JP2002520056A - Androgen receptor coactivator - Google Patents

Abstract

  (57) [Summary] Disclosed are androgen-related proteins, designated ARA54, ARA55, ARA24 and Rb. These proteins interact with the androgen receptor to alter androgen receptor-mediated transcriptional activation. Some of these proteins interact with the androgen receptor in an androgen-dependent manner. On the other hand, some other proteins will induce transcriptional activation in the presence of other ligands, such as E2 or HF. Also disclosed are methods for detecting androgen or anti-androgen activity using these proteins in a transient assay for detecting mammalian protein complexes.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] Prior art androgens regulate the development and proper functioning of mammalian male reproductive systems (including prostate and epididymis) Make up a class of hormones. Androgens also affect the physiology of many non-reproductive systems, including muscle, skin, pituitary, lymphocytes, hair growth and brain. Androgens exert their effects by altering gene expression levels of specific genes in a process mediated by the binding of androgens to androgen receptors. Androgen receptors, which are members of the steroid receptor superfamily, play an important role in male sexual differentiation and prostate cell proliferation. Androgen binding by the androgen receptor allows the androgen receptor to interact with the androgen-sensitive element (ARE). AREs are DNA sequences found in genes whose expression is regulated by androgens.

[0002] Androgen-mediated regulation of gene expression is a complex process that probably requires a large number of coactivators (Adler et al., Proc. Natl. Acad. Sci. USA,
89: 6319-9325 (1992)). A fundamental question in the area of steroid hormone biology is how transcription specifically activated by androgens is activated in vivo when several different receptors recognize the same DNA sequence. That is. For example, the androgen receptor (AR), glucocorticoid receptor (GR), and progesterone receptor (PR) all recognize the same sequence, but activate different transcriptional potentials. Some speculate that accessory factors may selectively interact with the androgen receptor and determine the specificity of gene activation by the androgen receptor.

[0003] Prostate cancer is the most common malignant neoplasm in elderly men in the United States. Standard treatments include treatment in which the testicles of the patient are surgically or chemically removed in conjunction with administration of an antiandrogen (eg, 17β estradiol (E2) or hydroxyflutamide (HE)). However, most prostate cancers treated with androgen ablation and antiandrogens progress from an androgen-dependent to an androgen-independent state, leading to a high recurrence within 18 months (Crawford, Br.
J. Urology 70: suppl.1, (1992)). The mechanism by which prostate cancer cells acquire resistance to hormone therapy is unknown. One hypothesis presented so far is that during treatment A
Mutations occur in R that alter the sensitivity of the receptor to other steroid hormones or antiandrogens (eg, E2 and HF), thereby leading to progression from androgen-dependent to androgen-independent prostate cancer. . This hypothesis is supported by a transient transfection assay. This assay indicated that anti-androgens may have agonistic activity to stimulate mutant AR (mAR) -mediated transcription.

[0004] Recently, A1B1 has been identified as an estrogen receptor coactivator expressed on ovarian and breast cancer cells at higher levels than non-cancerous cells (Anzick et al., Science 277: 965). -968 (1997)). This result suggests that steroid hormone receptor co-factors may play an important role in the development of certain diseases (eg, hormone-sensitive tumors). The identification, isolation and characterization of genes encoding factors required for the regulation of gene expression by the androgen receptor will aid in the development of screening assays to evaluate the efficacy of drugs in treating prostate cancer.

[0005] INVENTION It is an object the present invention provides isolated polynucleotides encoding the coactivator for human androgen receptor, ARA54 polypeptide, ARA55 polypeptide,
And a polynucleotide comprising a sequence encoding a polypeptide selected from the group consisting of an ARA24 polypeptide and an Rb polypeptide. Another feature of the invention is the use of ARA54 polypeptide, ARA55 polypeptide, A
A genetic construct comprising a promoter functional in prokaryotic or eukaryotic cells operably linked to a polynucleotide comprising a sequence encoding a polypeptide selected from the group consisting of RA24 and Rb polypeptides.

