Note: Descriptions are shown in the official language in which they were submitted.
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CELL LINES AND CELL-BASED ASSAYS FOR IDENTIFICATION OF
ANDROGEN RECEPTOR MODULATORS
Introduction
This application claims the benefit of priority from
U.S. Provisional Patent Application 60/214,392, filed June
28, 2000.
Field of the Invention
The invention relates to cell lines and methods for
using these cell lines in the identification of compounds
having biological activity. Tn particular, the invention
relates to muscle cell lines stably transfected with an
androgen receptor and reporter gene useful in the
identification of compounds which are modulators of the
androgen receptor.
Backcrround of the Invention
The androgen receptor (AR) is a member of the
steroid nuclear-receptor superfamily of ligand-dependent
transcription factors and is widely distributed among
reproductive and nonreproductive tissues, including the
prostate and seminal vesicles, male and female genitalia,
skin, testis, ovary, cartilage, sebaceous glands, hair
follicles, sweat glands, cardiac muscle, skeletal and
smooth muscle, gastrointestinal vesicular cells, thyroid
follicular cells, adrenal cortex, liver, pineal, and
numerous brain cortical and subcortical regions, including
spinal motor neurons (Negro-Vilar, A. JCE&M 1999
54(10):3459-62). As with the other members of the steroid
receptor family, AR has several functional domains
including a DNA binding domain (DBD), and a 261 residue
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ligand-binding domain (LBD) (Mw = 30,245 Da) that contains
the androgen binding site, and is responsible for switching
on the androgen function. The cDNA and amino acid
sequences of human and rat androgen receptors have been
described (Pros. Natl. Acad. Sci. U.S.A. 1988 85: 7211-
7215 ) .
AR is an important target in multiple areas of drug
discovery and patient therapy. In Oncology, for example,
inhibitors (antagonists or partial antagonists) of the
androgen receptor function are useful for the treatment of
androgen dependent prostate cancer while agonists or
partial agonists of the AR are applicable to the treatment
of breast cancer. For metabolic and endocrine diseases
disorders, for example, agonists or partial agonists of the
androgen receptor function are useful for the treatment of
age-related diseases and conditions of Cachexia in several
disease states including, but not limited to, AIDS.
Functional AR has also been identified in various bone
Cells and androgen administration has beneficial effects on
skeletal development and maintenance in men and women.
Progress of androgen therapy has been limited by the
inability to separate desirable androgeniC activities from
undesirable or dose limiting side effects. However, recent
advances in the development of selective estrogen receptor
modulators (SERMS) with a great degree of tissue
selectivity in targeting the estrogen receptor while
eliminating undesired side effects has resulted in the
suggestion of SARMs, selective androgen receptor modulators
(Negro-Vilar, A. JCE&M 1999 54(10):3459-62; Reid et al.
Investigational New Drugs 1999 17:271-284).
General assays and methods for detecting the
transCriptional activity of an intracellular receptor when
exposed to a known ligand or unknown compound have been
described. For example, U.S. Patent 5,071,773 describes an
assay for identifying hormone intracellular receptors,
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ligands for these receptors, and proteins capable of
transcriptionally activating the hormone intracellular
receptors. The assays involve use of a cell containing DNA
encoding a hormone response element such as a promoter
linked to an operative reporter gene and DNA encoding the
intracellular receptor protein. When the cell is exposed
to the hormone or a ligand, a hormone intracellular
receptor complex forms and is delivered to an appropriate
DNA binding region, thereby activating the hormone response
element, which in turn leads to expression of the product
encoded by the reporter gene. Activation of the reporter
gene is detected in accordance with known procedures for
detection of the reporter gene.
U.S. Patent 6,017,924 discloses non-steroidal
compounds characterized as high affinity, high specificity
agonists, partial agonists (i.e. partial activators and/or
tissue-specific activators) and antagonists for androgen
receptors based upon a "cis-trans" or "co-transfection"
assay. Non-steroidal compounds characterized as high
affinity, high specificity agonists, partial agonists (i.e.
partial activators and/or tissue-specific activators) and
antagonists for androgen receptors via the "cis-trans" or
"co-transfection" assay are also described in WO 01/16108,
WO 01/16133, and WO 01/16139. This co-transfection assay
(Evans et al. Science 1988 240:889-95) is suggested to
provide a method for identifying functional agonists and
partial agonists which mimic, or antagonists which inhibit,
the effect of native hormones, and quantifying their
activity for responsive intracellular receptor proteins.
In this assay, CV-1 cells (African green monkey kidney
fibroblasts) are transiently transfected with the plasmid
pRShAR containing the human AR under the constitutive
control of the SV40 promoter and a reporter plasmid MTV-LUC
containing the cDNA of firefly luciferase under the control
of a mouse mammary tumor virus (MTV) long terminal repeat.
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The plasmid pRS-(3-Gal, coding for constitutive expression
of E. coli ~3-galactosidase, is included as an internal
control for evaluation of transfection efficiency and
compound toxicity.
