The present invention contemplates methods of preventing or inhibiting
breast cancer cell proliferation, compounds and compositions that
interfere with or block sex steroid or growth factor binding to
and induction of the Cyr61 gene and methods of screening ligands
that regulate Cyr61 protein expression. The present invention further
contemplates compounds that block Cyr61 activity. The invention
further relates to methods of diagnosing and staging patients with
cancers associated with an upregulation of Cyr61 expression. Assay
methods and kits are also disclosed.
7. An antibody which binds to one or more ligands of a sex steroid
receptor which regulates the promoter of the gene which encodes
8. An antibody as defined in claim 7, wherein said sex steroid
receptor is selected from the group consisting of estrogen receptor,
progesterone receptor, and androgen receptor.
9. An oligonucleotide which binds under high stringency conditions
to a polynucleotide encoding Cyr61.
10. An oligonucleotide as defined in claim 9, wherein said oligonulceotide
is non-naturally occurring.
11. A vector comprising an oligonucleotide as defined in claim
12. A pharmaceutical composition comprising a therapeutically effective
amount of at least one of (i) an antibody as defined in any of claims
1-8; (ii) an oligonucleotide as defined in any of claims 8-9; or
(iii) a vector as defined in claim 11.
13. A method for preventing or inhibiting breast cancer cell proliferation,
said method comprising administering to a subject a breast cancer
cell proliferation blocking or reducing amount of a Cyr61 neutralizing
14. A method as defined in claim 13, wherein said neutralizing
antibody blocks sex steroid induced synthesis of Cyr61 DNA and proliferation
of breast cancer cells.
15. A method as defined in claim 13, wherein said neutralizing
antibody blocks growth factor induced synthesis of Cyr61 DNA and
proliferation of breast cancer cells.
16. A method as defined in claim 15, wherein said growth factor
is selected from the group consisting of epidermal growth factor,
heparin binding epidermal growth factor, and basic fibroblastic
17. A method for diagnosing or staging breast cancer, said method
comprising determining the level of Cyr61 in a breast cancer cell
present in said breast cancer suspected of being positive for breast
cancer and comparing said level to the level of Cyr61 in normal
breast tissue, whereby an increase in the level of Cyr61 in said
suspect tissue over the level of Cyr61 in said normal tissue indicates
the presence of breast cancer in said suspect tissue.
18. A method as defined in claim 17, wherein said level of Cyr61
is determined by exposing said suspect and said normal tissues to
a Cyr61 neutralizing antibody and comparing the amount of antibody
bound by each tissue, wherein an increase in the level of bound
antibody by said suspect tissue over the level of bound antibody
by said normal tissue indicates the presence of breast cancer in
said suspect tissue.
19. A method for diagnosing or staging breast cancer, said method
comprising determining whether breast tissue suspected of being
positive for breast cancer is (i) ER/Cyr61 positive, (ii) PR/Cyr61
positive, (iii) ER/PR/Cyr61 positive, (iv) AR/Cyr61 positive, or
(v) PR/EGFR/Cyr61 positive; whereby the presence of (i), (ii), (iii),
(iv), or (v) indicates a likelihood that said suspect tissue is
20. A method of screening for a compound which inhibits or prevents
breast cancer cell proliferation, said method comprising determining
a first amount of Cyr61 expressed by breast cancer cells exposed
to said compound, wherein said breast cancer cells overexpress Cyr61;
and comparing said first amount of Cyr61 to a second amount of Cyr61
expressed by said breast cancer cells that have not been exposed
to said compound; whereby said first amount being less than said
second amount indicates that said compound may inhibit or prevent
breast cancer cell proliferation.
21. A method of screening for a compound which inhibits or prevents
breast cancer cell proliferation, said method comprising determining
whether said compound inhibits the interaction of sex steroid response
element of the Cyr61 promoter and a sex steroid receptor associated
with the Cyr61 promoter.
22. A method of screening for a compound which inhibits or prevents
breast cancer cell proliferation, said method comprising determining
whether said compound binds with a sex steroid receptor which regulates
the Cyr61 promoter.
23. A transgenic non-human animal comprising DNA which can be induced
to overexpress Cyr61 in breast tissue.
24. The transgenic non-human animal as defined in claim 23, wherein
the DNA is human.
25. A kit for diagnosing or staging breast cancer, said kit comprising
an antibody as defined in any of claims 1-8.
26. A kit for diagnosing or staging breast cancer, said kit comprising
an oligonucleotide as defined in claim 9 or 10.
27. A method for screening compounds that regulate Cyr61 mRNA transcription
through a receptor, said method comprising detecting a difference
in the level of Cyr61 mRNA in a population of cells sufficient to
transcribe a detectable amount of mRNA encoding Cyr61 contacted
with a test compound in comparison with the level of Cyr61 mRNA
in such a population that is not contacted with said test compound.
28. An assay method for detecting the presence of breast cancer,
said method comprising detecting the level of Cyr61 mRNA isolated
from breast cancer tissue, in comparison with the level of Cyr61
mRNA isolated from normal mammary tissue; wherein an upregulation
of Cyr61 mRNA compared to normal mammary tissue indicates the presence
of breast cancer.
29. An assay method for detecting the presence of breast cancer,
said method comprising detecting the level of Cyr61 protein isolated
from breast cancer tissue, in comparison with the level of Cyr61
protein isolated from normal mammary tissue; wherein an upregulation
of Cyr61 protein compared to normal mammary tissue indicates the
presence of breast cancer.
30. A method for preventing or inhibiting breast cancer cell proliferation,
said method comprising administering to a subject, an amount of
a compound effective to inhibit the interaction of a sex steroid
receptor with a sex steroid response element of the Cyr61 promoter.
31. A method as defined in claim 15, wherein said sex steroid is
selected from the group consisting of an estrogenic compound, a
progestational compound, and an androgenic compound.
32. A method as defined in claim 13 or 18, wherein said neutralizing
antibody is an antibody of claims 1-8.
33. A method as defined in claim 22, 23 or 30, wherein said steroid
receptor is selected from the group consisting of estrogen receptor,
progesterone receptor, and androgen receptor.
34. The method as defined in claim 31, wherein said estrogenic
compound is 17.beta.-estradiol
35. An antibody which binds to an epitope of Cyr61.
 The present application claims priority from U.S. Provisional
Patent Application Serial No. 60/213,182, filed Jun. 21, 200, and
U.S. Provisional Patent Application Serial No. 60/291,510, filed
May 16, 2001, which are hereby incorporated by reference in their
FIELD OF THE INVENTION
 The present invention relates to methods of preventing or
inhibiting breast cancer cell proliferation, compounds and compositions
that interfere with or block sex steroid or growth factor binding
to and induction of the Cyr61 gene, and compounds that block Cyr61
activity. The present invention also relates to methods of screening
ligands that regulate Cyr61 protein expression. The invention further
relates to methods of diagnosing and staging patients with cancers
associated with an upregulation of Cyr61 expression. The invention
also describes oligonucleotides, antisense constructs, antibodies,
neutralizing antibodies, and pharmaceutical compositions related
thereto. Transgenic animals are also contemplated by the present
BACKGROUND OF THE INVENTION
 Breast cancer is the leading cause of cancer death among
non-smoking women today (Adami, et al., Sem. In Cancer Biol., 1998,
8:255). Although a number of genetic and environmental factors have
been implicated in the development of mammary epithelial neoplasia,
tumorigenesis appears to be under hormonal regulation.
 An emerging group of growth factor-regulated immediate-early
genes that play a role in development, cell proliferation, and tumorigenesis
belongs to the CCN (CTGF/Cyr61/Cef10/NOVH) family. All CCN proteins
(1) display a high degree of conservation among family members and
across species; (2) are cysteine-rich and structurally similar to
extracellular matrix-associated molecules; (3) are composed of multifunctional
modular domains; and (4) have been shown to mediate a variety of
cell functions such as cell adhesion, cell migration, mitogenesis,
cell survival, and differentiation (Law and Lam, Experimental Cell
Res, 1999, 248:44).
 Cyr61 is a secreted, cysteine-rich heparin-binding protein
that associates with the cell surface and the extracellular matrix.
Specifically, Cyr61 has been shown to be involved in developmentally
regulated processes including, angiogenesis and chondrogenesis.
The Cyr61 protein possesses many biochemical features that resemble
the Wnt-1 protein and other growth factors (Yang and Law, Cell Growth
& Diff, 1991, 2:351). The human Cyr61 protein is 381 amino acids
in length with a molecular mass of about 42 kilo-daltons (kDa).
See FIG. 1 and PCT Application No. WO 97/339950. The Cyr61 gene
is localized in the short arm of chromosome 1 (1p22-31) (Charles
et al., Oncogene, 1991, 8:23; Jay et al., Oncogene, 1997, 14:1753),
and the gene was identified by differential hybridization screening
of a cDNA library of serum-stimulated BALB/c3T3 fibroblasts (See
FIG. 2 and Law and Nathans, P.N.A.S., 1987, 84:1182). Comparison
of the human and murine Cyr61 sequences indicates that they are
91% similar (PCT application No. WO 97/339950).
 The present inventors have found that regulation of Cyr61
expression and activities is useful in the prevention, diagnosis,
and treatment of breast cancer.
SUMMARY OF THE INVENTION
 The present invention provides methods for preventing or
inhibiting breast cancer cell proliferation. These methods comprise
administering to a subject, a Cyr61 neutralizing antibody which
blocks activities associated with the proliferation and/or growth
of breast cancer cells. The neutralizing antibody is used in effective
amounts sufficient to block or reduce Cyr61 activities associated
with breast cancer cell proliferation and/or growth. In one embodiment,
the antibody is conjugated with an anti-tumor agent. In one embodiment,
the neutralizing antibody blocks sex steroid induced synthesis of
Cyr61 DNA and proliferation of breast cancer cells. In an alternative
embodiment, the neutralizing antibody blocks growth factor induced
synthesis of Cyr61 DNA and proliferation of breast cancer cells,
where the growth factor is selected from the group consisting of
epidermal growth factor, heparin binding epidermal growth factor,
and basic fibroblastic growth factor.
 Further contemplated are methods for preventing or inhibiting
breast cancer cell proliferation which comprise administering to
a subject an amount of a compound effective to inhibit the interaction
of a sex steroid response element of the Cyr61 promoter and a sex
steroid receptor associated with the Cyr61 promoter, to block a
sex steroid receptor which regulates the Cyr61 promoter, to inhibit
the synthesis of DNA or mRNA encoding Cyr61, to inhibit the upregulation
of the expression of Cyr61, or to inhibit the binding of Cyr61 to
cognate receptor(s) or interacting protein(s). In a specific embodiment,
the sex steroid is an estrogenic compound, progestational compound,
or androgenic compound. In another embodiment, the sex steroid response
element is an estrogen response element or a progesterone/androgen
response element. In another embodiment, the expression of Cyr61
is upregulated by a growth factor such as, but not limited to, epidermal
growth factor, heparin binding epidermal growth factor, and basic
fibroblastic growth factor.