[0006] The present invention provides methods for screening candidate drug molecules for their ability to modulate androgen activity by promoting or suppressing the interaction of AR and ARE.
The method comprises the steps of: (a) operably linked to a polynucleotide comprising a sequence encoding a polypeptide selected from the group consisting of an ARA54 polypeptide, an ARA55 polypeptide, an ARA24 polypeptide and a retinoblastoma polypeptide. Providing a genetic construct comprising a promoter functional in eukaryotic cells; (b) providing a suitable eukaryotic cell with the construct of step a, and a construct comprising at least a portion of an androgen receptor sequence capable of being expressed. (C) culturing the cells in the presence of the candidate drug molecule; (d) assaying the androgen receptor-induced transcriptional activity.

[0007] An object of the present invention is to provide a gene construct capable of expressing a factor required for human androgen receptor coactivation. It is an object of the present invention to provide a method for assaying the ability of a candidate drug substance to modulate the effect of androgen receptor coactivator on gene expression. Other objects, features and advantages of the invention will be apparent from the following detailed description, and from the claims.

[0008] transactivation of genes by means androgen receptors for solving the problem is a complex system that requires a number of different coactivators. At present, it is unclear how many factors are involved in the regulation of androgen receptor-mediated gene expression. Here we report the identification and characterization of four androgen receptor coactivators. Incorporating one or more of these coactivators into an assay for androgenic and anti-androgenic activity is expected to increase the sensitivity of the assay. Information about these co-activators
It is important in the design of medicaments aimed at enhancing or inhibiting normal coactivator function. A preliminary assessment of the efficacy of a potential therapeutic can be performed by assaying the effect of the drug on the ability of the coactivator to enhance androgen receptor transactivation.

One feature of the invention is an isolated polynucleotide encoding a coactivator for the human androgen receptor, wherein the polynucleotide comprises ARA5
4 polypeptides, ARA55 polypeptides, ARA24 polypeptides and Rb
A sequence encoding a polypeptide selected from the group consisting of polypeptides. Another feature of the invention is a prokaryotic cell operably linked to a polynucleotide comprising a sequence encoding a polypeptide selected from the group consisting of ARA54 polypeptide, ARA55 polypeptide, ARA24 polypeptide and Rb polypeptide. Alternatively, it is a gene construct containing a promoter that functions in eukaryotic cells.

[0010] The present invention includes a method of screening candidate drug molecules for the ability to enhance or suppress AR and ARE to modulate androgenic action. The method comprises the steps of: (a) operably linked to a polynucleotide comprising a sequence encoding a polypeptide selected from the group consisting of an ARA54 polypeptide, an ARA55 polypeptide, an ARA24 polypeptide and a retinoblastoma polypeptide. Providing a genetic construct comprising a promoter that functions in a eukaryotic cell, comprising: (b) simultaneously cooperating a suitable eukaryotic cell with the construct of step a and a construct comprising at least a portion of an androgen receptor sequence capable of being expressed. (C) culturing the cells in the presence of the candidate drug molecule; (d) assaying the transcriptional activity induced by the androgen receptor gene.

The human androgen receptor is composed of a ligand binding domain (LBD), a DNA binding domain (DBD), a hinge domain containing a nuclear localization signal, and a transactivation domain within the highly variable N-terminus. Truncation or deletion of the LBD results in constitutive transactivation by the N-terminal domain. In certain cases, the progression of the prostate cancer from the androgen-dependent stage to the androgen-independent stage may be caused by mutations in the LBD that alter the ligand specificity of the mAR (Taplan et al., New Engl. J. Med. 332: 1
393-1398 (1995); Gaddipati et al., Cancer Res. 54: 2861-2864 (1994)). We investigated whether the differential steroid specificity of the wild-type (wt) AR and the mAR required that these receptors use different androgen receptor-associated proteins or coactivators.