Hydroxyflutamide, a known AR antagonist in most
tissues, has also been suggested to function as a selective
AR modulator (SARM) for effects on IL-6 production by
osteoblasts (Hofbauer et al. J. Bone Miner. Res. 1999
14:1330-1337). Selectivity of hydroxyflutamide was
assessed by evaluating the proliferation and
differentiation of a human fetal osteoblast cell line
(HFOB/AR-6) that expresses a mature osteoblast phenotype
and a physiological number of androgen receptors in the
presence of this compound.
Hydroxyflutamide and Casodex, both known to be full
AR antagonists in most tissues, have also been shown, in
AR-transfected PC3 cells, to activate MAP kinases Erk-1 and
Erk-2 in an AR dependent fashion similar to
dihydrotestosterone (DHT; Peterziel et. al. Oncogene 18,
6322-6329 (1999)).
The compound LGD2226, a non-steroidal AR agonist,
has also been characterized as a selective androgen
receptor modulator for use in the treatment of androgen-
related diseases such as osteoporosis, male hormone
replacement, male and female sexual dysfunction and
cachexia based upon its activity in the CV-1 assay
described supra (SCRIP - World Pharmaceutical New FILED 12
May 2000; WO 01/16108; WO 01/16133; and WO 01/16139).
U.S. Patent 5,952,488 describes a bioassay for
androgenic materials in cell culture wherein HeLa cells or
PC-3 cells are transiently transfected or stably integrated
with a DNA sequence cloned from the probasin (PB) gene
promoter region coupled to a CAT reporter gene.
U.S. Patent 5,506,102 describes methods and assays
useful in screening compounds for potential antagonists of
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steroid intracellular receptor mediated transcription
wherein cells are transfected with a first vector encoding
the intracellular receptor, a second vector encoding the
PR-A isoform of human progesterone receptor and a third
vector encoding a reporter gene.
Applicants have now developed for the first time
cell lines and assays in which an AR and reporter have been
stably transfected into muscle tissue cells.
Summary of the Invention
An object of the present invention is to provide
muscle cell lines comprising a mammalian androgen receptor
stably introduced into said muscle cells. These cell lines
are useful in functional transactivation assays to assess
the efficacy of compounds as androgen receptor modulators
in a muscle cell background.
Another object of the present invention is to provide
functional transactivation assays for use in assessing the
efficacy of compounds as androgen receptor modulators in a
muscle cell background via these stable C2C12 mouse
skeletal muscle cell lines comprising the mammalian
androgen receptor.
Yet another object of the present invention is to
provide androgen receptor modulators, and in particular
selective androgen receptor modulators, identified via
functional transactivation assays with stable C2C12 mouse
skeletal muscle cell lines comprising a mammalian androgen
receptor.
Detailed Description of the Invention
The present invention relates to muscle cell lines
stably introduced with a mammalian androgen receptor and
reporter gene.
Various muscle cells can be used in the present
invention. In a preferred embodiment, the muscle cell line
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comprises stable C2C12 mouse skeletal muscle cells.
However, other exemplary muscle cells useful in the present
invention include, but are not limited to, mouse G-7, G-8,
P19 and Sol8 cells, rat H9c2(2-1), L6 and L8 cells, and
human SJRH30(RMS13) cells.
The muscle cell lines of the present invention
further comprise a mammalian androgen receptor which is
stably introduced into the muscle cells. Androgen
receptors useful in the present invention have been
isolated from various mammalian species. These receptors
and their sequences have been described in detail in the
prior art. For example, see U.S. Patent 5,614,620. In
addition, rat androgen receptors are set forth in Genbank
Accession No. M23264 and J05454, as well as by Chang et al.
(Science 1988 240(4850):324-326). Mouse androgen receptors
are set forth in Genbank Accession No. M37890 and by Gaspar
et al. (Pros. Natl Acad. Sci. USA 1991 88:8606-8610) and He
et al. (Biochem. Biophys. Res. Commun. 1990 171(2):697-
704). A guinea pig androgen receptor has also been
described by He et al. (Biochem. Biophys. Res. Commun. 1990
171(2):697-704). He et al. (Biochem. Biophys. Res. Commun.
1990 171(2):697-704) also describes a dog androgen receptor
as does Genbank Accession No. AF197950. A hamster androgen
receptor is described by Shiba et al. (J. Dermatol. Sci.
2001 26(3):163-8. In addition, human androgen receptors
are set forth in Genbank Accession No. M34233 and by
Trapman et al. (Biochem. Biophys. Res. Commun. 1988
153(1):241-248) and Tilley et al. (Proc. Nat1 Acad. Sci.
USA 1989 86(1):327-331). In a preferred embodiment, the
cell lines of the present invention comprise a rat androgen
receptor.
The cell lines of the present invention are useful in
assessing the activity of compounds as androgen receptor
modulators in a muscle Cell background.
In one embodiment, the cell line comprises stable
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C2C12 mouse skeletal cells containing a full length rat
androgen receptor such as that set forth in GenBank
Accession No. M23264. This cell line is referred to herein
as Stable 1.