 The present invention also provides antisense constructs
such as an oligonucleotide which binds under high stringency conditions
to DNA or mRNA encoding Cyr61, a vector comprising such oligonucleotides,
and pharmaceutical compositions comprising a therapeutically effective
amount of such oligonucleotides or vectors. In one embodiment, the
oligonucleotide is non-naturally occurring.
 Also provided are antibodies which neutralize Cyr61 activity
and pharmaceutical compositions containing therapeutically effective
amounts of these antibodies. These antibodies may be polyclonal
or monoclonal, chimeric, humanly acceptable and conjugated to an
anti-tumor agent or antibiotic such as, for example, calicheamicin.
Special mention is made of antibodies which bind to amino acids
163-229 of SEQ ID NO: 2 (see FIG. 1) and antibodies which bind to
amino acids 371-381 of SEQ ID NO: 2 (see FIG. 1). Antibodies that
bind to one or more ligands of a sex steroid receptor, such as,
but not limited to the estrogen receptor, progesterone receptor,
and androgen receptor, which regulates the promoter of the gene
which encodes Cyr61, also are contemplated. An antibody which binds
to an epitope of Cyr61 is also disclosed. Pharmaceutical compositions
comprising an antibody also are contemplated.
 The present invention provides for compounds that inhibit
the interaction of a sex steroid response element of Cyr61 gene
and a sex steroid receptor. The steroid response element resides
within the Cyr61 promoter.
 The present invention also provides for antibodies that
may bind to one or more ligands of a sex steroid receptor which
regulates the promoter gene that encodes Cyr61. Pharmaceutical composition
containing therapeutically effective amounts of these antibodies
are also contemplated.
 Also provided are methods for diagnosing or staging breast
cancer. These methods comprise determining the level of Cyr61 in
a breast cell that is obtained from breast tissue suspected of being
positive for breast cancer and comparing that level to the level
of Cyr61 in normal breast tissue. An increase in the level of Cyr61
in the suspect tissue over the level of Cyr61 in the normal tissue
indicates the presence of breast cancer in the suspect tissue. The
level of Cyr61 in this method can be determined by exposing the
suspect and the normal tissues to an antibody as described above
and then comparing the amount of antibody bound by each tissue.
An increase in the level of antibody bound by the suspect tissue
over the level of antibody bound by the normal tissue indicates
the presence of breast cancer in the suspect tissue.
 Other methods for diagnosing or staging breast cancer comprises
determining whether breast tissue suspected of being positive for
breast cancer is (i) ER/Cyr61 positive, (ii) PR/Cyr61 positive (iii)
ER/PR/Cyr61 positive, (iv) AR/Cyr61 positive, or (v) PR/EGFR/Cyr61
positive. The presence of (i), (ii), (iii), (iv), or (v) indicates
a likelihood that said suspect tissue is cancerous and the aforementioned
results can be utilized to design specific treatment regimens.
 Methods of screening for a compound which inhibits or prevents
breast cancer cell proliferation are also provided. These methods
comprise determining a first amount of Cyr61 expressed by breast
cancer cells exposed to the compound, where the breast cancer cell
overexpresses Cyr61; and comparing the first amount of Cyr61 to
a second amount of Cyr61 expressed by the breast cancer cells that
has not been exposed to the compound. If the first amount is less
than the second amount, this is an indication that the compound
may inhibit or prevent breast cancer cell proliferation.
 Other methods of screening for a compound which inhibits
or prevents breast cancer cell proliferation involve determining
whether the compound inhibits the interaction of a sex steroid response
element of the Cyr61 promoter, the compound binds with a sex steroid
receptor which regulates the Cyr61 promoter, or the compound blocks
interaction with Cyr61 receptors or interacting proteins.
 Additionally, the present invention provides a transgenic
non-human animals comprising DNA, such as, for example, human DNA
which can be induced to overexpress Cyr61 in breast tissue.
 Kits for diagnosing or staging breast cancer are also provided.
These kits include antibodies or oligonucleotides as described above.
 Methods for screening for compounds that regulate Cyr61
mRNA transcription are also provided. Transcription may be regulated
by a receptor or a non-receptor mediated mechanism. These methods
include detecting a difference in the level of Cyr61 mRNA in a population
of cells sufficient to transcribe a detectable amount of mRNA encoding
 Assay systems for detecting the presence of breast cancer
are also provided in which the level of Cyr61 polynucleotide isolated
from breast cancer tissue is detected. An upregulation of Cyr61
mRNA compared to normal mammary tissue indicates the presence of
breast cancer. In an alternate embodiment, the level of Cyr61 protein
isolated from breast cancer tissue, is detected and an upregulation
of Cyr61 protein compared to normal mammary tissue indicates the
presence of breast cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
 FIGS. 1(a-b). An illustration of the amino acid sequence
of the human Cyr61 protein (SEQ ID NO: 2).
 FIGS. 2(a-c). An illustration of the cDNA (SEQ ID NO: 1)
encoding the human Cyr61 protein.
 FIGS. 3(a-b). An illustration of a Northern blot of total
RNA from T47D and MCF-7 adenocarcinoma cells demonstrating the regulation
of Cyr61 mRNA transcription by R5020 and 17.beta.-estradiol, respectively.
 FIG. 4. An illustration of a Northern blot of total RNA
from T47D adenocarcinoma cells demonstrating the effects of transcription
inhibitor actinomycin D and protein synthesis inhibitor cycloheximide
on progestin induced regulation of Cyr61 transcription.
 FIG. 5. An illustration of a Northern blot of total RNA
from MCF-7 adenocarcinoma cells demonstrating the effects of transcription
inhibitor actinomycin D and protein synthesis inhibitor cycloheximide
on estrogen induced regulation of Cyr61 transcription.
 FIG. 6. An illustration of a Western blot of proteins from
T47D adenocarcinoma cells demonstrating the regulation of Cyr61
protein expression by R5020.
 FIG. 7. A graph of the time course of mRNA induction in
T47D and MCF-7 cells after treatment with R5020 and 17.beta.-estradiol,
 FIGS. 8(a-b). An illustration of a Western blot of proteins
from T47D and MCF-7 adenocarcinoma cells demonstrating the upregulation
of Cyr61 protein levels.
 FIGS. 9(a-c). (a-b) Illustrations of Western blots from
ER+/PR+/EGFR+ and ER-/PR-/EGFR+ breast cancer tissues probed for
the upregulation of Cyr61. (c) A bar graph showing the level of
Cyr61 proteins in ER+/PR+/EGFR+ and ER-/PR-/EGFR+ breast cancer
cells compared to normal mammary tissue.
 FIGS. 10(a-f). An illustration of in situ hybridization
studies that indicate the localization of Cyr61 mRNA in breast cancer
 FIGS. 11(a-d). A bar graph comparing a test compound to
the total number of cells showing of the effects of Cyr61 neutralizing
antibodies on estrogen and epidermal growth factor induced cell
proliferation and DNA-synthesis in MCF-7 cells.
 FIGS. 12(a-b). A bar graph comparing a test compound to
the total number of cells showing the effects of Cyr61 neutralizing
antibodies on progestin and serum induced DNA synthesis in T47D
 FIGS. 13(a-b). A bar graph comparing a test compound to
DNA synthesis showing the effects of Cyr61 neutralizing antibodies
on EGF and HB-EGF stimulation of DNA synthesis in breast cancer
 FIGS. 14(a-e). (a and c) An illustration of Northern blots
demonstrating the effects of EGF in T47D and MCF-7 cells, respectively.
(b and d) An illustration of Northern blots demonstrating the effects
of EGF and R5020 in T47D cells (b) or 17.beta.-estradiol and EGF
treated MCF-7 cells (d). (e) A graph demonstrating the time course
of mRNA transcription in cells treated with EGF, EGF and R5020,
and EGF and 17.beta.-estradiol.
 FIGS. 15(a-d). Illustrations of Northern blots demonstrating
the effects of DHT in AR+ MDA-MB-453 (a and b) and ZR-75-1 (c and
d) cells. Kinetics of Cyr61 mRNA induction in MDA-MB-453 (a) and
ZR-75-1 (c) cells was evaluated after treatment with 1.0 nM DHT
at specified time points. Determination of the DHT EC50 for Cyr61
upregulation in cells was evaluated at specified concentrations.
 FIGS. 16(a-d). (a) Illustration of Western blots demonstrating
the time-dependent upregulation of Cyr61 protein in MDA-MB-453 cells
after 0, 0.5, 2.0, 4.0, 8.0 and 24.0 h treatment with 1 nM DHT.
(c) Illustration of Western blots demonstrating the dose-dependent
upregulation of Cyr61 protein in MDA-MB-453 cells at 0, 0.1, 1.0,
10.0, and 100.0 nM DHT for 0.5 h. (b and d) Bar graphs illustrating
Cyr61 protein levels. Numerical values are based on the relative
optical density (OD) of the band size and the total amount of Cyr61
was normalized to the level of cytokeratin. The fold expression
of Cyr61 was calculated by dividing the ratio of Cyr61/cytokeratin
in treated cells by untreated cells. Significant increase in levels
compared to untreated controls, p<0.0001.
 FIGS. 17(a-b). (a) Illustration of Northern blot of total
RNA isolated from MDAMB-453 cells following treatments with 1.0
nM DHT, 10 .mu.g/ml cyclohexamide (Chx.), 1 .mu.g/ml actinomycin
D (Act. D) or 100 nM 2-OH Flu for 0.5 h. (b) A bar graph illustrating
the fold expression of Cyr61 mRNA (as calculated by dividing of
Cyr61/GAPDH)* Significant increase in levels compared to untreated
 FIGS. 18(a-b). Illustration of Northern blot of total RNA
isolated from MDA-MB-231 cells following treatments with 1.0 nM
DHT (a) or 20 ng/ml EGF (b) at specified time points.
 FIGS. 19(a-c). (a-b) Illustration of Western blot analysis
of breast tumors (T) and autologous normal mammary controls (N)
tissue protein extracts of 4 representative patients (#1-4) that
were AR- (a) and 4 representative patients (# 5-8) that were AR+
(b). (c) Bar graph representing Cyr61 increase.
 FIGS. 20(a-b). Bar graphs illustrating the effects of anti-Cyr61
neutralizing antibodies on DHT and EGF-dependent DNA synthesis in
MDA-MB-453 cells.* p<0.0001.
 FIGS. 21(a-b). Bar graphs illustrating the effects of anti-Cyr61
neutralizing antibodies on DHT and EGF-dependent proliferation in
MDA-MB-453 cells.* p<0.0001.