As described in the examples, a human prostate cDNA library was screened by a yeast protein complex detection system (two-hybrid system) using mART887S as a search fishing bait. The sequences of two clones (designated ARA54 and ARA55) encoding putative coactivators are shown in SEQ ID NO: 1 and SEQ ID NO: 3, respectively. The deduced amino acid sequences of ARA54 and ARA55 are shown in SEQ ID NO: 2 and SEQ ID NO: 4, respectively. Furthermore, the DNA and amino acid sequence of ARA24 (SEQ ID NO: 5 and SEQ ID NO: 6, respectively) and the DNA and amino acid sequence of Rb (SEQ ID NO: 7 and SEQ ID NO: 8, respectively) are provided. These coactivators were further characterized as detailed below. Nucleotide additions, deletions and mutations (
SEQ ID NO: (naturally occurring or introduced in vitro):
It is likely that small changes of 1-8 will not affect coactivation by these expression products or polynucleotides.

[0013] Briefly, ARA54 interacts with AR in an androgen-dependent manner.
It is a 4 kDa protein. Co-expression of ARA54 and AR in a mammalian protein complex detection system has been shown to enhance reporter gene activity in an androgen-dependent manner. ARA54 functions as a relatively specific coactivator for AR-mediated transcription. However, ARA54 also functions as a universal coactivator of transcriptional activity for other steroid receptors via their cognate ligands and response elements. ARA54 is AR
And PR transcriptional activity was found to enhance 6-fold and 3-5-fold, respectively. In contrast, ARA54 has only minimal effects on glucocorticoid receptor (GR) and estrogen receptor (ER) in DU145 cells.

By co-expression of ARA54 with the known AR coactivators SRC-1 or ARA70, each of these coactivators contributes individually to maximize A
It has been shown that R-mediated transcriptional activity may be achieved. Furthermore, ARA
When 54 was co-expressed with SRC-1 or ARA70, enhanced AR-mediated transactivation was added, but not synergistic compared to that observed when only individual coactivators were present. Was. The C-terminal domain of ARA54 (amino acids 361-471 of SEQ ID NO: 1)
Acts as a dominant negative inhibitor for AR-mediated gene expression of the target gene. Co-expression of exogenous full-length ARA54 can reduce this inhibitory effect in a dose-dependent manner.

ARA54 enhanced the transactivation of wild type AR in the presence of DHT (10 ⁻¹⁰ to 10 ⁻⁸ M) about 3-5 fold. However, transactivation of wild-type AR
When E2 (10 ^-9 -10 ^-7 ) or HF (10 ^-7 -10 ^-5 ) was used as the ligand, only slight enhancement was obtained. Natl. Acad. Sci. USA, in press (1998) showing different sensitivities to E2 and HF (1998). ARA54 enhances transactivation by two mutant receptors (mARt877a and mARe708k). Capabilities were also examined. Mutant mARt877a (
It is found in many prostate tumors (23)), but not in E2 (10 ^-9 -10 ^-7 M).
And HF (10 ^-7 -10 ^-5 M), ARA54 is E3- or HF
-The mediation AR transactivation could be further enhanced. In contrast, the variant m
ARe708k (first identified in a yeast genetic screen (C. Wang, P.
hD Thesis of University of Wisconsin-Madison (1997)) showed ligand specificity and response to ARE54 as compared to that of wild type AR.

[0016] Any polypeptide that is substantially homologous to ARA54 and still exerts the same biological effect is expected to be able to function as an "ARA54 polypeptide". Using the sequence information disclosed herein, one of skill in the art would be able to obtain any ARA54 polypeptide by standard molecular biology techniques. The ARA54 polypeptide enhances AR transactivation in an androgen-dependent manner, enhances E2 or HF transactivation by the mutant receptor mARt877a, and further enhances the C-terminal domain of ARA54 (amino acids 361-471 of SEQ ID NO: 1). Is a polypeptide capable of reducing the suppression of AR-mediated gene expression caused by overexpression of the gene. The sequence information presented in this application can be used not only to identify allelic variants of the clone or sequence of the ARA54 gene, but also to identify corresponding genes of other mammalian species. It is also contemplated that truncations of the native coding region may be made to express smaller polypeptides that retain the same biological activity.