To generate the Stable 1 cell line containing the
full length rat androgen receptor (rAR), the C2C12 mouse
skeletal cell line (Yaffe D. and Saxel, O. Nature 1977
270:725-727) was transfected with a plasmid, pIRESneo/rAR,
encoding a bicistronic message containing a full length rAR
and the neomycin resistance gene (Jackson et al. Trends
Biochem. Sci. 1990 15:477-483; Jang et al. J. ZTirol. 1988
62:2636-2643). Specifically, 50 ~Cg pIRESneo/rAR were
transfected into C2C12 cells using LipofectAmine PlusT"'
reagent (Gibco BRL) with 250 ~,l plus reagent and 375 ~tl
lipofectamine reagent in 10 milliliters optiMEM media
(Gibco BRL) in accordance with the manufacturer's
instructions. Cells (0.75 x 105) in 10 milliliters growth
media (Dulbecco's modified Eagle medium (DMEM) high glucose
supplemented with loo FBS, 1X sodium pyruvate and 0.5X
antibiotic-antimycotic (all from Gibco BRL)), referred to
hereinafter as Stable 1 growth media, were plated onto each
of five 10-cm culture plates. The following day, the media
was removed from each dish and replaced with 4.5
milliliters optiMEM. Two milliliters of the transfection
mixture were then added to each dish. After a three hour
incubation, the transfection media was removed and replaced
with 6.5 milliliters of growth media. The cells were
allowed to grow for 24 hours in non-selection media. To
select for individual cells stably transfected with
neo/rAR, the cells were split 1:15 into Stable 1 growth
media supplemented with 800 ~g/ml 6418 and allowed to
propagate as separate clonal cell lines. After fourteen
days, a total of 80 resistant clones were isolated. Clones
exhibiting normal growth characteristics were transiently
transfected with the enhancer/promoter/reporter construct,
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pGL3/2X DR-1/luciferase. Stable 1 cells are identified as
clones showing a significant increase in luciferase
activity, as measured via the Steady-GloT"" Luciferase Assay
System (Promega), upon addition of 0.1 ~,M
dihydrotestosterone (DHT). In a preferred embodiment, the
Stable 1 cell line exhibits approximately a 12-fold
increase or greater in luciferase activity upon addition of
the DHT.
Stable 1 cells of the present invention comprising a
stable C2C12 mouse skeletal cell line containing a full
length rat androgen receptor were sent for deposit on June
12, 2001 to the American Type Culture Collection (ATCC),
10801 University Boulevard, Manassas, VA USA 20110-2209.
Twenty-five vials of Stable 1 cells, with an approximate
activity of 30,000 specific relative luminescence units
(RLUs) in the presence of 100 nM DHT in the transactivation
assay described infra, were shipped to the ATCC. The ATCC
Deposit Number for the Stable 1 cell line is XXX.
In another embodiment, the stable C2C12 mouse
skeletal cell line contains a full length rat androgen
receptor plus an enhancer/promoter/reporter construct.
This cell line is referred to herein as Stable 2.
Various enhancer/promoter constructs can be used in
construction of the Stable 2 cell line. In a preferred
embodiment, the enhancer comprises an androgen response
element (ARE). Exemplary AREs used in these constructs
include, but are not limited to, C3-l, PB-ARE, and DR1.
C3-1 is a consensus ARE/GRE (glucocorticoid receptor
response element) isolated from the C3 subunit promoter of
the gene for rat prostatic binding protein. 2XC3,
containing two C3-1 elements, comprises a consensus
enhancer sequence for AR and GR (Claessens et al. J. Biol.
Chem. 1996 271:19013-19016). PB-ARE is an androgen
receptor specific response element isolated from the
promoter of the rat probasin gene (Claessens et al. J.
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Biol. Chem. 1996 271:19013-19016). The DRl response
element is also androgen receptor specific, however, it was
derived synthetically from a pool of degenerate
oligonucleotides containing a consensus ARE/GRE using a
random sequence selection and amplification method. 1X DR-
1, containing 1 DR-1 element, and 2X DR-1, containing two
DR-1 elements, have both been reported as specific for AR
(Zhou et al. J. Biol. Chem. 1997 272:8227-8235). Each DR1
element consists of two AR core binding sites oriented as
an overlapping direct repeat (Zhou et al.. J. Biol. Chem.
1997 272:8227-8235).
Various promoters can also be used in these
constructs. Exemplary promoters include, but are not
limited to, SV40, CMV, beta-globin, and HSVtk. However, as
will be understood by those of skill in the art upon
reading this disclosure, other promoters useful in the
present invention can be routinely selected.