 FIGS. 22(a-d). Line graphs illustrating the effects of anti-Cyr61
neutralizing antibodies on R5020 and EGF-dependent DNA cell growth
in T47D cells. Cell proliferation experiments were performed in
quadruplicates and repeated three times. Numerical values represent
total cell numbers+SEM. *p<0.0001.
 FIGS. 23(a-b). Illustration of Western blot of stage II
invasive ductal breast tumors (T) and autologous normal mammary
controls (N) tissue protein extracts generated from 5 patients (#1-5)
that were PR-/EGFR+ and 5 patients (# 6-10) that were PR+/EGFR+.
DETAILED DESCRIPTION OF THE INVENTION
 The present invention describes methods of preventing or
inhibiting breast cancer cell proliferation, diagnosing or staging
breast cancer, and screening for compounds which inhibit or prevent
breast cancer cell proliferation. These methods evaluate sex steroid
and growth factor mediated regulation of Cyr61 transcription and
translation and levels in samples of interest. The present invention
also advantageously provides for screening assays and kits. The
assay system of the invention is suitable for high throughput screening,
e.g., screening thousands of compounds per assay.
 The present invention also provides Cyr61 nucleic acids,
including oligonucleotide primers, probes, and antisense constructs,
and Cyr61 regulatory sequences; Cyr61-specific antibodies; and related
methods of using these materials to detect the presence of Cyr61
proteins or nucleic acids and in screens for agonists and antagonists
of Cyr61 for breast cancer. The invention also describes pharmaceutical
compositions of these materials.
 The term "isolated" means that the referenced
material is removed from the environment in which it is normally
found. Thus, an isolated biological material can be free of cellular
components, i.e., components of the cells in which the material
is found or produced. In the case of nucleic acid molecules, an
isolated nucleic acid includes a PCR product, an isolated mRNA,
a cDNA, or a restriction fragment. In another embodiment, an isolated
nucleic acid is preferably excised from the chromosome in which
it may be found, and more preferably is no longer joined to non-regulatory,
non-coding regions, or to other genes, located upstream or downstream
of the gene contained by the isolated nucleic acid molecule when
found in the chromosome. In yet another embodiment, the isolated
nucleic acid lacks one or more introns. Isolated nucleic acid molecules
include sequences inserted into plasmids, cosmids, artificial chromosomes,
and the like. Thus, in a specific embodiment, a recombinant nucleic
acid is an isolated nucleic acid. An isolated protein may be associated
with other proteins or nucleic acids, or both, with which it associates
in the cell, or with cellular membranes if it is a membrane-associated
protein. An isolated organelle, cell, or tissue is removed from
the anatomical site in which it is found in an organism. An isolated
material may be, but need not be, purified.
 The term "purified" refers to material that has
been isolated under conditions that reduce or eliminate the presence
of unrelated materials, i.e., contaminants, including native materials
from which the material is obtained. For example, a purified protein
is preferably substantially free of other proteins or nucleic acids
with which it is associated in a cell; a purified nucleic acid molecule
is preferably substantially free of proteins or other unrelated
nucleic acid molecules with which it can be found within a cell.
Purity can be evaluated by chromatography, gel electrophoresis,
immunoassay, composition analysis, biological assay, and other methods
known in the art.
 A "sample" refers to a biological material which
can be tested for the presence of Cyr61 protein or Cyr61 nucleic
acids. Such samples can be obtained from subjects, such as humans
and non-human animals, and include tissue, especially mammary glands,
biopsies, blood and blood products; plural effusions; cerebrospinal
fluid (CSF); ascites fluid; and cell culture.
 The term "non-human animals" includes, without
limitation, laboratory animals such as mice, rats, rabbits, hamsters,
guinea pigs, etc.; domestic animals such as dogs and cats; and,
farm animals such as sheep, goats, pigs, horses, and cows.
 The term "ability to elicit a response" refers
to the ability of a ligand to agonize or antagonize receptor activity.
 The term "transformed cell" refers to a modified
host cell that expresses a functional protein expressed from a vector
encoding the protein of interest. Any cell can be used, but preferred
cells are mammalian cells.
 The term "assay system" is one or more collections
of such cells, e.g., in a microwell plate or some other culture
system. To permit evaluation of the effects of a test compound on
the cells, the number of cells in a single assay system is sufficient
to express a detectable amount of the regulated Cyr61 mRNA and protein
expression. The methods of the invention are suitable cells of the
invention that are particularly suitable for an assay system for
test ligands that modulate transcription and translation of the
 A "test compound" is any molecule, such as, for
example, a sex steroid that can be tested for its ability to modulate
Cyr61 expression and/or activity.
 The terms "cancer" or "tumors" refers
to group of cells that display uncontrolled division. In a specific
embodiment, the cancer is breast cancer and particularly infiltrating
ductal carcinomas. The term "cell proliferation" refers
to the growth of a cell or group of cells.
 The term "humanly acceptable" refers to compounds
or antibodies that are modified so as to be useful in treatment
of human diseases or disorders. In a specific embodiment, antibodies
(polyclonal or monoclonal) are modified so that they are humanly
acceptable. In one embodiment, this requires the antibodies to be
humanized or primatized.
 The use of italics generally indicates a nucleic acid molecule
(e.g., Cyr61 cDNA, gene, etc.); normal text generally indicates
the polypeptide or protein. Alternatively, whether a nucleic acid
molecule or a protein is indicated can be determined by the content.
 The term "amplification" of DNA refers to the
use of polymerase chain reaction (PCR) to increase the concentration
of a particular DNA sequence within a mixture of DNA sequences.
For a description of PCR see Saiki et al., Science, 239:487, 1988.
 The term "sequence-specific oligonucleotides"
refers to related sets of oligonucleotides that can be used to detect
allelic variations or mutations in the Cyr61 gene.
 The term "nucleic acid molecule" refers to the
phosphate ester form of ribonucleosides (RNA molecules) or deoxyribonucleosides
(DNA molecules), or any phosphoester analogs, in either single stranded
form, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA
and RNA-RNA helices are possible. The term nucleic acid molecule,
and in particular DNA or RNA molecule, refers only to the primary
and secondary structure of the molecule, and does not limit it to
any particular tertiary forms. Thus, this term includes double-stranded
DNA found, inter alia, in linear (e.g., restriction fragments) or
circular DNA molecules, plasmids, and chromosomes. In discussing
the structure of particular double-stranded DNA molecules, sequences
may be described according to the normal convention of giving only
the sequence in the 5' to 3' direction along the nontranscribed
strand of DNA (i.e., the strand having a sequence homologous to
the mRNA). A "recombinant DNA molecule" is a DNA molecule
that has undergone a molecular biological manipulation.
 The terms "polynucleotide" or "nucleotide
sequence" is a series of nucleotide bases (also called "nucleotides")
in DNA and RNA, and means any chain of two or more nucleotides.
A nucleotide sequence typically carries genetic information, including
the information used by cellular machinery to make proteins and
enzymes. These terms include double or single stranded genomic and
cDNA, RNA, any synthetic and genetically manipulated polynucleotide,
and both sense and antisense polynucleotide. This includes single-
and double-stranded molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA
hybrids, as well as "protein nucleic acids" (PNA) formed
by conjugating bases to an amino acid backbone. This also includes
nucleic acids containing modified bases, for example thiouracil,
thio-guanine and fluoro-uracil.
 The polynucleotides may be flanked by natural regulatory
(expression control) sequences, or may be associated with heterologous
sequences, including promoters, internal ribosome entry sites (IRES)
and other ribosome binding site sequences, enhancers, response elements,
suppressors, signal sequences, polyadenylation sequences, introns,
5'- and 3'-non-coding regions, and the like. The nucleic acids may
also be modified by many means known in the art. Non-limiting examples
of such modifications include methylation, "caps", substitution
of one or more of the naturally occurring nucleotides with an analog,
and internucleotide modifications such as, for example, those with
uncharged linkages (e.g., methyl phosphonates, phosphotriesters,
phosphoroamidates, carbamates, etc.) and with charged linkages (e.g.,
phosphorothioates, phosphorodithioates, etc.). Polynucleotides may
contain one or more additional covalently linked moieties, such
as, for example, proteins (e.g., nucleases, toxins, antibodies,
signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine,
psoralen, etc.), chelators (e.g., metals, radioactive metals, iron,
oxidative metals, etc.), and alkylators. The polynucleotides may
be derivatized by formation of a methyl or ethyl phosphotriester
or an alkyl phosphoramidate linkage. Furthermore, the polynucleotides
herein may also be modified with a label capable of providing a
detectable signal, either directly or indirectly. Exemplary labels
include radioisotopes, fluorescent molecules, biotin, and the like.
 The term "host cell" means any cell of any organism
that is selected, modified, transformed, grown, or used or manipulated
in any way, for the production of a substance by the cell, for example
the expression by the cell of a gene, a DNA or RNA sequence, a protein
or an enzyme. Host cells can further be used for screening or other
assays, as described infra.
 Generally, a DNA sequence having instructions for a particular
protein or enzyme is "transcribed" into a corresponding
sequence of RNA. The RNA sequence in turn is "translated"
into the sequence of amino acids which form the protein or enzyme.
An "amino acid sequence" is any chain of two or more amino
acids. Each amino acid is represented in DNA or RNA by one or more
triplets of nucleotides. Each triplet forms a codon, corresponding
to an amino acid. The genetic code has some redundancy, also called
degeneracy, meaning that most amino acids have more than one corresponding
 A "coding sequence" or a sequence "encoding"
an expression product, such as a RNA, polypeptide, protein, or enzyme,
is a nucleotide sequence that, when expressed, results in the production
of that RNA, polypeptide, protein, or enzyme, i.e., the nucleotide
sequence encodes an amino acid sequence for that polypeptide, protein
 The term "gene", also called a "structural
gene" means a DNA sequence that codes for or corresponds to
a particular sequence of amino acids which comprise all or part
of one or more proteins or enzymes, and may or may not include regulatory
DNA sequences, such as promoter sequences, which determine for example
the conditions under which the gene is expressed. Some genes, which
are not structural genes, may be transcribed from DNA to RNA, but
are not translated into an amino acid sequence. Other genes may
function as regulators of structural genes or as regulators of DNA
 A "promoter sequence" is a DNA regulatory region
capable of binding a secondary molecule which in a cell and initiating
transcription of a coding sequence.
 A coding sequence is "under the control" or "operatively
associated with" of transcriptional and translational control
sequences in a cell when RNA polymerase transcribes the coding sequence
into mRNA, which is then trans-RNA spliced (if it contains introns)
and translated into the protein encoded by the coding sequence.
 The terms "express" and "expression"
mean allowing or causing the information in a gene or DNA sequence
to become manifest, for example producing a protein by activating
the cellular functions involved in transcription and translation
of a corresponding gene or DNA sequence. A DNA sequence is expressed
in or by a cell to form an "expression product" such as
a protein. The expression product itself, e.g. the resulting protein,
may also be said to be "expressed" by the cell. An expression
product can be characterized as intracellular, extracellular or
secreted. The term "intracellular" means something that
is inside a cell. The term "extracellular" means something
that is outside a cell. A substance is "secreted" by a
cell if it appears in significant measure outside the cell, from
somewhere on or inside the cell.
