular assays as described in Webb et al. ( 1992).
"Estrogen antagonist" is defined as a compound that
substantially inhibits the effect of estrogen agonists as measured in a
standard assay for estrogenic activity, for example, cellular as described
in Webb et al. ( 1992)
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"A functional ER" is defined as a receptor capable of
transcriptional activation of endogenous or transfected genes as measured
by changes in RNA, protein and/or downstream biological events.
"A nonfunctional ER" is defined as a receptor incapable of
transcriptional activation of endogenous or transfected genes as measured
by changes in RNA, protein and/or downstream biological events. A "test
compound" includes but is not limited to any small molecule compound,
peptide, polypeptide, natural product, toxin with potential biological
activity.
A "ligand" is intended to include any substance that interacts with
a receptor. .
"Transfection" is defined as any method by which a foreign gene is
inserted into a cultured cell.
A "reporter" is defined as any substance that can be readily
measured and distinguished from other cellular components. The reporter
may be the transfected receptor DNA, the transcribed receptor mRNA, an
enzyme, a binding protein or an antigen.
A "cell" useful for the present purpose is one which has the ability
to respond to signal transduction through a given receptor.
"A "receptor binding assay" is an assay measuring the amount of
ligand specifically interacting with a receptor. The ligand can be a
radioligand (e.g. conjugated to 3H or 'z51), a fluorescinated ligand (either
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conjugated to a fluorochrome or possessing inherent fluorescence) or
otherwise labeled so as to be detectable.
Description of the Assay
The present invention relies on the discovery that the expression of
MT-II is selectively regulated by the interaction of a ligand with ER-Vii. .
In the methods of the invention, the regulation of MT-ll activity
may be used to provide a screening system that selectively detects both
estrogen agonist or antagonist functional activity of a ligand following
its interaction with ER-(3.
The methods typically comprise cultured cells that express
functional human ER-~i and no, or a diminished amount of ER-a.
Such cells include but are not limited to Saos-2 (ATCC HTB-85) and
LNCaPLN3. Preferred cells for this purpose include cells which over-
express ER-(3, such as the cells described below which are recombinantly
manipulated to over-express ER-Vii. The ER-(3 receptor may be modified in
any way, such as in length; these modifications may result in increased
ER-(3 selectivity and increased sensitivity of the assay.
One of skill will recognize that various recombinant constructs
comprising ER-~i can be used in combination with any cell or line which
lacks functional ER-a.
To screen a number of compounds for estrogen agonist activity,
cells expressing ER-/3 are exposed to a test compound or a control
solution (which is used to dissolve the test compound). The cells can be
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exposed while either growing in separate wells of a multi-well culture dish
or in a semi-solid nutrient matrix. After treatment for a suitable period of
time, MT-II mRNA is measured. An estrogen agonist will increase MT-II
mRNA when compared to treatment with the control solution alone.
To screen a number of compounds for estrogen antagonist activity,
cells expressing ER-~i are exposed to one or more estrogens in the
presence or absence of a test compound. The cells can be exposed while
either growing in separate wells of a multi-well culture dish or in a semi-
solid nutrient matrix. After treatment for a suitable period of time, MT-11
mRNA is measured. An estrogen antagonist will decrease MT-II mRNA
when compared to treatment with the estrogen solution alone.
Estrogenic or antiestrogenic compounds identified in the assays of
the invention can be used in standard pharmaceutical compositions for the
treatment of cancer, as components of oral contraceptives, or any other
application in which the modulation of estrogen activity is desired. The
pharmaceutical compositions can be prepared and administered using
methods well known in the art. The pharmaceutical compositions are
generally intended for parenteral, topical, oral or local administration for
prophylactic and/or therapeutic treatment. The pharmaceutical
compositions can be administered in a variety of unit dosage forms
depending upon the method of administration. For example, unit dosage
forms suitable for oral administration include powder, tablets, pills, and
capsules.Suitable pharmaceutical formulations for use in the present
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invention arefound in Remington's Pharmaceutical Sciences, Mack
Publishing Company, Philadelphia, Pa., 17th ed. (19851. A variety of
pharmaceutical compositions comprising compounds of the present
invention and pharmaceutically effective carriers can be prepared.
5 Aaplications
The methods and compositions of the present invention can be used
to identify compounds that interact with ER-~3 , either to stimulate or to
inhibit transcriptional activity . Such compounds include, without limitation,
co-activator proteins, as well as estrogens and other steroids, steroid-like
10 molecules, or non-steroid-like molecules that act as agonists or
antagonists.
Screening methods can also be used to identify tissue-specific estrogens.
Identification of ER-(3 -interactive compounds can be achieved by
cell-free or cell-based assays. In one set of embodiments, purified ER-(3 is
contacted with a labeled ligand, such as, e.g., 17-~i estradiol, in the
15 presence of test compounds to form test reactions, and in the absence of
test compounds to form control reactions. The labeled moiety may
comprise a radiolabel (such as, e.g., 3H or '251) or a fluorescent molecule.
Incubation is allowed to proceed for a sufficient time and under appropriate
conditions to achieve specific binding, after which binding of labeled ligand
to ER-(3 is measured (by monitoring, e.g., radioactivity, fluorescence, or
fluorescence polarization). In one embodiment, the (igand binding domain
of ER-(3 produced in E. coli is adsorbed to the wells of a microtiter dish and
incubated with f3H]-17(3 estradiol in the absence or presence of test
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compounds. Alternatively, soluble receptor is incubated with the labeled
ligand in the absence or presence of test compounds, and bound ligand is
separated from free ligand, either by filtration on glass fiber filters or by
using dextran-coated charcoal
Whole cell binding assays may also be used in which bound ligand is
separated from free ligand by rinsing. Cells used in these assays may either
contain endogenous receptor, or may overexpress the receptor subsequent
to stable or transient transfection or infection of an ER-j3 cDNA. Non-
limiting examples of suitable cells include COS cells, HeLa cells, CHO cells,
human umbilical vein endothelial cellsand yeast. Once a compound has
been identified as an ER-~ -interactive compound by its binding activity,
further in vivo and in vitro tests may be performed to determine the nature
and extent of activity, i.e., as an agonist or antagonist (see below).
ER-~i -interactive compounds may also be identified using cell-based
assays that measure transcriptional activation or suppression of
endogenous or transfected estrogen-responsive genes. For example,
agonists (such as, e.g., 17(3 -estradiol) block interleukin-1 (3 induction of
endogenous E-selectin in primary human umbilical vein endothelial cells that
express ER-/3 . Antagonists (such as, e.g., ICI-182780) block the agonist
activity of 17(3 -estradiol. Non-limiting examples of other suitable
endogenous estrogen-responsive promoter elements include those that
regulate endothelin-1 (ET-1 ); HDL receptor (scavenger receptor type II); and
enzymes involved in coagulation and fibrinolysis (such as, e.g., plasminogen
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activator inhibitor-1 and complement C31. Any promoter element that
responds to estrogen may be used as an appropriate target, including, e.g.,
the NFkB binding site or the apolipoprotein A1 gene enhancer sequence.