In a preferred embodiment, the enhancer/promoter
construct of the Stable 2 cell line comprises pGL3/2X DR-1
which carries the stronger SV40 promoter. 2XDR-1 was
reported to be an AR specific response element in CV-1
cells (Zhou et. al. J. Biol. Chem. 1997 272:8227-8235). It
was developed by random mutagenesis of an AR/GR consensus
enhancer sequence. Experiments described in detail in
Example 2 showed 2X DR-1 to exhibit better stimulation and
selectivity upon addition of DHT as compared to
enhancer/promoter constructs comprising AREs C3, 1X DR-l,
as well as PB-ARE. However, alternative enhancer/promoter
constructs which can be used to construct the Stable 2 cell
line of the present invention include, but are not limited
to, pGL3/2XC3, pGL3/1XDR-1, pGL3/PB-ARE, HSVtk/2XC3,
HSVtk/1XDR-1, HSVtk/2XDR-1 and HSVtk/PB-ARE. Construction
of these enhancer/promoter constructs is described in
detail herein in Example 1.
Various reporter genes can also be used in the
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construct. Examples include, but are not limited to,
luciferase, beta-galactosidase, secretory alkaline
phosphatase, beta-lactamase, numerous green fluorescence
proteins, and chloramphenicol acetyltransferase. In a
preferred embodiment, the reporter gene is luciferase.
To generate the Stable 2 cell line containing the
full length rat androgen receptor plus the
enhancer/promoter/reporter construct, pGL3/2X DR-
1/luciferase, cells of the Stable 1 cell line were
cotransfected with a plasmid containing the
enhancer/promoter/reporter construct and a plasmid
conferring resistance to hygromycin B (pcDNA3.l-/Hygro,
Invitrogen, Carlsbad, CA). Specifically, 60 ~,g pGL3/2XDR-1
luciferase and 15 ~.g pCDNA3.1-/Hygro were transfected into
Stable 1 cells using LipofectAMINE PlusT"' reagent (Gibco
BRL) with 300 ~,l plus reagent and 450 ~,l lipofectamine
reagent in 12 milliliters optiMEM media (GibcoBRL) in
accordance with the manufacturer's instructions. Cells
(6.0 x 105) in 10 milliliters of Stable 1 growth media
supplemented with 800 ~,g/ml 6418 were plated onto each of
six 10-cm culture plates. The following day, the media was
removed from each dish and replaced with 4.5 milliliters
optiMEM. Two milliliters of the transfection mixture were
then added to each dish. After a three hour incubation,
the transfection media was removed and replaced with 6.5
milliliters of Stable 1 growth media. The cells were
allowed to grow overnight and then split 1:18 and 1:24 into
Stable 1 growth media supplemented with 800 ~,g hygromycin B
to select for individual cells stably transfected with the
enhancer/promoter/reporter construct as well as hygromycin
B resistance and then allowed to propagate as separate
clonal lines. After fourteen days, resistant clones were
isolated and clones exhibiting normal growth
characteristics were tested for luciferase activity in the
presence of 0.1 ~.M DHT. Clones with activities ranging
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from 3- to 12-fold increase over background were expanded.
Further characterization using standard reference compounds
DHT, fluoxymestrone, oxandrolone and medroxyprogesterone
acetate was performed on several of these clones. In a
preferred embodiment, the Stable 2 cell Line exhibits a 12X
increase or greater over background in luciferase activity
upon addition of the DHT with an ECSO in the sub-nanomolar
range. The expected activity was exhibited when the Stable
2 cells were exposed to the other reference compounds.
Stable 2 cells of the present invention comprising a
stable C2C12 mouse skeletal cell line containing a full
length rat androgen receptor and a stably transfected
pGL3/2X DR-1/luciferase reporter were sent for deposit on
June 12, 2001 to the American Type Culture Collection
(ATCC), 10801 University Boulevard, Manassas, VA USA 20110-
2209. Twenty-five vials of Stable 2 cells, with an
approximate activity of 30,000 specific relative
luminescence units (RLUs) in the presence of 100 nM DHT in
the transactivation assay described infra, were shipped to
the ATCC. The ATCC Deposit Number for the Stable 2 cell
line is XXX.
The present invention also relates to functional
transactivation assays developed to assess the activity of
compounds as androgen receptor modulators in a muscle cell
background via detection of expression of a reporter gene.
By demodulator", for purposes of the present invention, it
is meant to be inclusive of agonists, partial agonists,
antagonists, and/or partial antagonists of AR.
In these assays, efficacy of a compound as an
androgen receptor agonist or partial agonist is assessed by
contacting either Stable 1 cells transiently transfected
with an androgen response element, a promoter and a
reporter gene or Stable 2 cells with a compound and
detecting reporter gene expression in the cells in the
presence of the compound. An increase in reporter gene
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expression in the cells in the presence of the compound, as
compared to control cells not contacted with or exposed to
the compound, is indicative of the compound being an
androgen receptor agonist or partial agonist in muscle
cells.