 The term "transfection" means the introduction
of a foreign nucleic acid into a cell. The term "transformation"
means the introduction of a "foreign" (i.e. extrinsic
or extracellular) gene, DNA or RNA sequence to a host cell, so that
the host cell will express the introduced gene or sequence to produce
a desired substance, typically a protein or enzyme coded by the
introduced gene or sequence. The introduced gene or sequence may
also be called a "cloned" or "foreign" gene
or sequence, may include regulatory or control sequences, such as
start, stop, promoter, signal, secretion, or other sequences used
by a cell's genetic machinery. The gene or sequence may include
nonfunctional sequences or sequences with no known function. A host
cell that receives and expresses introduced DNA or RNA has been
"transformed" and is a "transformant" or a "clone."
The DNA or RNA introduced to a host cell can come from any source,
including cells of the same genus or species as the host cell, or
cells of a different genus or species.
 The terms "vector", "cloning vector"
and "expression vector" mean the vehicle by which a DNA
or RNA sequence (e.g. a foreign gene) can be introduced into a host
cell, so as to transform the host and promote expression (e.g. transcription
and translation) of the introduced sequence. Vectors include plasmids,
phages, viruses, etc.
 A common type of vector is a "plasmid", which
generally is a self-contained molecule of double-stranded DNA, usually
of bacterial origin, that can readily accept additional (foreign)
DNA and which can readily introduced into a suitable host cell.
A plasmid vector often contains coding DNA and promoter DNA and
has one or more restriction sites suitable for inserting foreign
DNA. A large number of vectors, including plasmid and fungal vectors,
have been described for replication and/or expression in a variety
of eukaryotic and prokaryotic hosts. Non-limiting examples include
pKK plasmids (Clonetech), pUC plasmids, pET plasmids (Novagen, Inc.,
Madison, Wis.), pRSET or pREP plasmids (Invitrogen, San Diego, Calif.),
or pMAL plasmids (New England Biolabs, Beverly, Mass.), and many
appropriate host cells, using methods disclosed or cited herein
or otherwise known to those skilled in the relevant art. Recombinant
cloning vectors will often include one or more replication systems
for cloning or expression, one or more markers for selection in
the host, e.g. antibiotic resistance, and one or more expression
 A "cassette" refers to a DNA coding sequence or
segment of DNA that codes for an expression product that can be
inserted into a vector at defined restriction sites. The cassette
restriction sites are designed to ensure insertion of the cassette
in the proper reading frame. Generally, foreign DNA is inserted
at one or more restriction sites of the vector DNA, and then is
carried by the vector into a host cell along with the transmissible
vector DNA. A segment or sequence of DNA having inserted or added
DNA, such as an expression vector, can also be called a "DNA
 The term "expression system" means a host cell
and compatible vector under suitable conditions, e.g. for the expression
of a protein coded for by foreign DNA carried by the vector and
introduced to the host cell. Common expression systems include E.
coli host cells and plasmid vectors, insect host cells and Baculovirus
vectors, and mammalian host cells and vectors.
 The term "heterologous" refers to a combination
of elements not naturally occurring. For example, heterologous DNA
refers to DNA not naturally located in the cell, or in a chromosomal
site of the cell. Preferably, the heterologous DNA includes a gene
foreign to the cell. A heterologous expression regulatory element
is a such an element operatively associated with a different gene
than the one it is operatively associated with in nature.
 The terms "mutant" and "mutation" mean
any detectable change in genetic material, e.g. DNA, or any process,
mechanism, or result of such a change. This includes gene mutations,
in which the structure (e.g. DNA sequence) of a gene is altered,
any gene or DNA arising from any mutation process, and any expression
product (e.g. protein or enzyme) expressed by a modified gene or
DNA sequence. The term "variant" may also be used to indicate
a modified or altered gene, DNA sequence, enzyme, cell, etc., i.e.,
any kind of mutant.
 A nucleic acid molecule is "hybridizable" to another
nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when
a single stranded form of the nucleic acid molecule can anneal to
the other nucleic acid molecule under the appropriate conditions
of temperature and solution ionic strength (see Sambrook et al.,
supra). The conditions of temperature and ionic strength determine
the "stringency" of the hybridization. For preliminary
screening for homologous nucleic acids, low stringency hybridization
conditions, corresponding to a T.sub.m (melting temperature) of
55.degree. C., can be used, e.g., 5.times.SSC, 0.1% SDS, 0.25% milk,
and no formamide; or 30% formamide, 5.times.SSC, 0.5% SDS. Moderate
stringency hybridization conditions correspond to a higher T.sub.m,
e.g., 40% formamide, with 5.times. or 6.times.SCC. High stringency
hybridization conditions correspond to the highest Tm, e.g., 50%
formamide, 5.times. or 6.times.SCC. SCC is a 0.15M NaCl, 0.015M
Na-citrate. Hybridization requires that the two nucleic acids contain
complementary sequences, although depending on the stringency of
the hybridization, mismatches between bases are possible. The appropriate
stringency for hybridizing nucleic acids depends on the length of
the nucleic acids and the degree of complementation, variables well
known in the art. The greater the degree of similarity or homology
between two nucleotide sequences, the greater the value of Tm for
hybrids of nucleic acids having those sequences. The relative stability
(corresponding to higher T.sub.m) of nucleic acid hybridizations
decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For
hybrids of greater than 100 nucleotides in length, equations for
calculating T.sub.m have been derived (see Sambrook et al., supra,
9.50-9.51). For hybridization with shorter nucleic acids, i.e.,
oligonucleotides, the position of mismatches becomes more important,
and the length of the oligonucleotide determines its specificity
(see Sambrook et al., supra, 11.7-11.8). A minimum length for a
hybridizable nucleic acid is at least about 10 nucleotides; preferably
at least about 15 nucleotides; and more preferably the length is
at least about 20 nucleotides.
 In a specific embodiment, the term "standard hybridization
conditions" refers to a T.sub.m of 55.degree. C., and utilizes
conditions as set forth above. In a preferred embodiment, the T.sub.m
is 60.degree. C.; in a more preferred embodiment, the T.sub.m is
65.degree. C. In a specific embodiment, "high stringency"
refers to hybridization and/or washing conditions at 68.degree.
C. in 0.2.times.SSC, at 42.degree. C. in 50% formamide, 4.times.SSC,
or under conditions that afford levels of hybridization equivalent
to those observed under either of these two conditions.
 The term "oligonucleotide" refers to a nucleic
acid, generally of at least 10, preferably at least 15, and more
preferably at least 20 nucleotides, preferably no more than 100
nucleotides, that is hybridizable to a genomic DNA molecule, a cDNA
molecule, or an mRNA molecule encoding a gene, mRNA, cDNA, or other
nucleic acid of interest. Oligonucleotides can be labeled, e.g.,
with .sup.32P-nucleotides or nucleotides to which a label, such
as biotin, has been covalently conjugated. In one embodiment, a
labeled oligonucleotide can be used as a probe to detect the presence
of a nucleic acid. In another embodiment, oligonucleotides (one
or both of which may be labeled) can be used as PCR primers, either
for cloning full length or a fragment of Cyr61, or to detect the
presence of nucleic acids encoding Cyr61. Generally, oligonucleotides
are prepared synthetically, preferably on a nucleic acid synthesizer.
 As mentioned above, the term "cancer" refers to
cells that display uncontrolled proliferation or division. The degree
to which a cancer has spread beyond its original location is referred
to as the "stage" of the cancer. Lower stages, such as
stages I and II, are generally more confined to their site or region
of origin than advanced stages (III and IV). See, e.g., The Merck
Manual, 15.sup.th Edition, Merck, Sharp, & Dohme Research Laboratories
 Breast cancers refer to a class of cancers that are associated
with development in the breast of women and men. The most common
type of breast cancer is invasive ductal carcinoma. It occurs most
frequently in women in their 50's and appears to spread from the
breast into the lymph nodes. Estrogen receptors (ER), progesterone
receptors (PR), and androgen receptors (AR) are molecules within
many breast cancer cells. The cancer along with the increased levels
of Cyr61 in breast cancer cells along with the presence or absence
of ER, PR, or AR have prognostic and predictive value and can be
used as a basis for designing treatment regimens. Presence of these
receptor molecules within the cancer cells is referred to as estrogen
receptors positive (ER+), progesterone receptors positive (PR+),
and/or androgen positive (AR+) tumors, while absence is referred
to as estrogen receptors negative (ER-), progesterone receptors
negative (PR-) and/or androgen receptors negative (AR-).
Antibodies and Antisense Constructs
 The present invention describes neutralizing antibodies
that may be used to block the activity of Cyr61 in cells and specifically
in cancer cells such as breast cancers. According to the invention,
Cyr61 polypeptides produced recombinantly or by chemical synthesis,
and fragments or other derivatives, may be used as an immunogen
to generate antibodies that recognize the Cyr61 polypeptide or portions
thereof. Such antibodies include, but are not limited to, polyclonal,
monoclonal, humanized, primatized, chimeric, single chain, Fab fragments,
and an Fab expression library. An antibody that is specific for
human Cyr61 may recognize a wild-type or mutant form of Cyr61. Preferred
neutralizing antibodies are produced to, but not limited to, the
amino acids 163-229 and 371-381 in SEQ ID NO. 2 (See FIG. 1).
 The invention also describes pharmaceutical compositions
that modulate the transcription of the Cyr61 gene by sex steroid
receptors (ovarian and testicular) and growth factors. These receptors
recognize consensus sex steroid responsive elements (ERE for estrogen
receptors, PRE/ARE for progesterone receptors and androgen receptors)
in the promoter region of the Cyr61 gene. In addition to the DNA
binding region, the steroid receptors also contain at least two
regions that initiate gene transcription. The estrogen receptor
recognizes an estrogen response element (ERE) having the DNA sequence
including, but not limited to, 5'-GGTCAxxxTGACC-3' (SEQ ID NO:3)
and the progesterone receptor and androgen receptor recognize a
progesterone receptor/androgen receptor element (PRE/ARE) having
the DNA sequence including, but not limited to, 5'TGTACAxxxTGTTCT-3'
(SEQ ID NO:4); where x represents any nucleotide.
 Pharmaceutical compositions that prevent ER, PR, and AR
binding to the Cyr61 promoter are contemplated in this invention.