The polynucleotide sequence of ARA54 (SEQ ID NO: 3) is derived from the mouse hic5 (
Hydrogen peroxide induced clone) (B. Pugh, Curr. Opin. Cell Biol. 8: 303-311 (1996)
)) Shows high homology with the C-terminus, and expression of ARA54 is similar to that of hic5.
Triggered by Co-transfection assays of transcriptional activation, described in detail below, showed that ARA55 binds to both wild-type AR and mART887S in a ligand-dependent manner and enhances AR transcriptional activity.
ARA55 enhanced transcriptional activation by wild-type AR in the presence of 10 ^-9 M DHT or T, but not 10 ^-9 M E2 or HF. In contrast, ARA
55 is mART8 in the presence of DHT, testosterone (T), E2 or HF
The transcriptional activation by 87S can be enhanced. ARA55 did not enhance the transcriptional activation of mARe708k in the presence of E2, but can enhance transcription in the presence of DHT or T. The C-terminal domain of ARA55 (amino acids 251-444 of SEQ ID NO: 3)
Sufficient for binding to R, but does not enhance transcriptional activation by AR.

[0018] The present invention is not limited to the particular ARA55 polypeptide disclosed in SEQ ID NO: 4. It is contemplated that any ARA55 polypeptide could be used in the practice of the present invention. "ARA55 polypeptide" refers to the transactivation of wild type AR, mutant receptor mARt877a, in the presence of DHT, E2 or HF.
Alternatively, transactivation of the intact receptor mARe708k is performed by DHT or THT.
Refers to a polypeptide that can be enhanced in the presence of Such polypeptides include truncated forms as well as allelic variants and the corresponding genes of other mammalian species.

The N-terminal domain of the AR contains a polymorphic polyglutamine (Q) stretch and a polymorphic polyglycine (G) stretch, which are the observed human AR cDNAs.
Is the cause of length variability. The length of the polyQ region (usually 11-33 residues in length) is inversely proportional to prostate cancer risk and directly proportional to SBMA or renal disease (La Spa
da et al., Nature (London) 352: 77-79 (1991)). The incidence of more severe, more distant metastases and lethal prostate cancer is higher in men with shorter AR polyQ stretches.

As described in the Examples, experiments performed to identify those with potential coactivators that interact with the AR polyQ region have shown that a coactivator called ARA24 that interacts with the polyQ region. Was isolated.
The sequence of the ARA24 clone and its predicted translation product are shown in SEQ ID NO: 5 and SEQ ID NO: 6. The ARA24 clone has an ORF that is identical to the published ORF of human Ran (an abundant ras-like small GTPase) (Beddow et al., Proc. Natl. Acad.
Sci. USA, 92: 3328-3332 (1995)). Overexpression of ARA24 in the presence of DHT enhances AR-mediated transcriptional activation over that seen in cells transfected with AR alone. Furthermore, antisense ARA24 (ARA24as)
Expression reduces DHT-induced transcriptional activation. An ARA24 polypeptide is a polypeptide that interacts with the AR polyQ region. A feature of ARA24 polypeptides is their ability to enhance transactivation when overexpressed in eukaryotic cells that contain some endogenous ARA24.
Expression of antisense RNA reduces AR receptor transactivation.

[0021] Androgen receptor mutations do not account for all cases of androgen-dependent tumors. Because some androgen-dependent tumors are wild-type AR
Is held. A significant proportion of androgen-insensitive tumors have reduced retinoblastoma protein (Rb) expression (Bookstein et al., Science 247: 712-715 (1990)) and truncated Rb protein expression (Bookstein et al., Proc. Natl. Acad. Sci. USA, 87: 776.
2-7766 (1990)) or loss of the Rb allele (Brooks et al., Prostate 26: 3).
5-39 (1995)). Prostate cancer cell line DU145 has an abnormal short mRNA transcript of Rb exon 21 (Sarkar et al., Prostate 21: 145-1).
52 (1992)), and transfection of wild-type Rb gene into DU145 cells was shown to suppress the malignant phenotype (Bookstein et al., Proc. Natl. Ac).
ad. Sci. USA, 87: 7762-7766 (1990)).