Efficacy of a compound as an androgen receptor
antagonist or partial antagonist is assessed by a
competition assay wherein the ability of a compound to
prevent the induction of expression of a reporter gene by
DHT in Stable 1 or Stable 2 cells is determined. In a
preferred embodiment, approximately 1 nM DHT is used in the
assay to induce expression of the reporter gene. A
decrease in reporter gene expression in the presence of the
compound as compared to control cells exposed to DHT, but
not contacted with the compound, is indicative of the
compound being an androgen receptor antagonist or partial
antagonist in muscle cells. These assays can be used to
determine the concentration at which the compound inhibits
DHT induction by 50%, also referred to as the ICso.
More specifically, a first assay of the present invention,
referred to herein as Androgen Receptor Transactivation
Assay (ARTA) Stable 1, uses the Stable 1 cell line, which
stably expresses the full length rat androgen receptor but
requires the transient transfection of an
enhancer/promoter/reporter construct. In this assay,
Stable 1 cells are plated, preferably in a 96 well format,
at approximately 5,000 to 10,000 cells/well, preferably
6,000 cells/well, in high glucose DMEM without phenol red
(Gibco BRL, Cat. No.: 21063-029) containing 10o charcoal
and dextran treated FBS (HyClone Cat. No.: SH30068.02), 50
mM HEPES Buffer (Gibco BRL, Cat. No.: 15630-080), 1X MEM Na
Pyruvate (Gibco BRL, Cat. No.: 11360-070), 0.5X Antibiotic-
Antimycotic, and 800 ~.g/ml Geneticin (Gibco BRL, Cat. No.:
10131-035). Once the cells have adhered and acclimated and
reached optimal confluency for transfection, approximately
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twenty-four hours after plating, the cells are transfected
with an enhancer/promoter/reporter construct such as
pGL3/2XDR-1/luciferase.
Preferably, the transfection is performed using
LipofectAMINE PlusT"' Reagent (Gibco BRL, Cat. No.: 10964-
013). In this preferred embodiment, pGL3/2XDR-1/luciferase
DNA (approximately 5 ng/well) and a carrier, such as Salmon
Sperm DNA (50 ng/well) or a generic plasmid DNA, are
diluted with 5 ~,l/well Opti-MEM media (Gibco BRL, Cat. No.:
31985-070). To this, 0.5 ~,1/well Plus reagent is added.
This mixture is incubated for 15 minutes at room
temperature. In a separate vessel, 0.385 ~1/well
LipofectAMINE reagent is diluted with 5 ~,l/well Opti-MEM.
The DNA mixture is then combined with the LipofectAMINE
mixture and incubated for an additional 15 minutes at room
temperature. During this time, the media from the cells is
removed and replaced with 60 ~,l/well of Opti-MEM. To this
is added 10 ~.l/well of the DNA/LipofectAMINE transfection
mixture. The cells are incubated for 4 hours. The
transfection mixture is removed from the cells and replaced
with 90 ~tl of the high glucose DMEM described supra.
Other transfection methods which can be used in the
present invention include, but are not limited to, DEAE-
dextran, calcium phosphate, direct microinjection,
electroporation, and biolistic particle delivery.
Compounds to be tested for activity in this assay are
then placed in each well. In a preferred embodiment, 10 ~1
of appropriate compound dilution is placed in each well.
It is preferred that a range of concentrations of compound,
i.e. from about 0.001 nM to 3000 nM, be tested. It is also
preferred that initial dilutions of compounds be made in
dimethylsulfoxide or ethanol and that subsequent dilutions
be made in assay media. Twenty-four hours later activity
of the compound is detected via a detection system such as
the Steady-GloTMLuciferase Assay System (Promega, Cat. No.:
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E2520), or via other luciferin substrates (Tropix or
Packard Biosciences) according to the manufacturers'
instructions.
A second assay of the present invention, referred to
herein as ARTA Stable 2, uses the Stable 2 cell line,
derived from Stable 1 which stably expresses both rat
androgen receptor and an ARE enhancer/promoter/reporter
construct. The enhancer/promoter/reporter construct used
in this system preferably comprises pGL3/2XDR-1/luciferase.
In the ARTA Stable 2 assay, Stable 2 cells are
plated, preferably in 96 well format, at approximately
5,000 to 10,000 cells/well, preferably 6,000 cells/well, in
high glucose DMEM without phenol red (Gibco BRL, Cat. No.:
21063-029) containing 10% charcoal and dextran treated FBS
(HyClone Cat. No.: SH30068.02), 50 mM HEPES Buffer (Gibco
BRL, Cat. No.: 15630-080), 1X MEM Na Pyruvate (Gibco BRL,
Cat. No.: 11360-070), 0.5X Antibiotic-Antimycotic, 800
~.g/ml Geneticin (Gibco BRL, Cat. No.: 10131-035) and 800
~,g/ml Hygromycin B (Gibco BRL, Cat. No.: 10687-010).
Approximately 24 hours later, the media on the cells is
removed and replaced with 90 ~l fresh assay media.
Compounds to be tested for activity in this assay are
then placed in each well. In a preferred embodiment, a 10
~.1 aliquot of compound at a concentration ranging from
about 0.001 nM to 3000 nM, is placed in each well. It is
preferred that initial dilutions of a compound be made in
dimethyl sulfoxide or ethanol and subsequent dilutions be
made in assay media. After 24 hours, activity is detected
via the Steady-GloT"" Luciferase Assay System(Promega, Cat.