Antibodies to the amino acid sequences of the sex steroid receptors
that recognize the specific consensus sequences are contemplated
in this invention. As mentioned above, polypeptides produced recombinantly
or by chemical synthesis, and fragments or other derivatives, may
be used as an immunogen to generate antibodies that recognize the
steroid receptor polypeptide regions that comprise the gene binding
sequence. Polyclonal, monoclonal, humanized, primatized, chimeric,
single chain, Fab fragments, and an Fab expression library antibodies
 Various procedures known in the art may be used for the
production of polyclonal antibodies to polypeptides, derivatives,
or analogs. For the production of antibody, various host animals,
including but not limited to rabbits, mice, rats, sheep, goats,
etc, can be immunized by injection with the polypeptide or a derivative
(e.g., fragment or fusion protein). The polypeptide or fragment
thereof can be conjugated to an immunogenic carrier, e.g., bovine
serum albumin (BSA) or keyhole limpet hemocyanin (KLH). Various
adjuvants may be used to increase the immunological response, depending
on the host species, including but not limited to Freund's (complete
and incomplete), mineral gels such as aluminum hydroxide, surface
active substances such as lysolecithin, pluronic polyols, polyanions,
peptides, oil emulsions, KLH, dinitrophenol, and potentially useful
human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium
 Monoclonal antibodies directed toward a Cyr61 polypeptide,
fragment, analog, or derivative thereof, may be prepared by any
technique that provides for the production of antibody molecules
by continuous cell lines in culture may be used. These include but
are not limited to the hybridoma technique originally developed
by Kohler and Milstein Nature 256:495-497, 1975), as well as the
trioma technique, the human B-cell hybridoma technique (Kozbor et
al., Immunology Today 4:72, 1983; Cote et al., Proc. Natl. Acad.
Sci. U.S.A. 80:2026-2030, 1983), and the EBV-hybridoma technique
to produce human monoclonal antibodies (Cole et al., in Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985).
"Chimeric antibodies" may be produced (Morrison et al.,
J. Bacteriol. 159:870, 1984; Neuberger et al., Nature 312:604-608,
1984; Takeda et al., Nature 314:452-454, 1985) by splicing the genes
from a non-human antibody molecule specific for a polypeptide together
with genes from a human antibody molecule of appropriate biological
 In the production and use of antibodies, screening for or
testing with the desired antibody can be accomplished by techniques
known in the art, e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbant
assay), "sandwich" immunoassays, immunoradiometric assays,
gel diffusion precipitin reactions, immunodiffusion assays, in situ
immunoassays (using colloidal gold, enzyme or radioisotope labels,
for example), western blots, precipitation reactions, agglutination
assays (e.g., gel agglutination assays, hemagglutination assays),
complement fixation assays, immunofluorescence assays, protein A
assays, and immunoelectrophoresis assays, etc.
 The foregoing antibodies can be used in methods known in
the art relating to the localization and activity of the polypeptide,
e.g., for Western blotting, imaging the polypeptide in situ, measuring
levels thereof in appropriate physiological samples, etc. using
any of the detection techniques mentioned above or known in the
art. Such antibodies can also be used in assays for ligand binding,
e.g., as described in U.S. Pat. No. 5,679,582. Antibody binding
generally occurs most readily under physiological conditions, e.g.,
pH of between about 7 and 8, and physiological ionic strength. The
presence of a carrier protein in the buffer solutions stabilizes
the assays. While there is some tolerance of perturbation of optimal
conditions, e.g., increasing or decreasing ionic strength, temperature,
or pH, or adding detergents or chaotropic salts, such perturbations
will decrease binding stability.
 In a specific embodiment, antibodies that agonize or antagonize
the activity of Cyr61 polypeptide can be generated. In particular,
intracellular single chain Fv antibodies can be used to regulate
(inhibit) Cyr61. Such antibodies can be tested using the assays
described below for identifying ligands.
 In another specific embodiment, antibodies of the present
invention are conjugated to a secondary component, such as, for
example, a small molecule, polypeptide, or polynucleotide. The conjugation
may be produced through a chemical modification of the antibody,
which conjugates the antibody to the secondary component. The conjugated
antibody will allow for targeting of the secondary component, such
as, for example, an anti-tumor agent to the site of interest. The
secondary component may be of any size or length. Examples of anti-tumor
agents include, but are not limited to, chemotherapeutic agents,
toxins, radioactive isotopes, mitotic inhibitors, cell-cycle regulators,
and anti-microtubule disassembly compounds. The secondary component
may be an antibiotic including, but not limited to, calicheamicin.
An example of an anti-microtubule disassembly compounds is taxol
(Wani et al., J. Amer. Chem. Soc., 1971, 93:2325-2327; Horwitz,et
al., Nature, 1979, 277:665).
 A further aspect of this invention relates to the use of
antibodies, as discussed supra, for targeting a pharmaceutical compound.
In this embodiment, antibodies against Cyr61 are used to present
specific compounds to cancerous cells. The compounds, preferably
an anti-tunor agent or an anti-cancer agent, when conjugated to
the antibodies are referred to as targeted compounds or targeted
agents. Methods for generating such target compounds and agents
are known in the art. Exemplary publications on target compounds
and their preparation are set forth in U.S. Pat. Nos. 5,053,934;
5,773,001; and 6,015,562.
 Any desired agent (known as an anti-tumor agent) having
activity against cancer cells may be employed in generating the
targeted agent. Examples of such compounds are discussed in U.S.
Pat. No. 6,015,562. See specifically U.S. Pat. Nos. 4,971,198; 5,079,233;
4,539,203; 4,554,162; 4,675,187; and 4,837,206. These publications
refer to anti-tumor agents and antibiotics which may be used as
the pharmaceutical compound of the target.
 The present invention provides antisense nucleic acids (including
ribozymes), which may be used to inhibit expression of Cyr61, particularly
to suppress Cyr61 effects on cell proliferation. An "antisense
nucleic acid" is a single stranded nucleic acid molecule or
oligonucleotide which, on hybridizing under cytoplasmic conditions
with complementary bases in an RNA or DNA molecule, inhibits the
latter's role. If the RNA is a messenger RNA transcript, the antisense
nucleic acid is a countertranscript or mRNA-interfering complementary
nucleic acid. As presently used, "antisense" broadly includes
RNA-RNA interactions, RNA-DNA interactions, ribozymes and RNase-H
mediated arrest. Antisense nucleic acid molecules can be encoded
by a recombinant gene for expression in a cell (e.g., U.S. Pat.
Nos. 5,814,500 and 5,811,234), or alternatively they can be prepared
synthetically (e.g., U.S. Pat. No. 5,780,607). Also contemplated
are vectors which include these oligonucleotides or antisense constructs.
 A "sex steroid" refers to a class of hormonal
substances that may be secreted from reproductive organs and glands.
Sex steroids include, but are not limited to, estrogenic compounds,
progestational compounds, and androgenic compounds.
 Estrogenic compounds are described, for example, in the
11th edition of "Steroids" from Steraloids Inc., Wilton
N. H. Non-steroidal estrogens described therein are included, as
well. Other compounds included are derivatives, metabolites, and
precursors. Also included are mixtures of more than one compound.
Examples of such mixtures are provided in Table II of U.S. Pat.
No. 5,554,601 (see column 6). Examples of estrogens either alone
or in combination with other agents are provided, e.g., in U.S.
Pat. No. 5,554,601.
 .beta.-estrogen is the .beta.-isomer of estrogenic compounds.
.alpha.-estrogen is the .alpha.-isomer of estrogen components. The
term "estradiol" is either .alpha.- or .beta.-estradiol
unless specifically identified. The term "E2" is synonymous
 Preferably, a non-feminizing estrogenic compound is used
herein. Such a compound has the advantage of not causing uterine
hypertrophy and other undesirable side effects, and thus, can be
used at a higher effective dosage. Examples of non-feminizing estrogen
include Raloxifene (Evista; Eli Lilly), Tamoxifen (Nolvadex; Astra
Zeneca), and other selective estrogen receptor modulators.
 Progestational compounds are described, for example, in
the 9.sup.th edition of "The Pharmacological Basis of Therapeutics"
from McGraw-Hill, New York, N.Y. Progestin compounds, for example,
include progestins containing the 21-carbon skeleton and the 19-carbon
(19-nortestosterone) skeleton. Non-steroidal progestin compounds,
derivatives, precursors, and metabolites are also contemplated herein.
 Androgenic compounds are described, for example, in the
9.sup.th edition of "The Pharmacological Basis of Therapeutics".
Androgens include, for example, testosterones containing the 17-carbon
skeleton. Non-steroidal testosterone compounds, derivatives, precursors,
and metabolites also are contemplated herein.
 In addition, certain compounds, such as the androgen testosterone,
can be converted to estradiol in vivo.
 Growth factors are a class of proteins that are involved
in stimulation of cell division. These proteins interact with cell
surface receptors to induce transcription factors to promote cell
survival. Growth factor receptors signal through the Ras pathway,
a highly conserved signal transduction pathway. The Ras pathway
functions to promote cell survival in radiation therapy, and genetic
changes in this pathway which produce constitutively activate intracellular
survival pathways are often associated with the development of cancer.
 Growth factors include, for example, small molecule compounds
that interact with growth factor receptors to produce the same effects
as observed with growth factor peptides. Other compounds included
are derivatives, metabolites, and precursors of endogenous growth
factors. In specific embodiments of the present invention, specific
growth factors that are used include, but are not limited to, epidermal
growth factor, heparin binding epidermal growth factor, and basic
fibroblastic growth factor.
 Any cell assay system that allows for assessing functional
activities of sex steroid, non-steroid, and growth factor receptor
agonists and antagonists is contemplated by the present invention.
In a specific embodiment, the assay can be used to identify compounds
that interact with specific isoforms of sex steroid receptors to
regulate Cyr61 transcription and translation, which can be evaluated
by assessing the effects of a test compound, which modulates Cyr61
mRNA transcription and Cyr61 translation.
 Any convenient method permits detection of the expressed
product. For example, the invention provides Northern blot analysis
for detecting Cyr61 mRNA product. The methods comprise, for example,
the steps of fractionating total cellular RNA on an agarose gel,
transferring RNA to a solid support membrane, and detecting a DNA-RNA
complex with a labeled DNA probe, wherein the DNA probe is specific
for a particular nucleic acid sequence of Cyr61 under conditions
in which a stable complex can form between the DNA probe and RNA
components in the sample. Such complexes may be detected by using
any suitable means known in the art, wherein the detection of a
complex indicates the presence of Cyr61 in the sample.
 Typically, immunoassays use either a labeled antibody or
a labeled antigenic component (e.g., that competes with the antigen
in the sample for binding to the antibody). Suitable labels include
without limitation enzyme-based, fluorescent, chemiluminescent,
radioactive, or dye molecules. Assays that amplify the signals from
the probe are also known, such as, for example, those that utilize
biotin and avidin, and enzyme-labelled immunoassays, such as ELISA
In Vitro Screening Methods
 Candidate agents are added to in vitro cell cultures of
host cells, prepared by known methods in the art, and the level
of Cyr61 mRNA and/or protein are measured. Various in vitro systems
can be used to analyze the effects of a new compound on Cyr61 transcription
and translation. Preferably, each experiment is performed more than
once, such as, for example, in triplicate at multiple different
dilutions of compound.
