Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
1999-07-15
2004-03-02
Eyler, Yvonne (Department: 1646)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C435S069100, C536S023500, C530S350000
Reexamination Certificate
active
06699714
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
Not applicable.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not applicable.
BACKGROUND OF THE INVENTION
Androgens constitute a class of hormones that control the development and proper function of mammalian male reproductive systems, including the prostate and epididymis. Androgens also affect the physiology of many non-reproductive systems, including muscle, skin, pituitary, lymphocytes, hair growth, and brain. Androgens exert their effect by altering the level of gene expression of specific genes in a process that is mediated by binding of androgen to an androgen receptor. The androgen receptor, which is a member of the steroid receptor super family, plays an important role in male sexual differentiation and in prostate cell proliferation. Binding of androgen by the androgen receptor allows the androgen receptor to interact with androgen responsive element (AREs), DNA sequences found on genes whose expression is regulated by androgen.
Androgen-mediated regulation of gene expression is a complicated process that may involve multiple co-activators (Adler et al.,
Proc. National Acad. Sci. USA
89:6319-6325, 1992). A fundamental question in the field of steroid hormone biology is how specific androgen-activated transcription can be achieved in vivo when several different receptors recognize the same DNA sequence. For example, the androgen receptor (AR), the glucocorticoid receptor (GR), and the progesterone receptor (PR) all recognize the same sequence but activate different transcription activities. Some have speculated that accessory factors may selectively interact with the androgen receptor to determine the specificity of gene activation by the androgen receptor.
Prostate cancer is the most common malignant neoplasm in aging males in the United States. Standard treatment includes the surgical or chemical castration of the patient in combination with the administration of anti-androgens such as 17 &bgr; estradiol (E2) or hydroxyflutamide (HF). However, most prostate cancers treated with androgen ablation and anti-androgens progress from an androgen-dependant to an androgen-independent state, causing a high incidence of relapse within 18 months (Crawford,
Br. J. Urology
70: suppl. 1, 1992). The mechanisms by which prostate cancer cells become resistant to hormonal therapy remain unclear. One hypothesis that has been advanced is that over the course of treatment, a mutation in the AR occurs which alters the receptor's sensitivity to other steroid hormones or anti-androgens, such as E2 and HF, thereby causing the progression from androgen-dependent to androgen-independent prostrate cancer. This hypothesis is supported by transient transfection assays in which it has been shown that anti-androgens may have an agonistic activity that stimulates mutant AR (mAR)-mediated transcription.
Recently, A1B1 was identified as estrogen receptor coactivator that is expressed at higher levels in ovarian cancer cell lines and breast cancer cells than in noncancerous cells (Anzick, et al.
Science
277:965-968, 1997). This result suggests that steroid hormone receptor cofactors may play an important role in the progression of certain diseases, such as hormone responsive tumors.
The identification, isolation, and characterization of genes that encode factors involved in the regulation of gene expression by androgen receptors will facilitate the development of screening assays to evaluate the potential efficacy of drugs in the treatment of prostate cancers.
BRIEF SUMMARY OF THE INVENTION
The present invention includes an isolated polynucleotide that encodes a co-activator for human androgen receptor, the polynucleotide comprising a sequence that encodes a polypeptide selected from the group consisting of an ARA54 polypeptide, an ARA55 polypeptide, an ARA24 polypeptide, and an Rb polypeptide.
Another aspect of the present invention is a genetic construct comprising a promoter functional in a prokaryotic or eukaryotic cell operably connected to a polynucleotide that encodes a polypeptide selected from the group consisting of an ARA54 polypeptide, an ARA55 polypeptide, an ARA24 polypeptide and an Rb polypeptide.
The present invention provides a method for screening candidate pharmaceutical molecules for the ability to promote or inhibit the interaction of ARs and AREs to modulate androgenic activity comprising the steps of:
(a) providing a genetic construct comprising a promoter functional in a eukaryotic cell operably connected to a polynucleotide comprising a sequence that encodes a polypeptide selected from the group consisting of an ARA54 polypeptide, an ARA55 polypeptide, an ARA24 polypeptide, and a retinoblastoma polypeptide;
(b) cotransforming a suitable eukaryotic cell with the construct of step a, and a construct comprising at least a portion of an expressible androgen receptor sequence;
(c) culturing the cells in the presence of a candidate pharmaceutical molecule; and
(d) assaying the transcriptional activity induced by the androgen receptor.
It is an object of the present invention to a provide a genetic construct capable of expressing a factor involved in co-activation of the human androgen receptor.
It is an object of the present invention to provide a method for evaluating the ability of candidate pharmaceutical molecules to modulate the effect of androgen receptor coactivators on gene expression.
Other objects, features, and advantages of the present invention will become apparent upon reading the specification and claims.
DETAILED DESCRIPTION OF THE INVENTION
Transactivation of genes by the androgen receptor is a complicated system that involves many different coactivators. It is not currently known just how many factors are involved in androgen receptor-mediated regulation of gene expression. The identification and/or characterization of four androgen receptor coactivators is reported herein. Inclusion of one or more of these coactivators in an assay for androgenic and antiandrogenic activity is expected to increase the sensitivity of the assay. Information about these coactivators is valuable in the design of pharmaceutical agents intended to enhance or interfere with normal coactivator function. A preliminary assessment of the efficacy of a potential therapeutic agent can be made by evaluating the effect of the agent on the ability of the coactivator to enhance transactivation by the androgen receptor.
One aspect of the present invention is an isolated polynucleotide that encodes a co-activator for human androgen receptor, the polynucleotide comprising a sequence that encodes a polypeptide selected from the group consisting of an ARA54 polypeptide, an ARA55 polypeptide, an ARA24 polypeptide and an Rb polypeptide.
Another aspect of the present invention is a genetic construct comprising a promoter functional in a prokaryotic or eukaryotic cell operably connected to a polynucleotide that encodes a polypeptide selected from the group consisting of an ARA54 polypeptide, an ARA55 polypeptide, an ARA24 polypeptide and an Rb polypeptide.
The present invention includes a method for screening candidate pharmaceutical molecules for the ability to promote or inhibit the ARs and AREs to result in modulation of androgenic effect comprising the steps of:
(a) providing a genetic construct comprising a promoter functional in a eukaryotic cell operably connected to a polynucleotide comprising a sequence that encodes a polypeptide selected from the group consisting of an ARA54 polypeptide, an ARA55 polypeptide, an ARA24 polypeptide, and a retinoblastoma polypeptide;
(b) cotransforming a suitable eukaryotic cell with the construct of step a, and a construct comprising at least a portion of an expressible androgen receptor sequence;
(c) culturing the cells in the presence of a candidate pharmaceutical molecule; and
(d) assaying the transcriptional activity induced by the androgen receptor gene.
The human androgen receptor is comprised of a ligand binding domain (LBD), a DNA binding domain (DBD), a hinge domain containing nuclear loc
Chernyshev Olga N.
Eyler Yvonne
Needle & Rosenberg P.C.
University of Rochester
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