In one set of embodiments, appropriate host cells are transfected
with an expression vector encoding ER-p and the transfectants are
incubated with or without one or more estrogens I in the presence or
absence of test compounds. ER-~i activity is assessed by measuring
transcriptional activation. This may be achieved by detection of mRNA
(using, e.g., Northern blot analysis, reverse transcriptase polymerase chain
reaction, RNase protection assays) and/or by detection of the protein
(using, e.g., immunoassays or functional assays). if activation of the target
sequence initiates a biochemical cascade, downstream biological events
may also be measured to quantify ER-(3 activity. ER-(3 -interactive
compounds are identified as those that positively or negatively influence
target sequence activation.
In another set of embodiments, appropriate host cells (preferably,
mammalian 1 are co-transfected with an expression vector encoding ER-(3
and a reporter piasmid containing a reporter gene downstream of one or
more ERE s. Transfected cells are incubated with or without an estrogen
in the presence or absence of test compounds, after which ER-(3 activity is
determined by measuring expression of the reporter gene. In a preferred
embodiment, ER-(3 activity is monitored visually. Non-limiting examples of
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18
suitable reporter genes include luciferase, chloramphenicol acetyl
transferase (CAT/, and green fluorescence protein.
Preferably, the methods of the present invention are adapted to a
high-throughput screen, allowing a multiplicity of compounds to be tested in
a single assay. Candidate estrogens and estrogen-like compounds include
without limitation diethylstilbesterol, genistein, and estrone. Other ER-(3 -
interactive compounds may be found in, for example, natural product
libraries, fermentation libraries (encompassing plants and microorganisms),
combinatorial libraries, compound files, and synthetic compound libraries.
For example, synthetic compound libraries are commercially available from
Maybridge Chemical Co. fTrevillet, Cornwall, UK), Comgenex (Princeton,
NJ), Brandon Associates (Merrimack, NH), and Microsource (New Milford,
CT). A rare chemical library is available from Aldrich Chemical Company,
Inc. (Milwaukee, WI). Alternatively, libraries of natural compounds in the
form of bacterial, fungal, plant and animal extracts are available from, for
example, Pan Laboratories (Bothell, WA) or MycoSearch (NC), or are readily
produced . Additionally, natural and synthetically produced libraries and
compounds are readily modified through conventional chemical, physical,
and biochemical means fBlondelle et al., 1996). ER-(3 binding assays
according to the present invention are advantageous in accommodating
many different types of solvents and thus allowing the testing of
compounds from many sources.
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Compounds identified as ER-(3 agonists or antagonists using the
methods of the present invention may be modified to enhance potency,
efficacy, uptake, stability, and suitability for use in therapeutic
applications,
etc. These modifications are achieved and tested using methods well-
known in the art.
Examples
The present invention is further described by the following
examples. The examples are provided solely to illustrate the invention by
reference to specific embodiments. These exemplifications, while
illustrating certain specific aspects of the invention do not portray the
limitations or circumscribe the scope of the invention.
EXAMPLE 1: CONSTRUCTION OF ER-Q RECOMBINANT ADENOVIRUS
The coding sequence of human ER-/3 was obtained as described in
co-owned and co-assigned pending US Serial 08/906,365 entitled "Novel
Human Estrogen Receptor j3" filed August 5, 1997, and in Bhat et. al.,
(19981. Essentially, human testis Poly A+ RNA (1 ~.g, Clontech, Palo Alto
CA) was mixed with 0.5 pg oligo dT primer (GIBCO-BRL, Gaithersburg MD)
in a total volume of 10 p.l. The mixture was heated at 70°C for 10
minutes, and, after cooling on ice, was supplemented with 500 ~M of each
deoxynucleoside triphosphate, 1 X cDNA synthesis buffer, and 10 mM DTT
to a final reaction volume of 20 ~.1. The mixture was incubated at 42°C
for
2.5 minutes and then supplemented with 1-2 units reverse transcriptase
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(GIBCO-BRL, Gaithersburg MD), after which it was incubated at 45°C for
minutes and 50°C for 5 minutes. One-tenth of this mixture
(approximately 2 p.11 containing the cDNA template was then used in PCR
amplification of ~R-(3 using forward and reverse primers as described below.
5 The PCR primers designated in Serial No. 08/906,365 (supra) were
used to amplify ER-~i sequences in a reaction containing the following
components: 2 ~I of the cDNA template described above; 1 X PCR buffer;
200 ~M of each deoxynucleoside triphosphate, 2 units of Hot Tub DNA
polymerase (Amersham, Arlington Heights IL), and 1 pg of each of the
10 forward and reverse primers. The reaction mixture was heated to 95°C
for
2 minutes, annealed at 52°C for 1 minute, and amplified using 36
cycles,
followed by an incubation at 72°C for 1.5 minutes.
A fragment of approximately 1500 by in length was produced. The
fragment digested with Hindlll and Xbal (which cleave at sites present in
15 the forward and reverse primer sequences, respectively, but not in the main
body of the amplified cDNA sequence) and cloned into the corresponding
sites of the pcDNA3 expression vector (Invitrogen, Carlsbad CA). This
asymmetric cloning strategy places the 5' end of ER-(3 cDNA under the
control of the viral CMV promoter in pcDNA3. This clone was designated
20 "truncated hER(3" or hER(3T.
To verify the amino terminal and upstream sequence of human ER(3,
two independent approaches were taken, as described below.
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(1) 10 p.l of a human ovary 5'-Stretch cDNA library
(Clontech, Palo Alto CA) was mixed with 50 ul of 1 X K solution ( 1 X PCR
Buffer (GIBCO-BRL, Gaithersburg MD), 2.5 mM MgCl2, 0.5% Tween-20,
100 ~g/ml Proteinase K), and the reaction mixture was incubated at 56°C
for 2 hours, then at 99°C for 10 minutes. Five p.L of this reaction
mixture
were then used as template in a nested PCR reaction using the PCR primers
designated in Serial No. 08/906,365 (supra). The reaction contained 1 X
Klentaq PCR reaction buffer (40 mM Tricine-KOH, 15 mM KOAc, 3.5 mM
Mg(OAc)2, 75 p.g/ml bovine serum albumin); 0.2 p.M of each dNTP; 0.2 pM
10 of each of the above primers, and 1 unit of Klentaq Polymerase Mix
(Clontech, Palo Alto CA). Touchdown PCR conditions were as follows: 5
cycles of 94°C for 2 seconds and 72°C for 4 minutes, followed by
30
cycles of 94°C for 2 seconds and 67°C for 3 minutes.
Excess nucleotides and primers were removed from first round PCR
reactions by purification using Wizard PCR columns (Promega, Madison
WI). A second-round PCR reaction was then performed using 2 p.l of the
purified first-round reaction mixture using the PCR primers designated in
Serial No. 08/906,365 (supra). The PCR reaction and cycling conditions
were identical to those employed in the first round. The products were
20 cloned into pCR2.1 (Invitrogen, Carlsbad CA ) and three resulting clones
were sequenced. All three clones (designated L1, L2, and L3) contained
ER(3 inserts of different lengths, all of which were homologous to ER(3 and
to each other.