Rb functions in controlling cell division and differentiation (RA Weinberg, Cell
81: 323-330 (1995); Kranenburg et al., FEBS Lett. 367: 103-106 (1995)). In resting cells, low phosphorylated Rb prevents cells from entering the cell division cycle inappropriately. Phosphorylation of Rb by cyclin-dependent kinases reduces Rb-mediated growth suppression and allows cell division (Dowdy et al., Cell 73: 499-511 (1993); Chen et a
l., Cell 58: 1193-1198 (1989)). Conversely, Rb dephosphorylation during G1 progression induces growth suppression or cell differentiation (Chen et al. (1989); Mihara et al., Science 246:
1300-1303 (1989)). In dividing cells, Rb is dephosphorylated at the end of mitosis and the onset of G1 (Ludlow et al., Mol. Cell. Biol. 13: 367-372 (1993)). This dephosphorylation activates Rb for the subsequent G1 phase of the cell cycle,
During this G1 phase, Rb exhibits a growth inhibitory effect.

We examined the role of Rb in transactivation of AR as detailed in the Examples. Rb expresses wild type AR or mARs8 in the presence of DHT, E2 or HF.
77t of transcriptional activity and, in the presence of DHT, mAre70
It was found that 8k transcriptional activity could be induced. We have also found that the transcriptional activities of Rb and ARA70 act synergistically to enhance the transcriptional activity of Ar. The sequence of the cloned Rb gene and the deduced amino acid sequence of its ORF are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively. Rb polypeptide is a polypeptide substantially homologous to SEQ ID NO: 8, which interacts with the N-terminal domain of AR and acts synergistically with ARA70 in enhancing transactivation by AR.

In the examples, various eukaryotic cell types (including yeast, prostate cells containing mutant AR and prostate cells lacking AR) are used to evaluate the ability of putative androgen coactivator to enhance transactivation by AR. did. It is contemplated that any eukaryotic cell can be used in the method of the invention in an assay for AR activity. This feature allows the assay to be freely designed. Cells were transfected by the calcium phosphate method, as described below. The method of the invention could be performed by any means of transfection. These means include, for example, electroporation or particle bombardment.

Changes in transactivation levels due to AR can be assessed by any means. These means include measuring the change in the level of mRNA for one gene under AR control, or quantifying the amount of a specific expressed protein whose expression is under AR control using an antibody specific to the protein. Are included. The simplest method is to evaluate transactivation by AR using a reporter gene. As used herein, a reporter gene is a gene under the control of the androgen receptor, which encodes a protein that is sensitive to quantitation by colorimetric or fluorescent assays. In the examples below, the chloramphenicol acetyltransferase or luciferase gene was used as the reporter gene. This gene may be present on the host chromosome, or may be introduced into the host cell by co-transfection with the coactivator gene. The following non-limiting examples are for illustrative purposes only.

[0026] Human prostate library being built pACT2 yeast expression vectors of Example plasmid (from Dr. S.Elledge) is, GAL4 activation domain fused to a human prostate cDNA (GAL4AD
, Amino acids 768-881). pSG5wtAR was reported previously (Yeh & Chang, Proc. Natl. Acad. Sci. USA,
93: 5517-5521 (1996)). pGAL0-AR (wild type) was obtained from Dr. Chen (University of Massachusetts). pGAL0 contains the GAL4 DNA binding domain (DBD). For construction of pAS2-wtAR or -mAR, the C-terminal fragment of wild type AR (amino acids 595-918), mARt877s (Dr. SP Balk, Beth
Israel Hospital, Boston, MA) or mARe708k (H. Shim, Hyogo Med
ical College, Japan) was inserted into the pAS2 yeast expression vector (Clontech). Another AR variant (mARv888m) (from a patient with androgen insensitivity syndrome)
Was constructed according to a previous report (Mowszowicz, et al., Endocrine 1: 203-209 (1993)).

An ARA70 fusion was constructed using pGAL4-VP16. pGAL4-
VP16 contains a GAL4DBD linked to the acidic activation domain of VP16. pCMX-Gal-N-RB and pCMX-VP16-AR are fragments of Rb (aa370-928) and AR (aa590-918) with pCMX-g
It was constructed by inserting into al-N and pCMX-VP16, respectively. The sequence of the junction of the construct was confirmed by sequencing.