No.: E2520) or via other luciferin substrates (Tropix or
Packard Biosciences) according to the manufacturers'
instructions.
An agonist or partial agonist, for purposes of the
present invention, is defined as any compound that achieves
500 of the maximal activity of DHT at a concentration less
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than or equal to 3000 nM (3 ~,M) in the transactivation
assay of the present invention.
An antagonist or partial antagonist, for purposes of
the present invention, is defined as any compound that is
able to inhibit by 50% the maximal activity of 1 nM DHT at
a concentration less than or equal to 3000 nM in the
transactivation assay of the present invention.
The assays of the present invention are particularly
useful in identifying specific or selective androgen
receptor modulators or SARMs. By "SARM" it is meant an
androgen receptor modulator exhibiting a difference-in-kind
of the modulation effected in one type of tissue, i.e.
tumors, containing the androgen receptor relative to the
modulation effected in other tissues, i.e. nontumor
tissues, containing the androgen receptor.
In this embodiment, the agonist or antagonist
activity of a potential SARM is measured in an assay of the
present invention to ascertain activity of the compound in
a muscle cell background. Activity of the potential SARM
can also be measured in other nontumor cells lines such as
primary rat prostate epithelial and stromal cells, primary
guinea pig smooth muscle cells, primary smooth-muscle cells
from immature (I-PSMC) or adult (A-PSMC) rat penis, primary
rabbit smooth muscle cell line, prostatiC smooth muscle
cell line PS-1, prostatiC smooth muscle cell line PSMC1,
mouse bone cell cultures and osteoblasts cells and primary
rat seminal vesicle lines SVC-1 and SCV-2. Such cell lines
are described in the following exemplary references and the
references contained therein: Nemeth et. al. J. Andrology
19, 718-724 (1998), Zhuang et. al. J. Steroid Biochem. Mol.
Biol. 41, 693-696 (1992), Zhang et. al. Prostate 30,
117-129 (1997), Ricciardelli et. al. J. Endocrinol. 140,
373-383 (1994), Gonzalez-Cadavid et. al. Mol. Cell.
Endocrinol. 90, 219-229 (1993), Sadeghi-Nejad et. al. Int.
J. Impotence Res. 10, 165-169 (1998), Gerdes et. al.
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Endocrinology 139, 3569-3577 (1998), Sarah et. al. J. Cell.
Physiol. 185, 416-424 (2000), Chen et. al., FEBS Letters
491, 91-93 (2001) and Tajana et. al. EMBO J. 3, 637-644
(1984).various methods for identifying SARMs having
antagonist activity against hormone-dependent tumors while
exhibiting no activity, or more preferably agonist activity
against other nontumor tissues containing the androgen
receptor can be used.
The agonist or antagonist activity of the potential
SARM is then also ascertained in hormone-dependent tumors
via screening for inhibition of growth in hormone-dependent
tumor cell lines. Examples of hormone-dependent tumor cell
lines which can be used for screening potential SARMs
include, but are not limited to, human breast tumor cell
line MDA MB453, human breast tumor cell line ZR-75-1,
murine breast line Shionogi, rat prostate adenocarcinoma
line Dunning R-3327, human prostate tumor cell line MDA PCa
2a and PCa 2b, human prostate cell line LNCap, human
prostate tumor cell line CWR22, human prostate tumor cell
line LuCaP 35 and LuCaP 23.12, human prostate cell line
LAPC-4 and LAPC-9, human prostate tumor cell line PC-295,
human prostate tumor cell line PC-310, and human
osteosarcoma cell line MG-63. These experimental human and
murine prostate and breast cell lines are well accepted by
those of skill in the art as indicative of the pharmacology
of human hormone-dependent tumors, such as prostate cancer.
Examples of the relationship of such models to the human
disease state can be found in, but are not limited to, the
following references and the references contained therein,
Jacques et. al. Endocrinology 140, 416-421 (1999); Yeap et.
al. Endocrinology 140, 3282-3291 (1999), Sharma et. al.
Oncogene 18, 5349-5355 (1999), Isaacs, J. T. Urol. Oncol.
2, 115-116 (1996), Bentei et. al. In Vitro Cell Dev. Biol.
35, 655-662 (1999), Suzuki et. al. J. Steroid Biochem. Mol.
Biol. 37, 559-567 (1990), Peehl, D. M. Urol. Oncol. 2,
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100-102 (1996), Wytske et. al. Urol. Oncol. 2, 122-125
(1996), Leland, C. W. K. Urol. Oncol. 2, 126-128 (1996),
Buhler et. al. The Prostate 43, 63-70 (2000), Navone et.
al. Clin. Cancer Res. 6, 1190-1197 (2000), Etreby et. al.
The Prostate 42, 99-106 (2000), Jongsma et. al. Cancer Res.