 The host cell screening system of the invention permits
two kinds of assays: direct activation assays (agonist screen) and
inhibition assays (antagonist screen). An agonist screen involves
detecting changes in the level of expression of the gene by the
host cell contacted with a test compound; generally, gene expression
increases. If the Cyr61 gene is expressed, the test compound has
stimulated Cyr61 transcription via receptor interaction.
 An antagonist screen involves detecting expression of the
reporter gene by the host cell when contacted with an Cyr61 regulatory
compound. If Cyr61 expression is decreased, the test compound is
a candidate antagonist. If there is no change in expression of the
reporter gene, the test compound is not an effective antagonist.
 The assay system described here also may be used in a high-throughput
primary screen for agonists and antagonists, or it may be used as
a secondary functional screen for candidate compounds identified
by a different primary screen, e.g., a binding assay screen that
identifies compounds that interact with the receptor and affect
In Vivo Testing Using Transgenic Animals
 Transgenic animals, and preferably mammals, can be prepared
for evaluating the molecular mechanisms of Cyr61. Preferably, for
evaluating compounds for use in human therapy, the animals are "humanized"
with respect to Cyr61. Such mammals provide excellent models for
screening or testing drug candidates. The term "transgenic"
usually refers to animal whose germ line and somatic cells contain
the transgene of interest, i.e., Cyr61. However, transient transgenic
animals can be created by the ex vivo or in vivo introduction of
an expression vector of the invention. Both types of "transgenic"
animals are contemplated for use in the present invention, e.g.,
to evaluate the effect of a test compound on Cyr61 or Cyr61 activity.
 Thus, human Cyr61, "knock-in" mammals can be prepared
for evaluating the molecular biology of this system in greater detail
than is possible with human subjects. It is also possible to evaluate
compounds or diseases on "knockout" animals, e.g., to
identify a compound that can compensate for a defect in Cyr61 or
Cyr61 activity. Both technologies permit manipulation of single
units of genetic information in their natural position in a cell
genome and to examine the results of that manipulation in the background
of a terminally differentiated organism.
 Although rats and mice, as well as rabbits, are most frequently
employed as transgenic animals, particularly for laboratory studies
of protein function and gene regulation in vivo, any animal can
be employed in the practice of the invention.
 A "knock-in" mammal is a mammal in which an endogenous
gene is substituted with a heterologous gene (Roemer et al., New
Biol. 3:331, 1991). Preferably, the heterologous gene or regulation
system is "knocked-in" to a locus of interest, either
the subject of evaluation(in which case the gene may be a reporter
gene; see Elefanty et al., Proc Natl Acad Sci USA 95:11897,1998)
of expression or function of a homologous gene, thereby linking
the heterologous gene expression to transcription from the appropriate
promoter. This can be achieved by homologous recombination, transposon
(Westphal and Leder, Curr Biol 7:530, 1997), using mutant recombination
sites (Araki et al., Nucleic Acids Res 25:868, 1997) or PCR (Zhang
and Henderson, Biotechniques 25:784, 1998). See also, Coffman, Semin.
Nephrol. 17:404, 1997; Esther et al, Lab. Invest. 74:953, 1996;
Murakami et al., Blood Press. Suppl. 2:36, 1996.
 A "knockout mammal" is an mammal (e.g., mouse)
that contains within its genome a specific gene that has been inactivated
by the method of gene targeting (see, e.g., U.S. Pat. Nos. 5,777,195
and 5,616,491). A knockout mammal includes both a heterozygote knockout
(i.e., one defective allele and one wild-type allele) and a homozygous
mutant. Preparation of a knockout mammal requires first introducing
a nucleic acid construct that will be used to suppress expression
of a particular gene into an undifferentiated cell type termed an
embryonic stem cell. This cell is then injected into a mammalian
embryo. A mammalian embryo with an integrated cell is then implanted
into a foster mother for the duration of gestation. Zhou, et al.
(Genes and Development, 9:2623-34, 1995) describes PPCA knock-out
 The term "knockout" refers to partial or complete
suppression of the expression of at least a portion of a protein
encoded by an endogenous DNA sequence in a cell. The term "knockout
construct" refers to a nucleic acid sequence that is designed
to decrease or suppress expression of a protein encoded by endogenous
DNA sequences in a cell. The nucleic acid sequence used as the knockout
construct is typically comprised of (1) DNA from some portion of
the gene (exon sequence, intron sequence, and/or promoter sequence)
to be suppressed and (2) a marker sequence used to detect the presence
of the knockout construct in the cell. The knockout construct is
inserted into a cell, and integrates with the genomic DNA of the
cell in such a position so as to prevent or interrupt transcription
of the native DNA sequence. Such insertion usually occurs by homologous
recombination (i.e., regions of the knockout construct that are
homologous to endogenous DNA sequences hybridize to each other when
the knockout construct is inserted into the cell and recombine so
that the knockout construct is incorporated into the corresponding
position of the endogenous DNA). The knockout construct nucleic
acid sequence may comprise (1) a full or partial sequence of one
or more exons and/or introns of the gene to be suppressed, (2) a
full or partial promoter sequence of the gene to be suppressed,
or (3) combinations thereof. Typically, the knockout construct is
inserted into an embryonic stem cell (ES cell) and is integrated
into the ES cell genomic DNA, usually by the process of homologous
recombination. This ES cell is then injected into, and integrates
with, the developing embryo. However, the invention does not require
any particular method for preparing a transgenic animal.
 Generally, for homologous recombination, the DNA will be
at least about 1 kilobase (kb) in length and preferably 3-4 kb in
length, thereby providing sufficient complementary sequence for
recombination when the construct is introduced. Transgenic constructs
can be introduced into the genomic DNA of the ES cells, into the
male pronucleus of a fertilized oocyte by microinjeciton, or by
any methods known in the art, e.g., as described in U.S. Pat. Nos.
4,736,866 and 4,870,009, and by Hogan et al., Transgenic Animals:
A Laboratory Manual, 1986, Cold Spring Harbor. A transgenic founder
animal can be used to breed other transgenic animals; alternatively,
a transgenic founder may be cloned to produce other transgenic animals.
 Included within the scope of this invention is a mammal
in which two or more genes have been knocked out or knocked in,
or both. Such mammals can be generated by repeating the procedures
set forth herein for generating each knockout construct, or by breeding
to mammals, each with a single gene knocked out, to each other,
and screening for those with the double knockout genotype.
 Regulated knockout animals can be prepared using various
systems, such as the tet-repressor system (see U.S. Pat. No. 5,654,168)
or the Cre-Lox system (see U.S. Pat. Nos. 4,959,317 and 5,801,030).
Cloning and Expression of Cvr61
 The present invention contemplates analysis and isolation
any antigenic fragments of Cyr61 from any source, preferably human.
It further contemplates expression of functional or mutant Cyr61
protein for evaluation, diagnosis, or therapy.
 Conventional molecular biology, microbiology, and recombinant
DNA techniques within the skill of the art may be employed in the
use of this invention. Such techniques are explained fully in the
literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular
Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein "Sambrook
et al., 1989"); DNA Cloning: A Practical Approach, Volumes
I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M.
J. Gait ed. 1984); Nucleic Acid Hybridization [B. D. Hames &
S. J. Higgins eds. (1985)]; Transcription And Translation [B. D.
Hames & S. J. Higgins, eds. (1984)]; Animal Cell Culture [R.
I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press,
(1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984);
F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology,
John Wiley & Sons, Inc. (1994).
Methods of Diagnosis-Upregulation
 According to the present invention, upregulation of Cyr61
mRNA or protein can be detected to diagnose a Cyr61 associated disease,
such as increased susceptibility to breast cancers. The various
methods for detecting such upregulation of Cyr61 mRNA or protein
expression are well known in the art and have been discussed earlier.
Methods of detection include, but are not limited to, Northern blots,
in situ hybridization studies, Western blots, ELISA, radioimmunoassay,
"sandwich" immunoassays, immunoradiometric assays, gel
diffusion precipitation reactions, immunodiffusion assays, in situ
immunoassays (using colloidal gold, enzyme or radioisotope labels,
for example),precipitation reactions, complement fixation assays,
immunofluorescence assays, protein A assays, and immunoelectrophoresis
Nucleic Acid Assays
 The DNA may be obtained from any cell source. DNA is extracted
from the cell source or body fluid using any of the numerous methods
that are standard in the art. It will be understood that the particular
method used to extract DNA will depend on the nature of the source.
Generally, the minimum amount of DNA to be extracted for use in
the present invention is about 25 pg (corresponding to about 5 cell
equivalents of a genome size of 4.times.10.sup.9 base pairs). Sequencing
methods are described in detail, supra.
 In another alternate embodiment, RNA is isolated from biopsy
tissue using standard methods well known to those of ordinary skill
in the art such as guanidium thiocyanate-phenol-chloroform extraction
(Chomocyznski et al., Anal. Biochem., 162:156, 1987). The isolated
RNA is then subjected to coupled reverse transcription and amplification
by polymerase chain reaction (RT-PCR), using specific oligonucleotide
primers that are specific for a selected site. Conditions for primer
annealing are chosen to ensure specific reverse transcription and
amplification; thus, the appearance of an amplification product
is diagnostic of the presence of a particular genetic variation.
In another embodiment, RNA is reverse-transcribed and amplified,
after which the amplified sequences are identified by, e.g., direct
sequencing. In still another embodiment, cDNA obtained from the
RNA can be cloned and sequenced to identify a mutation.
 In an alternate embodiment, biopsy tissue is obtained from
a subject. Antibodies that are capable of specifically binding to
Cyr61 are then contacted with samples of the tissue to determine
the presence or absence of a Cyr61 polypeptide specified by the
antibody. The antibodies may be polyclonal or monoclonal, preferably
monoclonal. Measurement of specific antibody binding to cells may
be accomplished by any known method, e.g., quantitative flow cytometry,
enzryme-linled or fluorescence-linked immunoassay, Western analysis,
 Immunoassay technology, e.g., as described in U.S. Pat.
Nos. 5,747,274 and 5,744,358, and particularly solid phase "chromatographic"
format immunoassays, are preferred for detecting proteins in blood
or blood fractions.
 The test compounds, salts thereof, antibodies, and antisense
constructs may be formulated into pharmaceutical compositions. The
pharmaceutical composition comprises a therapeutically, inhibiting
preventing, or blocking effective amount of at least one of the
above. This can be an amount effective to inhibit a sex steroid,
a growth factor, or other factors that can increase Cyr61 expression
or activity or the Cyr61 gene. The pharmaceutical compositions also
typically include a pharmaceutically acceptable carrier (or dosing
vehicle), such as ethanol, glycerol, water, and the like. Examples
of such carriers and methods of formulation are described in Remington's
Pharmaceutical Sciences, 18th edition (1990), Mack Publishing Company.