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(2) A Marathon Ready thymus cDNA kit (Clontech, Palo Alto
CA) for 5' rapid amplification of cDNA ends (RACE) was also used to
isolated ER~i 5' clones. In the first round of a nested PCR reaction, 5 ~I of
human thymus Marathon-ready cDNA (Clontech, Palo Alto CA) was used as
template and using the primers designated in Serial No. 08/906,365
(supra). The PCR reaction and cycling conditions were identical to those
described in (1) above.
Excess nucleotides and primers were removed from the first round
PCR reactions by purification over Wizard PCR columns (Promega, Palo Alto
CA). A second round PCR reaction was performed using 2 ~I of the purified
first round reaction using primers designated in Serial No. 08/906,365
(supra). The second round PCR reaction and cycling conditions were
identical to those employed in the first round. The products were cloned
into the pCR2.1 vector (Invitrogen, Carlsbad CA) and two clones were
sequenced. The two clones contain insert sequences of different lengths
that are homologous to ER~3, to each other, and to the sequences isolated
from a human ovary cDNA library as described above.
All of the ER(3 sequences isolated by methods (1) and (2) above
contained 110 nucleotides corresponding to hER~iT sequences, as well as
228 additional nucleotides at the 5' end.
The hER(3T and 5' end sequences were joined and the resulting cDNA was
cloned into pCDNA3 (Invitrogen, Carlsbad CA ) under the control of the
cytomegalovirus IE promoter; this expression vector was designated "long
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hER-Vii" or hER(3L . Full length hER~iL cDNA sequence encodes a polypeptide
having 530 amino acid residues.
The hERpL sequence contained an optimal translation initiation sequence
CCACC immediately upstream to the initiation codon and the sequence
was under the control of the cytomegalovirus IE promoter. The coding
sequence of hER~iL was then transferred into an Ad50E1 a vector plasmid
containing adenovirus sequences from map unit 0-17 with an deletion of
E1 a region between map units 1.4 -9.1 (Davis AR et. al., 1985,
Gluzman,Y. et. a1..1982). The hERpL transcription unit in Ad50E1 a
plasmid contained cytomegalovirus 1 E promoter, Ad5 tripartite leader,
coding sequence of hER~ and SV40 late polyadenylation signal
sequences. hER~iL in Ad5 DE1 a plasmid was then linearized with BstEll
enzyme and transfected along with Clal A fragment of Ad5 virus with E3
region deletion (80-88 map unitsl into 293 cells (transformed primary
human embryonic kidney, ATCC CRL 15731. Viral plaques generated by
homologous recombination were isolated, amplified and characterized by
restriction DNA analysis and cell lysis assay in A549 cells (human lung
carcinoma, ATCC CCL 185). Confirmatory tests indicated that the
recombinant Ad5 hER~3L virus contained the expected DNA fragments and
was replication defective. The virus was further purified by re-plaguing.
The isolated plaques were amplified, tested and used as a seed stock to
generate large amounts of the virus in 293 cells. The virus was titered in
293 cells by plaque assay and the stock contained 1.28 X10 9 PFU/ml.
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EXAMPLE 2: ASSESSMENT OF ENDOGENOUS LEVELS OF ER MRNA IN SAOS-2
AND LNCAPLN3 CELLS
Unless otherwise noted, cell culture reagents were obtained from
Gibco BRL (Gaithersburg MD). LNCaPLN3 cells were grown in RPMI 1640
medium supplemented with 10% FBS, 2 mM GIutaMAX-1, 100 U/ml
penicillin g, and 100 Nglml streptomycin sulfate. Saos-2 cells (ATCC,
Manassas VA) were maintained in monolayer culture using McCoy's 5A
medium supplemented with 10% fetal bovine serum (FBS), 2 mM
GIutaMAX-1, 100 U/ml penicillin g, 100 Ng/ml streptomycin sulfate.
RT-PC R
Total RNA was isolated from LNCaPLN3 and Saos-2 cells using
TRlzol (Gibco BRL, Gaithersburg MD) according to the manufacturer's
directions. The samples were then treated with RNase-free DNase I
(Gibco BRL, Gaithersburg MD1 at 1 unitlNg for 30 minutes at 37°C. RNA
was purified from the reaction using RNeasy columns (Qiagen, Hilden
Germany) and amount recovered estimated by UV spectrophotometry.
Reverse transcription reactions were performed on 0.5 Ng of RNA
in a 20 NI reaction. For ER-a the reaction contained 1 x PCR Buffer (Gibco
BRL, Gaithersburg MD), 5 mM MgCl2, 1.25 NM ERa-specific reverse
primer (5'-CCAGCAGCATGTCGAAGATC-3', SEQ ID No. 1 ), 0.5 NM
GAPDH-specific reverse primer (5'-CACCCTGTTGCTGTAGCCAAATTC-3',
SEQ ID No. 21, 0.5 mM dNTPs, 20 units of RNasin (Promega, Madison
WI) and 200 units of Superscript II Reverse Transcriptase (Gibco BRL,
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Gaithersburg MD). The ER-~3 reaction contained the same components as
ER-a with the following exceptions: 2.5 mM MgCl2 and 1.25 NM ER~i-
specific reverse primer (5'-GCAGAAGTGAGCATCCCTCTTTG-3', SEQ ID
No. 31. A duplicate reaction which was identical in all reagents except
5 that it did not contain Superscript II Reverse Transcriptase was performed
for each sample as a negative control to ensure that the RNA samples
were not contaminated by DNA. Reactions were incubated at 42°C for
15 minutes followed by 5 minutes at 99°C and 5 minutes on ice prior to
amplification.
10 PCR was initiated by adding 80Ni of master mix containing ER-a-
specific forward primer (5'-GGAGACATGAGAGCTGCCAAC-3', SEQ ID
No. 4) or ER-(3-specific forward primer (5'-CAGCATTCCCAGCAATGT
CAC-3', SEQ ID No. 5) and GAPDH-specific forward primer (5'-
GACATCAAGAAGGTGGTGAAGCAG-3', SEQ ID No. 6) directly to the
15 20NI reverse transcriptase reaction. Final concentration of reagents in the
ER-a 100p1 PCR reaction was as follows: 0.25 NM each ER-specific
primer, 0.1NM each GAPDH primer, 1 x PCR Buffer (Gibco BRL,
Gaithersburg MD), 0.2 mM dNTPs, 2 mM MgCl2 and 0.5 units of Taq
DNA Palymerase (Gibco BRL, Gaithersburg MD1. The ER-(3 reaction
20 contained the same amount of reagents except for 1 mM MgCl2. Two
step PCR was carried out in a PE 9600 for 25 cycles as follows: 95°C
for
sec, 64°C for 1.5 min. Samples were incubated at 64°C for 10 min
after amplification.
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Twenty microliters of each sample were separated using a 1.5%
agarose gel and transferred to Hybond-N + (Amersham Pharmacia,
Piscataway NJ) by capillary alkali Southern Blotting in 0.4 N NaOH, 0.6M
NaCI. Blots were pre-hybridized at 42°C for 30 min in Rapid-Hyb
buffer
(Amersham Pharmacia, Piscataway NJ). Oligonucleotide probes specific
for ER-a (5'-TGAACCAGCTCCCTGTCTGCCAGGTTGGT-3', SEQ ID No.