60, 741-748 (2000), Jongsma et. al. Amer. J. Path. 154,
543-551 (1999), Ye et. al. Clin. Cancer Res. 5, 2171-2177
(1999), Navone et. al. Clin. Cancer Res. 3, 2493-2500
(1997), Klein et. al. Nature Medicine 3, 402-408 (1997),
Chen et. al. Cancer Res. 58, 2777-2783 (1998), and Craft
et. al. Cancer Res. 59, 5030-5036 (1999).
Preferred SARMs identified via assays of the present
invention are those exhibiting antagonist activity in
tumors versus agonist activity in other, nonmalignant
tissues containing the androgen receptor. SARMs identified
in accordance with these assays as agonists of androgen
receptors in muscle tissue are useful in inhibiting muscle
wasting and cachexia oftentimes observed in patients
suffering from cancer or AIDS.
The following nonlimiting examples are provided to
further illustrate the present invention.
EXAMPLES
Example 1: Construction of Plasmids
Androgen Receptor Plasmid pIRESneo/rAR
The rat androgen receptor (GenBank Accession No.
M23264) was subcloned as a NotI fragment into the NotI site
of pIRESneo (Clontech Laboratories, Palo Alto, CA).
ARE/Luciferase Reporter Plasmids
A series of luciferase reporter constructs containing
known androgen receptor response elements (AREs), C3, DR-1
and PB-ARE were prepared in the pGL3-Promoter vector
(Promega Corporation, Madison, WI).
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pGL3/1XDR-1/Luciferase
Equimolar amounts of the complementary
oligonucleotide DR-1(F) and DR-1(R) were annealed and then
ligated into the XhoI digested pGL3-Promoter plasmid
(Promega Corporation). The oligonucleotide DR-1(F) has the
sequence:
5'-TCGAGTCCTGAAGGAACGGAACAGACTGA-3' (SEQ ID N0:1). The
oligonucleotide DR-1(R) has the sequence:
5'-TCGATCAGTCTGTTCCGTTCCTTCAGGAC-3' (SEQ ID N0:2).
pGL3/2XDR-1 Luci ferase
A second DR-1 response element was inserted upstream
of the existing DR-1 element in pGL3/1XDR-1/Luciferase by
annealing equimolar amounts of the complementary
oligonucleotide 1XDR-1(F) and 1XDR-1(R) and then ligating
into SacI/XhoI digested pGL3/1XDR-1/Luciferase plasmid.
The oligonucleotide 1XDR-1(F) has the sequence:
5'-CGTCCTGAAGGAACGGAACAGACTGA-3' (SEQ ID N0:3). The
oligonucleotide 1XDR-1(R) has the sequence:
5'-TCGATCAGTCTGTTCCGTTTTTCCTTCAGGACGAGCT-3' (SEQ ID N0:4).
pGL3/2XC3-1/Luciferase
Equimolar amounts of the complementary
oligonucleotides C3-1(F) and C3-1(R) were annealed and then
ligated to each other. Gel purified dimers were then
ligated into the XhoI digested pGL3-Promoter plasmid
(Promega Corporation). The oligonucleotide C3-1(F) has the
sequence:
5'-TCGAGTACATAGTACGTGATGTTCTCAA-3' (SEQ ID N0:5). The
oligonucleotide C3-1(R) has the sequence:
5'-TCGATTGAGAACATCACGTACTATGTAC-3' (SEQ ID N0:6).
pGL3/2XPB-ARE/Luciferase
Equimolar amounts of the complementary
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oligonucleotide PB-ARE-2F and PB-ARE-2R were annealed and
then ligated to each other. Gel purified dimers were then
ligated into the XhoI digested pGL3-Promoter plasmid
(Promega Corporation). The oligonucleotide sequence of PB-
ARE-2F has the sequence:
5'-TCGAGTAATAGGTTCTTGGAGTACTTTACGG-3' (SEQ ID N0:7). The
oligonucleotide sequence of PB-ARE-2R has the sequence:
5'-TCGACCGTAAAGTAACTCCAAGAACCTATTAC-3' (SEQ ID NO:8).
pGL3/HSVtk
This vector was prepared by replacing the SV40
promoter in pGL3-Promoter plasmid (Promega Corporation)
with the HSVtk (Herpes Simplex Virus Thymidine Kinase)
promoter from pRL-TK (Promega Corporation). Both promoters
are contained within Bgl II/Hind III fragments and were
easily exchanged by ligating the HSVtk fragment from pRL-TK
into the Bgl II/Hind II digested pGL3-Promoter vector.
pGL3/HSVtk/IXDR-1/Luciferase, pGL3/HSVtk/2XDR-1/Luciferase,
pGL3/HSVtk/2XC3-1/Luciferase and pGL3/HSVtk/2XPB-
ARE/Luciferase
These constructs containing the HSVtk promoter in
place of the SV40 promoter were prepared by replacing the
SV40 promoter in the respective parent plasmid with the
HSVtk promoter from pRL-TK as described for pGL3/HSVtk.