The present invention also discloses pharmaceutical compositions
that are composed on antibodies, neutralizing antibodies, conjugated
antibodies, and antisense constructs. These pharmaceutical compositions
comprise a therapeutically, inhibiting preventing, or blocking effective
amount of at least one component. This can be an amount of the component
effective to interact with the sex steroid or EGF receptor, the
gene, or the protein. Conjugated antibodies can direct the secondary
component to the targeted site.
 The pharmaceutical composition may also include other additives,
such as a flavorant, a sweetener, a preservative, a dye, a binder,
a suspending agent, a colorant, a disintegrant, an excipient, a
diluent, a lubricant, a plasticizer, or any combination of any of
the foregoing. Suitable binders include, but are not limited to,
starch; gelatin; natural sugars, such as glucose and beta-lactose;
corn sweeteners; natural and synthetic guns, such as acacia, tragacanth,
and sodium alginate; carboxymethylcellulose; polyethylene glycol;
waxes; and the like. Suitable lubricants include, but are not limited
to, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate,
sodium acetate, sodium chloride and the like. Suitable disintegrators
include, but are not limited to, starch, methyl cellulose, agar,
bentonite, xanthan gum and the like.
 Suitable salts of the test compounds include, but are not
limited to, acid addition salts, such as those made with acids,
such as hydrochloric, hydrobromic, hydroiodic, perchloric, sulfuric,
nitric, a phosphoric, acetic, propionic, glycolic, lactic pyruvic,
malonic, succinic, maleic, fumaric, malic, tartaric, citric, benzoic,
carbonic cinnamic, mandelic, methanesulfonic, ethanesulfonic, hydroxyethanesulfonic,
benezenesulfonic, p-toluene sulfonic, cyclohexanesulfamic, salicyclic,
p-aminosalicylic, 2-phenoxybenzoic, and 2-acetoxybenzoic acid; and
salts made with saccharin. Other suitable salts of the compounds
include, but are not limited to, alkali metal salts, such as sodium
and potassium salts; alkaline earth metal salts, such as calcium
and magnesium salts; and salts formed with organic ligands, such
as quaternary ammonium salts.
 Representative salts include, but are not limited to, acetate,
benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate,
borate, bromide, calcium edetate, camsylate, carbonate, chloride,
clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate,
esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate,
hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate,
iodide, isothionate, lactate, lactobionate, laurate, malate, maleate,
mandelate, mesylate, methylbromide, methylnitrate, methylsulfate,
mucate, napsylate, nitrate, N-methylglucamine ammonium salt, oleate,
pamoate (embonate), palmitate, pantothenate, phosphate/diphosphate,
polygalacturonate, salicylate, stearate, sulfate, subacetate, succinate,
tannate, tartrate, teoclate, tosylate, triethiodide and valerate
salts of the compounds of the present invention.
 The present invention includes prodrugs of the test compounds.
Prodrugs include, but are not limited to, functional derivatives
of the test compounds of the present invention which are readily
convertible in vivo into the compounds of the present invention.
Conventional procedures for the selection and preparation of suitable
prodrug derivatives are described, for example, in "Design
of Prodrugs", ed. H. Bundgaard, Elsevier, 1985.
 The pharmaceutical compositions may be formulated as unit
dosage forms, such as tablets, pills, capsules, boluses, powders,
granules, sterile parenteral solutions or suspensions, sterile I.V.,
sterile I.M., elixirs, tinctures, metered aerosol or liquid sprays,
drops, ampoules, autoinjector devices or suppositories for oral,
parenteral, intranasal, occular, mucosal, transdermal, bucal, topical,
sublingual or rectal administration, or for administration by inhalation
or insufflation, for example. The unit dosage form may be in a form
suitable for sustained or delayed release, such as, for example,
an insoluble salt of the compound, e.g. a decanoate salt, adapted
to provide a depot preparation for intramuscular injection.
 Solid unit dosage forms may be prepared by mixing the compound
of the present invention with a pharmaceutically acceptable carrier
and any other desired additives to form a solid preformulation composition.
Examples of suitable additives for solid unit dosage forms include,
but are not limited to, starches, such as corn starch; lactose;
sucrose; sorbitol; talc; stearic acid; magnesium stearate; dicalcium
phosphate; gums, such as vegetable gums; and pharmaceutical diluents,
such as water. The solid preformulation composition is typically
mixed until a homogeneous mixture of the compound of the present
invention and the additives is formed, i.e., until the compound
is dispersed evenly throughout the composition, so that the composition
may be readily subdivided into equally effective unit dosage forms.
The solid preformulation composition is then subdivided into unit
dosage forms of the type described above.
 Tablets or pills can also be coated or otherwise compounded
to form a unit dosage form which has prolonged action, such as time
release and sustained release unit dosage forms. For example, the
tablet or pill can comprise an inner dosage and an outer dosage
component, the latter being in the form of an envelope over the
former. The two components can be separated by an enteric layer
which serves to resist disintegration in the stomach and permits
the inner component to pass intact into the duodenum or to be delayed
in release. The compound may be released immediately upon administration
or may be formulated such that the compound is released in a sustained
manner over a specified time course, such as, for example, 2-12
 Liquid unit dosage forms include, but are not limited to,
aqueous solutions, suitably flavoured syrups, aqueous or oil suspensions,
and flavoured emulsions with edible oils, such as cottonseed oil,
sesame oil, coconut oil or peanut oil, as well as elixirs and similar
pharmaceutical vehicles. Suitable dispersing and suspending agents
for aqueous suspensions include, but are not limited to, synthetic
and natural gums, such as tragacanth, acacia, alginate, dextran,
sodium carboxymethylcellulose, methylcellulose, polyvinyl-pyrrolidone
 Suitable pharmaceutically acceptable carriers for topical
preparations include, but are not limited to, alcohols, aloe vera
gel, allantoin, glycerine, vitamin A and E oils, mineral oil, PPG2
myristyl propionate, and the like. Such topical preparations may
be liquid drenches, alcoholic solutions, topical cleansers, cleansing
creams, skin gels, skin lotions, and shampoos in cream or gel formulations
(including, but not limited to aqueous solutions and suspensions).
Typically, these topical preparations contain a suspending agent,
such as bentonite, and optionally, an antifoaming agent. Generally,
topical preparations contain from about 0.005 to about 10% by weight
and preferably from about 0.01 to about 5% by weight of the compound,
based upon 100% total weight of the topical preparation.
 Pharmaceutical compositions of the present invention for
administration parenterally, and in particular by injection, typically
include an inert liquid carrier, such as water; vegetable oils,
including, but not limited to, peanut oil, cotton seed oil, sesame
oil, and the like; and organic solvents, such as solketal, glycerol
formal and the like. A preferred liquid carrier is vegetable oil.
These pharmaceutical compositions may be prepared by dissolving
or suspending the compound of the present invention in the liquid
carrier. Generally, the pharmaceutical composition for parenteral
administration contains from about 0.005 to about 10% by weight
of the compound of the present invention, based upon 100% weight
of total pharmaceutical composition.
 The compounds of the present invention can also be administered
in the form of liposome delivery systems, such as small unilamellar
vesicles, large unilamellar vesicles and multilamellar vesicles.
Liposomes can be formed from a variety of phospholipids, such as
cholesterol, stearylamine or phosphatidylcholines.
 Compounds of the present invention may also be delivered
by the use of monoclonal antibodies as individual carriers to which
the compound molecules are coupled. The compounds of the present
invention may also be coupled with soluble polymers as targetable
drug carriers. Such polymers include, but are not limited to, polyvinyl-pyrrolidone,
pyran copolymer, polyhydroxypropylmethacryl-amidephenol, polyhydroxyethylaspartamidephenol-
, and polyethyl-eneoxideopolylysine substituted with palmitoyl residues.
Furthermore, the compounds of the present invention may be coupled
to biodegradable polymers for controlling the release of the compound,
for example, polylactic acid, polyepsilon caprolactone, polyhydroxy
butyric acid, polyorthoesters, polyacetals, polydihydro-pyrans,
polycyanoacrylates and cross-linked or amphipathic block copolymers
 The pharmaceutical compositions of the present invention
may be administered to an animal, preferably a human being, in need
thereof to inhibit Cyr61 transcription or expression such as, for
example, through activation of a steroid or growth factor receptor,
or the like.
 The effective amounts of the active agents of the pharmaceutical
composition of the present invention may vary according to a variety
of factors such as the individual's condition, weight, sex and age
and the mode of administration. This amount of test compound can
be determined experimentally by methods well-known in the art such
as by establishing a matrix of dosages and frequencies and assigning
a group of experimental subjects to each point in the matrix.
 The compound of the present invention may be administered
alone at appropriate dosages defined by routine testing in order
to obtain optimal activity while minimizing any potential toxicity.
In addition, co-administration or sequential administration of other
active agents may be desirable.
 The daily dosage of the compounds of the present invention
may be varied over a wide range. For oral administration, the pharmaceutical
compositions are preferably provided in the form of scored or unscored
tablets for the symptomatic adjustment of the dosage to the patient
to be treated. The dosage amount may be adjusted when combined with
other active agents as described above to achieve desired effects.
On the other hand, unit dosage forms of these various active agents
may be independently optimized and combined to achieve a synergistic
result wherein the pathology is reduced more than it would be if
either active agent were used alone.
 Advantageously, the pharmaceutical compositions may be administered
in a single daily dose, or the total daily dosage may be administered
in divided doses of two, three or four times daily.
 For combination treatment with more than one active agent,
where the active agents are in separate dosage formulations, the
active agents can be administered concurrently, or they each can
be administered at separately staggered times.
 The present invention will be better understood by reference
to the following Examples, which are provided by way of exemplification
and not by way of limitation.
Estrogen and Progesterone Upregulate Cyr61 Transcription
 T47D (progesterone responsive cells), MCF-7 (estrogen responsive
cells), and MDA-MB-231 adenocarcinoma cell lines were obtained from
ATCC (Rockville, Md.) and propogated in DMEM/F12/Ham's-10 media
(GIBCO, Rockville, Md.) supplemented with 10% fetal bovine serum
(FBS), 100 U/mL penicillin, 100 .mu.g/mL streptomycin, and 2 mM
Glutamax (GIBCO BRL; Rockville, Md.). For steroid treatments, adenocarcinoma
cells were cultured in phenol-red free DMEM/F12 media supplemented
with 2% charcoal stripped FBS (Clonetics, Inc; San Diego, Calif.).