7), ER-p l5'-CCGCATACAGATGTGATAACTGGCGATGGA-3', SEQ ID No.
8) and GAPDH (5'-GCTGTTGAAGTCACAGGAGACAACCTGGT-3', SEQ ID
No. 9) fragments were end-labeled with 32P-y ATP using Polynucleotide
Kinase (Gibco BRL, Gaithersburg MD). Probes were added to the blot at
3.0 x 105 CPM/ml and incubated at 42°C for 1 hour. ER and GAPDH
hybridizations were done independently. Blots were washed once in 2x
SSC, 0.1 % SDS at room temperature for 15 min then twice in 0.2x SSC,
0.1 % SDS at 42°C for 15 min. Blots were then exposed to film.
Saos-2 cells expressed endogenous ER-~i but not ER-a mRNA when
assessed by PCR (Figure 11. As a positive control, GAPDH mRNA was
coamplified in these reactions. Whereas both cell lines contained GAPDH
mRNA, only the Saos-2 cells contained ER-Vii. Similar results were
obtained for the LNCaPLN3 cells (data not shownl.
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EXAMPLE 3: DISCOVERY THAT ER:f3 UPREGULATES MT-II- IN SAGS-2 AND
LNCAPLN3 CELLS
Cell culture and infection
Because message levels for ER-(3 were iow, Saos-2 and LNCaPLN3
cells were engineered to overexpress hER~3L by transient infection with a
recombinant adenovirus (see Example 11 prior to differential display.
LNCaPLN3 and Saos-2 cells were cultured as described above.
Sixteen hours prior to infection, the cells were plated in phenol red-free
RPMI 1640 medium supplemented with 10% charcoal/dextran-treated
("stripped") FBS (HyClone, Logan UT1, 2 mM GIutaMAX-1, 100 U/ml
penicillin g, 100 Ng/ml streptomycin sulfate. This medium was used for
the remainder of the experiment.
Cells were infected with a 1 /20 dilution of an Ad5 hER(3L virus (see
Example 1 ) using 2% stripped FBS phenol red-free medium with
antibiotics and GIutaMAX-1 for 2 hours at 37° C. Medium containing
virus was aspirated and the cells were washed with medium. Fresh
medium was added and the cells allowed to recover overnight at 37°C.
The following day, cells were treated with 10 nM 173-estradiol or
vehicle for 24 hours and total RNA prepared for differential display using
the TRlzol reagent (Gibco BRL, Gaithersburg MD). To remove residual
DNA, samples were treated with RNase-tree DNase I (Gibco BRL,
Gaithersburg MD) at 1 unit/Ng for 30 minutes at 37°C. RNA was
purified
from the reaction using RNeasy columns (Qiagen, Hilden Germany) and
amount recovered estimated by UV spectrophotometry.
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Rapid Analysis of Differential Expression (RADE)
Reverse transcription (RT)
Following DNase I treatment, six micrograms total RNA was
incubated with 1 x RT buffer 125 mM Tris-CI, pH 8.3, 37.6 mM KCI, 3 mM
MgCl2 and 5 mM DTT, from Genhunter, Nashville TN), 20 NM dNTP's (dA,
C, G and TTP 2'- deoxynucleoside 5' triphosphates, From Gibco BRL
(Gaithersburg MD), 0.2 NM HT"C (oligonucleotide AAGCTTTTTTTTTTTC,
SEQ ID No. 15) in a final volume of 600 NL. This reaction mixture was
incubated at 65°C for five minutes to denature secondary structures,
followed by a ten minute incubation at 37°C. At this time 30 NI
Superscript II reverse transcriptase (200U/NI, Gibco BRL, Gaithersburg
MD) was added to the reaction and incubation proceeded for 1 hr at
37°C. The enzyme was inactivated by heating at 75°C for five
minutes.
An aliquot of this reaction was then used for the second strand synthesis
by PCR.
Polymerase chain reaction fPCRJ
To 2NI of the RT reaction was added, 1 x PCR buffer ( 10 mM Tris-
CI, pH 8.4, 100 mM KCI, 1.5 mM MgClz and 0.001 % gelatin), 2 NM
dNTP's, 15 nM "P dATP (NEN, Boston MA), 1 unit AmpIiTaq DNA
polymerase iPerkin-Elmer, Norwalk CT) and 1NM arbitrary primer
5'AAGCTTGCCATGG-3' for a total reaction volume of 20NI. This reaction
mixture was then thermocycled using the following parameters:
92°C for 2 min, 1 cycle
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92°C for 15 sec, 40°C for 2 min, 72°C for 30 sec, 40
cycles
72°C for 5 min
Gel alectrophoresis
Duplicate samples of PCR products were separated by gel
electrophoresis on a 6% denaturing polyacrylamide gel (5.7% acrylamide,
0.3% bisacrylamide, 42% urea and 51 % H20) in 1 x TBE buffer (0.1 M
Tris, 0.09 M Boric Acid, 1 mM EDTA) for three hours at 2000 volts. The
gel was then transferred to fitter paper (Schleicher & Schuell, Keene NH),
dried under vacuum at 80°C for one hour and exposed to X-ray film for
twenty four hours.
One band, designated 6a, was apparently upregulated in both the
Saos-2 and LNCaPLN3 cells by 17-~3 estradiol (Figure 2). The developed
film was then superimposed over the dried gel and the band's corners
were marked using a 22 gauge syringe needle. The gel slice within these
boundaries excised with a razor blade and immersed in 100 NI HzO. This
sample was boiled in a water bath for fifteen minutes, centrifuged for two
minutes and the supernatant solution transferred to a new tube. Added to
this sample was 5 NI of 10 mg/ml glycogen, 10NI of 3 M sodium acetate
and 450,u1 of 100% ethanol. The sample was mixed, allowed to
precipitate overnight at -20°C and centrifuged for ten minutes at 10
000
g. The supernatant solution was removed, the pellet washed with 200 NI
of 85% ethanol, dried and resuspended in 10 NI H20. A 3 NI aliquot was
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used in a reamplification PCR reaction in the presence of 1 x PCR buffer,
20 NM dNTP's, 0.2 NM arbitrary primer and 0.2 NM oligonucleotide HT"C
and 2 units AmpIiTaq polymerase using the same cycling parameters as
the PCR reaction above. The resulting product was then used as probe in
5 a Northern hybridization assay to confirm regulation and cloned into a
bacterial piasmid for sequence analysis.
Fragment cloning
Band 6a was cloned using the TA cloning kit (Invitrogen, Carlsbad
CA). After lysis, colonies were screened by PCR for the correct insert
10 size. Colonies were lysed in 20 mM Tris-HCI, pH $, 50 mM KCI, 2.5 mM
MgCl2, 0.5% Tween 20 and 100,ug/ml Proteinase K by incubating for 30
min at 56°C then 10 min at 99°C to inactivate the Proteinase K.