PYX-ARA24 is obtained by transforming the ARA24 gene into a yeast expression plasmid pYX243.
(Ingenus) by placing it under the control of the gal-1 promoter. A
R poly-Q stretch and its flanking region (AR amino acids 11-208)
Was ligated to a PAS2 yeast expression plasmid for use as a search bait in a protein complex detection system. For the protein complex detection liquid culture system β-gal assay, AR cDNAs with poly Q stretches of various lengths spanning the same AR poly Q region were constructed in the same manner as pAS2 as our search fishing plasmid. . Poly Q length is 1, 25, 4
Transforming yeast Y190 with all 9 of these AR search bait plasmids,
It turned out to have no autonomous activity. pCMV-antisense ARA24 / Ra
n (ARA24as) expression plasmid is 334 bp B of ARA24 / Ran.
The glII fragment was constructed by inserting it into the pCMV vector in the antisense orientation. The fragment extends from the 5 'untranslated region of ARA24 / Ran to the translation initiation codon (nucleotides 1-334 of SEQ ID NO: 5). MMT
V-CAT and MMTV-Luc reporter genes were used for AR transactivation assays. pSG5-AR and pSV-βgal are under the control of the SV40 promoter and the β-globulin gene intron-1 enhancer. p6R-ARQ1, p6R-ARQ25, p6R-ARQ49 are Dr
Provided by Roger L. Meisfield (Chamberlain et al., Nucleic Acids Re
s. 22: 3181-3186 (1994)).

[0029] pSG5-GAL4DBD-ARA24 was prepared by inserting the coding sequence of the Gal4DBD-ARA24 hybrid protein into the pSG5 vector. pVP16-ARN-Q1, pVP16-ARN-Q25, pVP16-A
RN-Q35 and pVP16-ARN-Q49 were prepared by inserting the N-terminal domain (aa34-555) of each polyQAR into a pVP16 vector (Clontech).
It was expressed as a P16AD hybrid protein. The GAL0AR plasmid (containing GAL4DBD fused to the E region of human AR) was provided by Dr. D. Chen. The pSG5-CAT reporter plasmid (Clontech) contains 5 GAL4
It contains a binding site upstream of the ElbTATA box (linked to the CAT gene).

[0030] pSG5-AR and pSG5-ARA70 are described above (Yeh & Chang, Proc.
Natl. Acad. Sci. USA, 93: 5517-5521 (1996)). Two variants of the AR gene (mAT877 from prostate cancer, mutation of codon 877 from Thr to Ala; and mAR708 from partial androgen insensitivity syndrome (PIAS), mutation of codon 708 from Glu to Lys) S. B respectively
alk (Beth Israel Hospital, Boston) and H. Shima (Hyogo Medical College,
Japan). The clone used in the protein complex detection system to evaluate the role of Rb in AR transactivation was the Rb fragment (aa371-928)
No. 4 DBD. Similarly, almost full length (aa36-
The AR (nAR) and AR-LBD (aa590-918) fragments of 918) were ligated to the transcriptional activator VP16.

[0031] For Yeast Associated with Ligand Binding Domain by Yeast Protein Complex Detection System
To identify ARA coactivators that interact with the prostate cDNA library screening LBD, pACT2
-Prostate cDNA library was used to transform Y190 yeast cells into plasmid pAS2mAR
(MART877S) (the latter containing GAL4DBD (aa1-147) fused to the C-terminal domain of this mAR). Transformants were selected by growth on SD plates (-3SD plates) containing 3-aminotriazole (25 mM) and DHT (100 nM) lacking histidine, leucine and tryptophan. Colonies were further filtered assayed for β-galactosidase activity. interacts with mtARt877s but GALA4T
Plasmid D obtained from a positive cDNA clone found not to react with R4
NA was isolated from yeast, amplified in E. coli, and the insert was confirmed by DNA sequencing. To identify clones that interact with the polyQ region of the N-terminal domain, AR
The polyQ stretch (amino acids 11-208) was inserted into the pAS2 yeast expression plasmid, which was co-transformed into Y190 yeast cells with a human brain cDNA library fused to the Ga14 activation domain. Transformants were selected for growth on SD plates lacking histidine, leucine and tryptophan and supplemented with 3-aminotriazole (40 mM).