 T47D and MCF-7 cells were treated for varying time points
(0-24 hours) with varying concentrations of test compounds. The
pure ER antagonist ICI 182, 986 was co-incubated at a concentration
of 1 .mu.M with 10 nM 17.beta.-estradiol in MCF-7 cells for 1.0
h prior to lysis in 10 nM guanidium isothiocynate detergent. The
PR antagonist RU486 was co-incubated at a concentration of 1 .mu.M
with 1 .mu.M R5020 in T47D cells for 4.0 h prior to lysis in 10
mM guanidium isothiocynate detergent.
 Total cellular RNA was isolated from T47D and MCF-7 breast
cancer cells by lysis in 250 .mu.l of 10 mM guanidium isothiocynate
detergent followed by 250 .mu.l of phenol/chloroform extraction
for 5 minutes at 25.degree. C. Subsequently, total cellular RNA
(20 .mu.l) was subjected to electrophoresis in an 1% agarose gel
containing IM formaldehyde in 10 mM MOPS buffer for 3 hours at 100
V at 25.degree. C. Separated RNA transcripts were transferred onto
nylon membranes by capillary electrophoresis in 10.times.SSC at
25.degree. C. for 18 hours, and subsequently prehybridized at 60.degree.
C. in RapidHyb hybridization solution (Amersham, Arlington Heights,
Ill.). A 0.41 kb human Cyr61 a cDNA fragment was radiolabeled with
[.alpha.-.sup.32P]-dCTP (3,000 Ci/mmol) using the rando-primer technique
(Rediprime II, Amersham) and used as the hybridization probe. The
radiolabeled probe (1.times.10.sup.6 cpm/ml) was hybridized to membranes
for 4 hours at 60.degree. C. Membranes were washed twice in 1.times.SSPE
(0.15 M NaCl, 1 .mu.M EDTA, and 0.01 M sodium phosphate, pH 7.4)
and 0.1% SDS for 15 minutes at 25.degree. C., followed by a final
wash in 0.1.times.SSPE and 0.1% SDS for 5 minutes at 60.degree.
C. Densiometric analysis of Cyr61 mRNA levels was accomplished with
Molecular Dynamics phosphorimager and image quantification software
(Amersham Pharmacia Biotech, Piscataway, N.J.). Relative levels
of Cyr61 were normalized to glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) after reprobing membranes with a .sup.32P-radiolabeled oligonucleotide
according to manufacturers protocol (endlabeling lcit, GibcoBRL,
 Statistical analysis was performed using SAS statistical
software (SAS Inc., Cary, N.C.) for significance using a one-way
analysis of variance (ANOVA). Multicomparison significance level
for the ANOVA was a p-value equal to or less than 0.05. If significance
was achieved, a Scheffe's F test was performed.
 Results are shown in FIGS. 3-7 and 14. FIG. 3 shows that
estrogen and progesterone receptor ligands induce up regulation
of Cyr61 mRNA in T47D and MCF-7 cells, respectively. FIG. 4 shows
that upregulation of Cyr61 by R5020 in T47D cells occurs at the
transcriptional level since R5020 effects were fully blocked by
the transcription inhibitor actinomycin D, but not the protein synthesis
inhibitor cyclohexamide. FIG. 5 shows that upregulation of Cyr61
by 17.beta.-estradiol in MCF-7 cells occurs at the transcriptional
level since 17.beta.-estradiol effects were fully blocked by the
transcription inhibitor actinomycin D, but not the protein synthesis
inhibitor cyclohexamide. FIG. 6 shows that R5020 induction of Cyr61
mRNA transcription occurred in a dose-dependent manner and was progesterone
receptor specific, since the progesterone receptor antagonist, RU486,
fully blocked the observed effects and other steroids had little
to no effect. FIG. 7 shows the time course of mRNA induction in
cells treated with estrogen and progesterone receptor ligands. Stimulation
of Cyr61 transcription by estrogen ligands occurred earlier than
Cyr61 transcription by progesterone ligands. FIG. 14 shows that
R5020 and 17.beta.-estradiol stimulates the effects of EGF on mRNA
transcription in T47D and MCF-7.
Estrogen and Progesterone Upregulate Cyr61 Protein Expression
 T47D and MCF-7 adenocarcinoma cell lines were maintained
and propagated as described in Example 1. Cells were treated for
varying time points (0-24 hours) with varying concentrations of
test compounds. Cells were homogenized in 50 mM Tris-HCl (pH=8.0)
with 250 mM NaCl, 1.0% Nonidet P-40, 1.0% Triton-X 100, 2.0% sodium
dodecyl sulfate, 0.5% deoxycholate, 1 mM ethylenediaminetetraacetic
acid, and a protease inhibitor cocktail containing 10 .mu.g/mL pepstatin,
aprotinin, and leupeptin each (Sigma; St. Louis, Mo.). Protein extracts
(20 .mu.g) were subjected to SDS-polyacrylamide gel electrophoresis
under reducing conditions in 10% bis-acrylamide gels at 100 V for
3 hrs at 25.degree. C. Proteins were electrophoretically transferred
to polyvinyl difluoride membrane (Immobilon-P, Biorad, Redding,
Calif., USA) in 500 mls of 100 mM Tris-Glycine/1-% MeOH buffer at
50 mA for 18 hrs. Membranes were blocked with 5% dry milk on TBS/0.1%
Tween-20 (TBST), and incubated with anti-Cyr61 pAb (10 .mu.g/ml)
for 1 hr. at room temperature. Following primary antibody incubation,
membranes were washed 4.times.10 mins. with 50 mls of 1.times.TBST
and subsequently incubated with 10 .mu.gs/ml donkey anti-rabbit
IgG antibody conjugated to horseradish peroxidase (HRP) for 1 hr.
at room temperature. Secondary antibody detection was determined
by an enhanced chemilurninescence detection kit (Amersham Biotech,
Piscataway, N.J.) at room temperature for 5 min. In order to normalize
protein levels, Cyr61 western blots were subsequently reprobed with
1 .mu.g/ml anti-cytokeratin monoclonal antibody (Sigma, Inc.; St.
Louis, Mo) and 1 .mu.g/ml of donkey anti-mouse secondary antibody
conjugated to HRP (Amersham Biotech, Piscataway, N.J.,) for 1 hr.
at room temperature. Cyr61 protein levels were quantified by densiometric
analysis using a Biorad molecular imager (Biorad Laboratories, Hercules,
Calif.). Numerical values were based on the relative optical density
(OD) of the band. Protein levels were normalized to the total level
of cytokeratin in each sample. Statistical analysis was performed
as described above. Results are shown in FIG. 8. FIG. 8 shows increased
Cyr61 protein expression is observed after stimulation of cells
with estrogen and progestin ligands.
Cyr61 is Upregulated in Human Breast Cancer Tumors Classified as
ER+, PR+ and/or EGFR+
 Breast tumor biopsies and matched normal mammary tissues
were obtained from Clinomics (Pittsfield, Mass.) following informed
patient consent and internal review borad approval. Patients (n=40)
were between the ages of 42-68 and diagnosed with stage 1I invasive
ductal carcinoma following histological examination. All breast
tumor classifications were performed by Clinomics, Inc. (Pittsfield,
Mass.) utilizing standard immunohistochemical techniques. Tumors
were classified as estrogen receptor (ER), progesterone receptor
(PR), and epidermal growth factor receptor (EGFR) positive (n-20)
or ER and PR negative and EGFR positive (n=20). Briefly, formalin-fixed
tissue sections were deparaffinized in 100% Xylene for 10 min at
room temperature and rehydrated through a 100%-30% EtOH gradient
at room temperature. Tissue sections were subsequently incubated
with either anti-ER (Santa Cruz Technologies, Santa Cruz, Calif.),
anti-PR (Santa Cruz Technologies, Santa Cruz, Calif.) or anti-EGFR
(Sigma Immunochernicals, St. Louis, Mo.) monoclonal antibodies for
1 hr. at room temperature. Tissue sections were washed 2.times.10
min at room temperature in Tris-Buffered Saline (TBS) and incubated
with goat anti-mouse secondary antibodies conjugated to horseradish
peroxidase (HRP) for 1 hr at room temperature. Sections were washed
again in TBS 2.times.10 min at room temperature and incubated with
a chromagenic substrate (Vector Laboratories, Burlingame, Calif.)
for colormetric detections. Tissue sections were counterstained
with hematoxylin (Sigma Inc., St. Louis, Mo.), dehydrated in graded
ethanol series, and mounted for viewing. Receptor positive tissues
were identified by brown percipatates that were associated with
the cell nucleus (ER and PR) or cell membranes (EGFR). Cyr61 protein
extraction and data analysis was performed as described in Example
 Results are shown in FIG. 9. FIG. 9 shows that tumors that
are classified as ER+/PR+/EGFR+ display a higher level of Cyr61
protein when compared to tumors that are classified as ER-/PR-/EGFR+.
Both ER+/PR+/EGFR+ and ER-/PR-/EGFR+ tumors displayed higher levels
of Cyr61 protein than normal mammary cells.
Differential Expression of Cyr61 in Breast Tumor Patients
 Breast tumor biopsies were fixed in 10% neutral-buffered
formalin. 0.28 kb human Cyr61 cDNA fragment was positionally cloned
into the EcoRI and HindIII sites of pGEM4Zf-plasmid (Promega Corp.;
Madison, Wis.) to generate pGEM4Zf-Cyr61. Radiolabeled .sup.35S-UTP
sense and antisense cRNA transcripts were transcribed in vitro with
T3 and T7 RNA polymerases, respectively, using Gemini Riboprobe
 In situ hybridization studies were performed utilizing processed
slides that were hybridized overnight with 100-150 .mu.l of antisense
or sense riboprobes at 4.7.times.10.sup.6 DPM/slide in 50% formamide
hybridization mixture including 5% dextran sulfate and 100 dithiothreitol
(DTT) at 55.degree. C. in a humidified chamber containing 50% formamide/600
mM NaCl. Slides were washed 3 times in 2.times.SSC (0.3 M NaCl,
0.03 M sodium citrate, pH=7.0)/10 mM DTT at room temperature. The
washes were followed by RNase A (20 .mu.g/ml) treatment for 30 minutes
at 37.degree. C. and then washed for 15 minutes in 0.1.times.SSC
at room temperature. Slides were further washed with 0.1.times.SSC
to remove nonspecific label and dehydrated with a graded series
of alcohol:ammonium acetate (70%, 95%, and 100%). Air-dried slides
were exposed to X-ray film (Amerasham Inc., Piscataway, N.J.) for
3 days for preliminary examination and then dipped in NTB2 nuclear
emulsion (Eastman Kodak; Rochester, N.Y.), which was diluted 1:1
with 600 mM ammonium acetate. Slides were exposed for 31 days in
light-tight, black dessicated boxes, photographically processed,
and then stained in cresyl violet and coverslipped.
 Results are shown in FIG. 10. FIG. 10 shows in situ hybridization
studies that show that Cyr61 levels are very low in normal breast
cell, but are abundant in invading luminal epithelial cells within