Of this
reaction 2,u1 was used in a PCR reaction of 20 mM Tris-HCI, pH $, 50 mM
KCI, 2.5 mM MgCl2, 75 NM dNTPs, 375 nM M-13 forward. and reverse
15 primers and 2.5 U of Taq polymerase (Gibco BRL, Gaithersburg MD). The
reactions were cycled as follows: 95°C for 30 sec; 64°C for 30
sec;
72°C for 45 sec for 30 cycles. One clone, designated 6a.2, was chosen
for sequencing.
Fragment sequencing
20 Clone 6a.2 was sequenced according to the ABI PRISM"" Dye
Terminator Cycle Sequencing Ready Reaction Kit with AmpIiTaq DNA
Polymerase using the recommended protocol from Applied Biosystems
(Foster City CA). Spin columns (AGTC) were employed to remove
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unincorporated dye-labeled nucleotides after cycle sequencing. Automated
DNA sequencing grade 4.75% polyacrylamide gels were run for all the
DNA sequencing samples using ABI 373 DNA sequencers. Sequencing
data was edited using Sequence Navigator and assembled using DNAStar
(DNAStar, Madison WI). The sequence was trimmed of BADE primers
and used in a BLAST search using Millennium Software (Boston MA). A
nucleotide homology search of clone 6a.2 sequence revealed a 98
identity with human MT-II-. Over the coding sequence, however, there
were no amino acid mismatches (Figure 3)
Confirmation of regulation using Northern blots and 4uantitative reverse
transcriptase c~olymerase chain reaction Ic~RT-PCR1:
To confirm the results obtained with the PCR amplification of
cellular mRNAs, Northern blots and qRT-PCR were used to assess the
effect of 17-(3 estradiol or other compounds on message levels. Unless
otherwise noted, all experiments use cells transiently overexpressing
hER~3L in response to adenovirus infection as described above, and were
treated with compound for 24 hours. In some cases cells transiently
overexpressing ER-a were used for comparison. Methods for adenovirus-
mediated overexpression of ER-a are identical to those described for ER-(3.
Northern blots
Poly A+ RNA was isolated from total RNA using Oligotex mRNA
Isolation kit (Qiagen, Hilden Germany) according to manufacturers
instructions. Six micrograms of mRNA or 10 pgs of total RNA were
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separated on a 1.5% agarose, 0.22 M formaldehyde, 10 mM HEPES, 1
mM EDTA gel. RNA was transferred to Hybond-N (Amersham Pharmacia,
Piscataway NJ) by capillary action in 20X SSC (Gibco BRL, Gaithersburg
MD ) overnight. After transfer, the membrane was UV-cross-linked and
dried at 80°C for 10 min. Northern Blots were pre-hybridized in Rapid
Hyb solution (Amersham Pharmacia, Piscataway NJ) for 30 minutes.
The insert from clone 6a.2 was isolated from the plasmid using
PCR as described above for colony screen. The PCR product was purified
from an agarose gel using Wizard Preps fPromega, Madison WI). Re-
amplified RADE fragments for band 6a or fragment from clone 6a.2 were
random primer labeled using Redi-prime kit (Amersham Pharmacia,
Piscataway NJ) according to manufacturers instructions. Unincorporated
nucleotides were removed using a Nap-5 column (Amersham Pharmacia,
Piscataway NJ) and incorporation of [32P]-dCTP measured by liquid
scintillation counting.
The probes were denatured at 100°C for 10 min and 1.5 x 106
CPM/ml of Rapid-Hyb hybridization solution was added to the membrane.
The blots were hybridized at 65°C for 5 hours and were washed as
follows: Once in 2X SSC, 0.1 % SDS at 65° C for 15 min; twice in 0.2X
SSC, 0.1 % SDS at 65°C for 15-30 min. Blots were exposed to film
and
to a Phosphorlmager screen (Molecular Dynamics, Sunnyvale CA). After
probing with 6a RADE fragment or 6a.2 cloned fragment, blots were
probed with a cDNA homologous to GAPDH as above. Hybridization
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33
signal from 6a.2 fragment was normalized to that of GAPDH on a
Phosphorlmager (Molecular Dynamics, Sunnyvale CA) to determine fold
induction.
QRT PCR - A MT-II fragment identical to clone 6a.2 except that it
contained a 63 by deletion was subcioned into pcDNA.3 (lnvitrogen,
Carlsbad CA). RNA was transcribed using the T7 Promoter Large Scale
Transcription Kit (Novagen, Madison Wil. After phenol-chloroform
extraction and ethanol precipitation the synthesized RNA was quantified
and analyzed using UV spectrophotometry and gel electrophoresis.
Reverse transcription reactions were performed on 200 ng and 300
ng of DNased Saos-2 total RNA plus a known amount of MT-II standard
RNA in a 20 NI reaction. The reaction contained 1 x PCR Buffer (Gibco
BRA, Gaithersburg MD1. 3.75 mM MgCl2, 1.25 NM MT-II-specific reverse
primer (5'-GGAATATAGCAAACGGTCAGGGTC-3', SECT ID No. 101, 0.5
mM dNTPs. 1 mM DTT, 20 units of RNasin (Promega) and 200 units of
Superscript II Reverse Transcriptase (Gibco BRL, Gaithersburg MD1.
Reactions were incubated at 42°C for 15 minutes followed by 5 minutes
at 99°C and 5 minutes on ice prior to amplification
PCR was initiated by adding 80NI of master mix containing MT-II
specific forward primer (5'-GGCTCCTGCAAATGCAAAGAG-3', SECT ID
No. 11) directly to the 20NI reverse transcriptase reaction. Final
concentration of reagents in the 100NI PCR reaction was as follows: 0.25
NM each MT-II -specific
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primer, 1 x PCR Buffer (Gibco BRL, Gaithersburg MD1, 0.1 mM dNTPs, 1.5
mM MgCl2 and 0.5 units of Taq DNA Polymerase (Gibco BRL,
Gaithersburg MD). Two step PCR was carried out in a PE 9600 (Perkin-
Elmer, Norwalk CT) for 40 cycles as follows: 95°C for 30 sec,
64°C for
5 1.5 min. Samples were incubated at 64°C for 10 min after
amplification.
PCR products were separated and analyzed on a reverse-phase ion-
pair high performance liquid chromatography DNASep column (Sarasep,
San Jose CA). The elution system was a gradient of acetonitrile in 0.1 M
triethylammonium acetate (Fluka, Ronkonkoma NY) at a flow rate of 0.7
ml/min. The acetonitrile gradient increased from 14.6% to 16.6°~ over 5
minutes. The amount of product from the standard and the native RNA
was determined by UV absorbance detection at 254 nm and signal was
analyzed by an on-line integrator. From the chromatograms, the ratio of
the area under each peak was used to determine the ratio of the amount
15 of input MT-II standard RNA to the amount of native MT-II message in the
Saos-2 RNA.