Amplification and Characterization of ARA Clones ARA54 (SEQ ID NO: 1) and ARA55 (SEQ ID NO: 1) found to interact with mARt877s
The full-length DNA sequence containing the two coactivators, designated SEQ ID NO: 3), was isolated by 5 'RACE PCR using a Marathon cDNA amplification kit (Clontech) according to the manufacturer's protocol. The missing 5 'coding region of the ARA54 gene was replaced with the gene-specific antisense primer of SEQ ID NO: 9 and the following PCR conditions: 94 ° C for 1 minute; 94 ° C for 5 seconds, followed by 72 ° C
For 5 minutes at 94 ° C., followed by 5 cycles of 3 minutes at 70 ° C .; 25 cycles of 5 seconds at 94 ° C., followed by 3 minutes at 68 ° C., from H1299 cells. PCR product
It was subcloned into a T7-blue vector (Novagen) and sequenced. ARA55 was converted from an HeLa cell line to an ARA55-specific antisense primer (SEQ ID NO: 10).
) And ARA54 were isolated by PCR using the PCR reaction conditions described. Using 5 'RACE-PCR, we have developed a novel protein of 474 amino acids in length.
1721 bp DNA having an open reading frame encoding (SEQ ID NO: 2)
A fragment (SEQ ID NO: 1) could be isolated from the H1299 cell line. The in-vitro translation product is a polypeptide with an apparent molecular weight of 54 ± 2 kDA, which is the calculated molecular weight (53
.8 kDa). Central portion of ARA54 (amino acid 220-265 of SEQ ID NO: 2), the zinc finger motif called RING finger, which is defined as _{CX 2 CX 9- 27 CXHX 2 CX} 2 CX 6-17 CX 2 C ( SEQ ID NO: 11) Contains the cysteine-rich region that forms. This region is a conserved domain among several human transcription factors or proto-oncogenic proteins, including BRCA1, RING1, PML and MEL-18 (Miki et al.,
Science 266: 66-71 (1994); Borden et al., EMBO J. 14: 1532-1541 (1995); Lo
vering et al., Proc. Natl. Acad. Sci. USA 90: 2112-2116 (1993); Blake et.
al., Oncogene 6: 653-657 (1991); Ishida et al, Gene 129: 249-255 (1993))
. In addition, ARA54 contains a second cysteine-rich motif with a B box-like structure located 43 amino acids downstream of the RING finger motif. However
ARA54 lacks the predicted coil domain at the C-terminus directly to the highly conserved B box domain in the RING finger-B box family.
Different from members of the RING finger-B box family. Therefore, ARA54
Will represent a new subgroup of this family.

[0033] Full-length human ARA55 has 91% homology with mouse hic5 and has an expected molecular weight of 55 kD.
Has an open reading frame encoding a 444 amino acid polynucleotide (SEQ ID NO: 4) having Human ARA55 has four LIM motifs in the C-terminal region. The LIM motif has a consensus sequence: CX ₂ CX _16-23 HX ₂ CX ₂ CX ₂ CX _16-21 CX ₂ (C, H, D) (SEQ ID NO: 1
2) is a cysteine-rich zinc-binding motif (Sadler, et al., J. Ce
ll Biol. 119: 1573-1587 (1992)). Although the function of the LIM motif is poorly defined, some data suggest that it plays a role in protein-protein interactions (Schmeichel & Beckerle, Cell 79: 211-219, 1994).
Of all the identified SR-related proteins, only ARA55 and thyroid hormone interacting protein 6 (Trip 6) (Lee, et al. Mol. Endocrinol. 9: 243-254 (1995)) have a LIM motif . Clones that showed a strong interaction with the poly-Q diet were identified and subsequently subjected to sequence analysis. This clone contains a 1566 bp insert (SEQ ID NO: 6) with an open reading frame encoding a 216 amino acid polypeptide with a calculated molecular weight of 24 kDa (SEQ ID NO: 6). GenBank sequence comparison showed that this clone was Ran / TC4 (
Abundant ras-like small GTPase found in a wide range of cell types and associated with nuclear plasma transfer
(Beddow et al., Proc. Natl. Acad-Sci. USA 92: 3328-3312, 1995)). Therefore, this factor was named ARA24 / Ran. The cDNA sequence of the ARA24 clone (SEQ ID NO: 5) (GenBank accession number AF052578) is longer than the known human Ran ORF in that it contains untranslated regions of 24 bp and 891 bp at 5′- and 3′-, respectively.