Example 2: Selection of Enhancer/Reporter Construct
Two sets of vectors with luciferase as a reporter
were constructed and tested in the C2C12 mouse skeletal
muscle cell line. The first variant of the luciferase
reporter construct carried a strong promoter, in this
specific example the SV40 promoter. The second variant of
the luciferase reporter construct carried a basal promoter,
in this specific example the HSVtk promoter. To select the
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most effective androgen response element (ARE) to drive
expression of the luciferase gene, both SV40 and HSVtk
promoters were coupled to four different AREs, C3, DR-1 (1X
and 2X) and PB-ARE. The C3 enhancer is a strong androgen
dependent regulatory element with a crossover activity with
Glucocorticoid Receptor (GR). Both DR-1 and PB-ARE are
considered to be specific androgen response elements.
A transient transactivation experiment was performed
in which CMVrAR was cotransfected with the aforementioned
enhancer/promoter/reporter construct (10:1 receptor to
enhancer/promoter/reporter) in C2C12 cells using
LipofectAMINE PlusT"" reagent (GibcoBRL) according to the
manufacturer's instructions was used to compare the
activities of the enhancer/promoter/reporter constructs.
Specifically, 10,000 cells/well were plated in growth media
(Dulbecco's modified Eagle Medium (DMEM) high glucose
supplemented with 10% FBS, 1X sodium pyruvate and 0.5X
antibiotic-antimycotic (all from GibcoBRL)). The next day,
the media was removed and replaced with optiMEM media
(Gibco BRL). The transfection mixture was prepared so that
10 ~,l added to each well resulted in 0.05 ~.g/well receptor,
0.005 ~,g/well enhancer/promoter/reporter construct and
0.385 ~,l/well lipofectamine. After three hours of
incubation, the transfection mixture was removed and
replaced with growth media which had been made using
charcoal/dextran FBS (Hyclone, Logan UT). The method of
detection used was the Steady-GloT"' Luciferase Assay System
(Promega Corporation) with counting performed on a Packard
TopCount (Packard Instrument Co. Downers Grove, IL).
Results showed that C3, DR-l, and PB-ARE/HSVtk-
luciferase reporter constructs had a lower background
signal as compared to C3, DR-1, and PB-ARE/SV40 luciferase
reporters. Addition of 1 ~.M testosterone gave a 3.5 fold
increase over background with C3/HSVtk/luciferase, and a
2.0-, 2.0- and 6.5-fold increase with 2XPB-ARE-
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HSVtk/luciferase, IXDR-1/HSVtk/luciferase and 2XDR-
1/HSVtk/luciferase, respectively. The constructs with the
greatest fold window of stimulation, C3 and DR-
1/HSVtk/luciferase, both showed a minimum 100-fold
selectivity of testosterone over dexamethasone when tested
in dose response experiments. Therefore, for reasons of
fold window of stimulation and selectivity, the 2XDR-1
construct was selected. Although in the transiently
transfected receptor system, the HSVtk promoter seemed
preferable due to the lower background, when tested in the
Stable 1 cell line the signal greatly diminished.
Therefore, the construct used in the production of the
Stable 2 cell line was pGL3/2X DR-1/luciferase which
carries the stronger SV40 promoter.
What is Claimed is:
1. A stable muscle cell line comprising muscle
Cells and a mammalian androgen receptor stably introduced
into said muscle cells.
2. The stable muscle cell line of claim 1 wherein
the muscle cells comprise C2C12 mouse skeletal muscle
cells.
3. The stable muscle cell line of claim 1 wherein
the mammalian androgen receptor comprises a rat androgen
receptor.
4. The stable muscle cell line of claim 1 further
comprising a stably transfected enhancer/promoter/reporter
construct.
5. The stable muscle cell line of claim ~ wherein
the enhancer comprises an androgen response element.
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SEQUENCE LISTING
<110> Ostrowski, Jacek
Driscoll, Joyce
Lupisella, John
Salvati, Mark
Bristol-Myers Squibb Company
<120> Cell Lines and Cell-Based Assays for Identification of
Androgen Receptor Modulators
<130> D0177 PCT
<140>
<141>
<150> 60/214,392
<151> 2000-06-28
<160> 8
<170> PatentIn Ver. 2.1
<210> 1
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
<400> 1
tcgagtcctg aaggaacgga acagactga 29
<210> 2
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
<400> 2
tcgatcagtc tgttccgttc cttcaggac 29
<210> 3
1
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<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
<400> 3
cgtcctgaag gaacggaaca gactga 26
<210> 4
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
<400> 4
tcgatcagtc tgttccgttt ttccttcagg acgagct 37
<210> 5
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
<400> 5
tcgagtacat agtacgtgat gttctcaa 28
<210> 6
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
<400> 6
tcgattgaga acatcacgta ctatgtac 28
<210> 7
2
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<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
<400> 7
tcgagtaata ggttcttgga gtactttacg g 31
<210> 8
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
<400> 8
tcgaccgtaa agtaactcca agaacctatt ac 32
3