In LNCaPLN3 cells, the magnitude of MT-II induction by 17-(3
estradiol is approximately 6 fold (Figure 4). In Saos-2 cells, 10 nM 17(3-
estradiol upregulates MT-II mRNA as much as 14 fold (Figure 4). This
20 upregulation in Saos-2 cells has been repeated in at least 20 experiments
and the range of induction is 3.5-14 fold. The EC5° of this regulation
is
4.6 t 2.7 nM as assessed by qRT-PCR (Figure 51. If Saos-2 cells are
treated with 10 nM 17(3-estradiol and RNA prepared at different times
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post treatment, the first discernible increase in MT-II message occurs
after 8 hours and the response peaks by 24 hours (Figure 61.
Since the samples prepared for differential display overexpressed
hER(3L, , MT-II regulation was assessed in native Saos-2 cells and those
5 overexpressing ERa. As illustrated in Figure 7 , endogenous levels of ER-
~3 or overexpressed levels of ER-a were ineffective at mediating 17(3-
estradiol's regulation of MT-II.
Other compounds were tested for their ability to upregulate MT-II
in Saos-2 cells. Ten nanomolar diethylstilbesterol (Sigma, St. Louis MO)
10 and 0.1 pM genistein (RBI, Natick MAI both increased MT-II mRNA. One
micromolar of the antiestrogen, ICI-182780 (Zeneca, Wilmington DE),
completely blocked induction by 17(3-estradiol and genistein but had no
effect when given alone (Figure 8). Co-treatment with 1 uM of the
antiprogesterone/antiglucocorticoid, RU486 (Ligand Pharmaceuticals, La
15 Jolla CA), did not block regulation by 17(3-estradiol (data not shownl.
EXAMPLE 4: TREATMENT OF SAOS-2 CELLS WITH CYCLOHEXIMIDE
To determine if the increase in MT-II mRNA requires new protein
synthesis, cycloheximide was used after hER(3L overexpression and
20 before treatment with 17-(3 estradiol to arrest translation.
Verification of cycloheximide's effect on protein synthesis
Cells were plated and infected with hER(3L virus as described
above, then pre-treated with 10 ~g/mL of cycloheximide or vehicle for one
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hour at 37°C. After this preincubation, 10 pg/mL of cycloheximide, 10
nM 17-(3 estradiol and 50 ~Ci/ml 35S-Methionine (NEN, Boston MA) in
methionine-deficient media was added and incubation continued at 37°C
for 8 or 24 hours.Cells were washed 3x in cold PBS and then scraped
off plates in 500 ~.I of PBS. . Cells were pelleted and resuspended in 200
~I of RIPA buffer (150 mM NaCI, 1 % NP-40, 0.5% DOC, 0.1 % SDS, and
50 mM Tris, pH 8.0). Methionine incorporation was measured by TCA
precipitation. Five and ten microliters of each sample were spotted on
individual Whatman filters. Filters were boiled in 10% trichloroacetic acid
for 10 minutes, then washed 3 times for 10 minutes in deionized water, 3
times for 10 minutes in 95% ethanol and once for 10 minutes in acetone.
The dried filters were placed in scintillation fluid and counted for 1
minute.
Treatment of cells with 17-~i estradiol
Cells were plated and infected with hER[3L virus as described
above. Cells were pretreated with 10 ug/mL of cycloheximide or vehicle
for one hour at 37°C. After this preincubation, 10 nM 17-~i estradiol
or
vehicle was added and incubation continued at 37°C for 8 or 24 hours.
RNA was isolated from the cells as described above and gene expression
evaluated by Northern blot analysisor qRT-PCR as described in Example 3.
Verification of functional ER-(3 protein after cvcloheximide treatment
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Duplicate cultures were prepared and treated as described in the
previous section. After the 8 hour incubation, cells were washed four
times with DMEM to remove 17-(3 estradiol. Cycloheximide (10 ug/mL)
was added to all samples as was 1 nM (3H]-estradiol. Some cells were
co-treated with 0.3 ~,M diethylstilbesterol to estimate nonspecific binding.
After incubation for 2.5 hr at 37°C, cells were washed with DMEM
and
lysed with 0.1 % sodium dodecyl sulfate. DPM were measured by liquid
scintillation counting.
The MT promoter contains glucocorticoid and metal response
elements, but EREs have not been described. It is possible the effect of
17-(3 estradiol on MT-II mRNA expression is indirect. After
overexpression of ER-(3, Saos-2 cells were treated with cycloheximide to
severely limit new protein synthesis (Figure 9A1. Although comparable
amounts of receptor protein were expressed with and without
cycloheximide treatment as measured by a whole cell binding assay
(Figure 9B), MT-II induction did not occur (Figure 9C,D).
EXAMPLE 5 : TREATMENT OF RATS WITH 17-Q ESTRADIOL
Because 17-(3 estradiol upregulates MT-II in LNCaPLN3 cells, a
prostate cancer cell line, we looked for a similar response in the rat
prostate.
All animals were treated according to institutional guidelines using
approved protocols. Adult (15-19 week, '375g) castrated Sprague-
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Dawley rats were purchased from Taconic Farms (Germantown NY). Ten
days after castration, rats were injected with vehicle, 16 ~.g 17-(3
estradiol, 16 wg diethylstilbesterol (Sigma, St. Louis MO), or 16 ~g 17-(3
estradiol plus 1.6 mg raloxifene (synthesized in-house) subcutaneously
once per day for three days. Approximately 24 hr after the last dose, rats
were euthanized by COZ asphyxiation and the prostate excised. Total
RNA was prepared and analyzed for MT-II mRNA as described above. In
another experiment, rats were dosed for 1, 2 or 3 days with 16 ~.g 17-(i
estradiol before prostate tissue was collected.
Metallothionein-II mRNA increased five-fold after 17-~i estradiol
treatment. A similar response occurred when rats were dosed with
diethylstilbesterol, but this upregulation was blocked by coadministration
with a 100 fold excess of raloxifene hydrochloride, an estrogen
antagonist (Figure 10). Although the initial experiments used tissue from
rats dosed for three days, the upregulation of MT-II in the rat prostate is
first seen after two days of dosing with 17-(3 estradiol (Figure 11 ).
EXAMPLE 6 : ERE-LUCIFERASE REPORTER ASSAY USING MCF-7 CELLS
Because MT-II regulation in Saos-2 cells is mediated by ER-(3 and
not ER-a, another assay was needed to compare selectivity of compounds
for these two receptors in cell based assays. MCF-7 is an estrogen-
responsive breast cancer cell line and, in our hands, expresses only ER-a
as measured by RT-PCR (data not shown). When MCF-7 cells are
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transiently infected with an ERE--reporter gene construct and treated with
17-~i estradiol, reporter gene activity can be measured.
MCF-7 HTB 22, ATCC , Manassas VA) cells are passaged twice a
week with growth medium [D-MEM/F-12 medium containing 10% (vlv)
heat-inactivated fetal bovine serum100 U/ml penicillin g, 100 p.g/ml
streptomycin sulfate, 2 mM GIutaMAX-1 ]. The cells are maintained in
vented flasks at 37oC inside a 5% C02/95% humidified air incubator.
One day prior to treatment, the cells are plated with growth medium at
25,000/well into 96 well plates and incubated at 37oC overnight.
The cells are infected for 2 hr at 37°C with 50 p.l/well of a 1:10
dilution of adenovirus 5-ERE-TK-luciferase (Bodine et. al., 1997) in
experimental medium (phenol red-free DMEM/F-12 medium containing
10% (v/v) heat-inactived charcoal-stripped fetal bovine serum, 100 U/ml
penicillin g, 100 wg/ml streptomycin sulfate, 2 mM GIutaMAX-1 , 1 mM
sodium pyruvatel. The wells are then washed once with 150 wl of
experimental medium. Finally, the cells are treated for 24 hr at 37oC in
replicates of 8 wells/treatment with 150 ~I/well of vehicle ( < 0.1 % v/v
DMSO) or compound that is diluted > 1000-fold into experimental
medium.
After treatment, the cells are lysed on a shaker for 15 min with 25
~,I/well of 1 X cell culture lysis reagent (Promega, Madison WI). The cell
lysates (20 ~1) are transferred to a 96 well luminometer plate, and
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luciferase activity is measured in a MicroLumat LB 96 P luminometer (EG
& G Berthold, Bad Wildbad Germany) using 100 wl/well of luciferase
substrate (Promega, Madison WI). Prior to the injection of substrate, a 1
second background measurement is made for each well. Following the
5 injection of substrate, luciferase activity is measured for 10 seconds after
a 1 second delay. After subtracting background subtracts the mean and
standard deviation are calculated.
Compounds which induce MT-II in Saos-2 cells and do not
stimulate reporter gene activity in MCF-7 cells are thus selective for ER-[3.
10 In addition, results from these two assays can also be used to select a
compound that is selective for ER-a as shown in Figure 12.
Discussion
Using differential display, it was unexpectedly discovered that ER-~i
15 increases MT-II mRNA in two cell lines treated with 17-~i estradiol. To our
knowledge, this is the first gene discovered to be regulated by this new
form of the ER. This response has been extensively characterized in the
human osteosarcoma cell line Saos-2. A variety of estrogens can
upregulate MT-II, and this response is blocked by cotreatment with the
20 estrogen antagonist 1CI-182780. The ECSO for 17-/3 estradiol is
approximately 5 nM and this response is mediated by ER-~i acting through
as yet unknown proteins.
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The action of ER-(3 on MT-II is not a general phenomenon as a
survey of over a dozen cell lines failed to reveal this induction in samples
other than Saos-2 and LNCaPLN3 cells. However, the fact that MT-II is
regulated by 17-[3 estradiol in the rat prostate strengthens the possibility
that this is a physiologically relevant response and not a artifact of
receptor overexpression in cancer cell lines. We are confident that this
prostate response is mediated by the ER for two reasons: A nonsteroidal
estrogen (diethylstilbesterol) upregulates MT-II and raloxifene, an estrogen
agonist/antagonist, blocks 17-~3 estradiol's action. However, at this time
it is not clear if this induction reflects activity of ER-(3 and/or ER-a.
Although ER-~3 is the predominant form of the ER in the rat prostate, ER-a
is also present. Current studies are underway to define the receptor type
responsible for this in vivo response.
Metallothioneins are low molecular weight, cysteine rich proteins
that bind metals such as cadmium, copper and zinc. Although the first
metallothionein was discovered over forty years ago (Vallee, 1957),
debate continues as to their function. Several proposals have been made
and these include protection form metal toxicity, regulation of zinc and
copper homeostasis and defense against oxidative stress. Regulation of
energy balance has also been implicated because, after reaching sexual
maturity, MT (-I and -1I) knockout mice become obese (Beattie et al,
1998).
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Recently, studies have detailed how MT may act to regulate zinc
homeostasis in cells. Using purified zinc-dependent enzymes such as E.
coli alkaline phosphatase, bovine carboxypeptidase A and sheep sorbitol
dehydrogenase two recent publications show how agents in the cellular
milieu can facilitate exchange between zinc complexed with MT and
(metal depleted) apoenzymes (Jacob et al, 1998; Jiang et al, 1998).
Citrate and glutathione can influence the direction of zinc transfer and
thus regulate enzyme activity depending on the redox state of the cell.
Although not an enzyme, the ER also requires zinc for activity, and ER
from MCF-7 cells can reversibly exchange zinc with purified MT in vitro
(Cano-Gauci et al, 19961.
A myriad of agents can regulate MT levels, including
glucocorticoids and metals such as cadmium (for review see Hamer,
19861. Estrogens are not a classical regulators of MT, but two intriguing
papers appear in the literature. First, a two week treatment of female
rats with 17-(3 estradiol upregulated a copper binding protein in intestinal
mucosa which reduced the amount of copper absorbed into the plasma.
Since the molecular weight of this protein was about 10K, the authors
suggest it is MT (Cohen et al, 19791. Recently, MT was identified in a
subtractive hybridization screen of uterine mRNAs regulated after a single
injection of 17a ethinyl estradiol Rivera-Gonzalez et al, 1998). Although
the isoform is not identified, a MT transcript increased three fold between
four and eight hours after 17-a ethinyl estradiol stimulation. In addition,
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it is not clear which receptor type effects this regulation because ER-a,
not ER-(3, is the most abundant ER in the uterus (Kuiper at al, 1996;
Couse et al, 1997).
Since the function of MT is only beginning to be elucidated and is
completely unknown for ER-~3, understanding the significance of their
association is impossible at this time. However, even if the connection
between these two proteins is unclear, the observation of regulation can
still be exploited to help design an ER-p or ER-a specific ligand. The fact
that genistein upregulates MT-II- in Saos-2 cells as well or better than
17~i-estradiol indicates an ER-p-selective compound can effectively direct
transcription. In addition, several structurally diverse compounds which
bind to ER-(3 can also upregulate this message in Saos-2 cells. When data
on a compound's ability to regulate MT-II in Saos-2 cells are coupled with
information about how it regulates reporter gene activity in an MCF-7 cell,
15 ER-(3 and ER-a selectivity can be assessed. Thus, when used together,
these two assays are tools to help design selective compounds for either
ER type. Finally, if the regulation in the prostate can also be shown to be
mediated by ER-(3, then the in vivo activity of compounds can be
assessed, providing another valuable tool for drug discovery.
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REFERENCES
Beattie JH, Wood AM, Newman AM, Bremner I, Choo KHA, Michalska
AE, Duncan JS, Trayhurn P. 1998. Obesity and hyperleptinemia in
metallothionein (-I and -II? null mice. Proc Natl Acad Sci (USA) 95:358-
363.
Bhat RA, Harnish DC, Stevis PE, Lyttle CR, Komm BS. 1998. A novel
human estrogen receptor ~3: Identification and functional analysis of
additional N-terminal amino acids. J Steroid Molec Biol, in press.
Blondelle SE, Houghten RA. 1996. Novel antimicrobial compounds
identified using synthetic combinatorial library technology. Trends
Biotechnol 14(21:60-65.
Bodine PVN, Green J, Harris HA, Bhat R, Stein GS, Lian JB, Komm B.
1997. Functional properties of a conditionally phenotypic, estrogen-
responsive, human osteoblast cell line. J Cell Biochem 65:368-387.
Cano-Gauci DF, Sarkar B. 1996. Reversible exchange between
metallothionein and the estrogen receptor zinc finger. FEBS Letters
386:1-4.
CA 02352203 2001-05-24
WO 00/37681 PCT/US99/29856
Cohen DI, Illowsky B, Linder MC. 1979. Altered copper absorption in
tumor-bearing and estrogen-treated rats. Am J Physiol 23613):E309-15.
Couse JF, Lindzey J, Grandien K, Gustafsson J, Korach, KS. Tissue
5 distribution and quantitative analysis of estrogen receptor-a (ERa) and
estrogen receptor-(3 (ERp) messenger ribonucleic acid in the wild type and
ERa-knockout mouse. 1997. Endocrinology 138(1 1 ):4613-4621.
Cowley SM, Hoare S, Mosselman S, Parker MG. 1997. Estrogen
10 receptors a and (3 form heterodimers on DNA. J Biol Chem
272(32):19858-19862.
Davis AR, Kostek B, Mason B, Hsiao CL, Morin J, Dheer SK, Hung P.
1985. Expression of hepatitis B surface antigen with a recombinant
15 adenovirus. Proc Natl Acad Sci (USA) 82:7560-7564.
Dotzlaw H, Leygue E, Watson PH, Murphy LC. 1996. Expression of
estrogen receptor-beta in human breast tumors. J Clin Endocrinol Metab
82(7):2371-2374.
Enmark E, Pelto-Huikko M, Grandien K, Lagercrantz S, Lagercrantz J,
Fried G, Nordenskjold M, Gustafsson J. 1997. Human estrogen receptor-
CA 02352203 2001-05-24
WO 00/37681 PCT/US99/29856
46
(3 gene structure, chromosomal localization, and expression pattern. J Clin
Endocrinol Metab 82(12):4258-4265
Green S, Kumar V, Walter KP, Chambon P. 1986. Structural and
5 functional domains of the estrogen receptor. Cold Spring Harbor
Symposia on Quantitative Biology LI:751-758.
Gluzman Y, Reichl H, Solnick D. 1982. Helper-free adenovirus type-5
vectors. In Eukaryotic Viral Vectors, Y Gluzman ed., (Cold Spring Harbor
Laboratory), pp 187, 192.
Hamer DH. 1988. Metallothionein. Ann Rev Biochem 55:913-951.
lafrati MD, Karas RH, Aronovitz M, Kim S, Sullivan TR, Lubahn DB,
O'Donnell Jr. TF, Korach KS, Mendelsohn ME. 1997. Estrogen inhibits
the vascular injury response in estrogen receptor-a-deficient mice. Nature
Med 3(5):545-548.
Jacob C, Maret W, Vallee BL. 1998. Control of zinc transfer between
thionein, metallothionein and zinc proteins. Proc Natl Acad Sci (USA)
95:3489-3494.
CA 02352203 2001-05-24
WO 00/3?681 PCT/US99/29856
47
Jiang L, Maret W, Valee BL. 1998. The glutathione redox couple
modulates zinc transfer from metallothionein to zinc-depleted sorbitol
dehydrogenase. Proc Natl Acad Sci (USA) 95:3483-3488.
Kuiper GGJM, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA.
1996. Cloning of a novel estrogen receptor expressed in rat prostate and
ovary. Proc Natl Acad Sci (USA) 93:5925-5930.Kuiper GGJM, Carlsson
B, Grandien K, Enmark E, Haggblad J, Nilsson S, Gustafsson, JA. 1997.
Comparison of the ligand binding specificity and transcript tissue
distribution of estrogen receptors a and (3. Endocrinology 138(3):863-
870.
Lau K, Leav I, Ho S. 1998. Rat estrogen receptor-a and -(3, and
progesterone receptor mRNA expression in various prostatic lobes and
microdissected normal and dysplastic epithelial tissues of the Noble rats.
Endocrinology 139( 1 ):424-427.
Lobo R. 1995. Benefits and risks of estrogen replacement therapy. Am J
Obstet Gynecol 173(3):982-989.
Lubahn DB, Moyer JS, Golding TS, Couse JF, Korach, KS. 1993.
Alteration of reproductive function but not prenatal sexual development
CA 02352203 2001-05-24
WO 00/37681 PCT/US99/29$56
48
after insertional disruption of the mouse estrogen receptor gene. Proc Natl
Acad Sci (USA) 90:11162-11166.
Mosselman S, Polman J, Dijkema R. 1996. ER(3: identification and
characterization of a novel human estrogen receptor. FEBS Letters
392:49-53.
Ogawa S, Inoue S, Watanabe T, Hiroi H, Orimo A, Hosoi T, Ouchi Y,
Muramatsu M. 1998. The complete primary structure of human estrogen
10 receptor (3 IhER(3) and its heterodimerization with ER a in vivo and in
vitro.
Biochem Biophys Res Comm 243:122-126.
Pan LC, Ke HZ, Simmons HA, Crawford DT, Chidsey-Fink KL, McCurdy
SP, Schafer JR, Kimbro KS, Taki M, Korach KS, Thompson DD. Estrogen
15 receptor-alpha knockout (ERKO) mice lose trabecular and cortical bone
following ovariectomy. 1997. J Bone Mineral Res 12 (Supplement
1 ):S134.
Pettersson K, Grandien K, Kuiper GGJM, Gustafsson J. 1997. Mouse
20 estrogen receptor [3 forms estrogen response element-binding
heterodimers with estrogen receptor-a. Mol Endocrinoi 1 1:1486-1496.
CA 02352203 2001-05-24
WO 00/37681 PCTlUS99/z9856
49
Rivera-Gonzalez R, Peterson DN, Tkalcevic G, Thompson DD, Brown TA.
1998. Estrogen-induced genes in the uterus of ovariectomized rats and
their regulation by droloxifene and tamoxifen. J Steroid Biochem Molec
Biol 64( 1 /2):13-24.
Sagraves R. 1995. Estrogen therapy for postmenopausal symptoms and
prevention of osteoporosis. J Clin Pharmacol 35:2s-10s.
Shughrue PJ, Komm B, Merchenthaler I. 1996. The distribution of
estrogen receptor-/3 mRNA in the rat hypothalamus. Steroids 61:678-
681.
Trembley GB, Tremblay A, Copeland NG, Gilbert DJ, Jenkins NA, Labrie
F, Giguere V. 1997. Cloning, chromosomal localization, and functional
analysis of the murine estrogen receptor (3. Mol Endocrinol 1 1:353-365.
Vallee BL. 1957. A cadmium protein from equine kidney cortex. J Am
Chem Soc 79:4813.
20 Vladusic EA, Hornby AE, Guerra-Vladusic FK, Lupu R. 1998. Expression
of estrogen receptor ~i messenger RNA variant in breast cancer. Cancer
Res 58:210-214.
CA 02352203 2001-05-24
WO 00/37681 PCTNS99/29856
Webb P, Lopez GN, Greene GL,. Baxter JD, Kushner PJ. 1992. The limits
of the cellular capacity to mediate an estrogen
response. Mol Endocrinology. 6(2):157-fi7.