Northern blotting Total RNA (25 μg) was fractionated on a 1% formaldehyde-MOPS agarose gel, transferred to a Hybond-N nylon membrane (Amersham), and prehybridized.
A probe corresponding to the 900 bp C-terminus of ARA55 or the ARA54-specific sequence can be used in vitro with a Random Primed DNA Labeling Kit (Boehringer-M).
annheim) was ³² P-labeled using the manufacturer's protocol and hybridized overnight. After washing, the blots were exposed and quantified on a Molecular Dynamics PhosphorImager. β-actin was used to monitor the amount of total RNA in each lane. Northern blot analysis showed the presence of a 2 kb ARA55 transcript in HeLa and prostate cells. This transcript was not detected in other tested cell lines including HepG2, H1299, MCF7, CHO, PC12, P19 and DU145 cells. The ARA54 transcript is H
1299 cells, found in prostate cancer cell lines PC3 and LNCaP.

Co-immunoprecipitation of AR and ARAs In -vitro translated full-length AR and ARA54 were combined with 10 ^-8 M DHT in modified RIPA buffer (50 mM Tris-HCL pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.1 mM % NP40, 1mM PMSF, aprotinin, leupeptin, pepstatin, 0.25% Na-deoxycholate, 0.
(25% gelatin) with or without shaking at 4 ° C. for 2 hours. The mixture was incubated with rabbit anti-His-tag polyclonal antibody for an additional 2 hours, protein A / G plus (PLUS) -agarose (Santa Cruz) was added and incubated at 4 ° C. for another 2 hours. The conjugated beads are washed four times with RIPA buffer, boiled in SDS sample buffer, analyzed by 8% SDS / PAGE, and stored on STORM 840.
(Molecular Dynamics). ARR54 and AR were found in the complex when immunoprecipitated in the presence of 10 ^-8 M DHT, but not in the absence of DHT. This result suggests that ARA54 interacts with AR in an androgen-dependent manner. Interaction between recombinant full-length human AR and ARA24 / Ran protein, co-immunoprecipitation,
This was further investigated by subsequent SDS-PAGE and Western blotting. The results of the co-immunoprecipitation assay indicate that ARA24 / Ran interacts directly with AR. The phosphorylation state of the bound guanine nucleotide for small GTPase does not affect this interaction.

AR-pull-down assay using GST-Rb Full-length Rb fused to glutathione-S-transferase (ST-Rb _1-928 ) was recently reported (Zarkowska & Mittnacht, J. Biol. Chem., 272: 12738-12746, 1997) and expressed in E. coli. B121pLys strain and purified. About 2 μg of the His-tag column purified baculovirus AR was added to GST-loaded glutathione-Sepharose beads in 1 ml NET-N (20 mM Tris-HCL (pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% (v / v )
Noniodet P-40) solution and incubated at 4 ° C. for 3 hours with gentle agitation. After pelleting the beads by low speed centrifugation, the clear supernatant is mixed with GST-Rb loaded glutathione-Sepharose beads in the presence or absence of 10 mM DHT and further mixed at 4 ° C with gentle stirring. Incubated for 3 hours. The pelleted beads were washed 5 times with NET-N, mixed with SDS-sample buffer, boiled and separated by electrophoresis in a 7.5% polyacrylamide gel. The Western blot of the gel was incubated with the anti-AR polyclonal antibody NH27 and developed with a secondary antibody conjugated to alkaline phosphatase. AR co-precipitated with GST-Rb, but not with GST alone. This indicates that AR and Rb are both involved in the complex.

Transfection studies Human prostate cancer DU145 or PC3 cells or human lung cancer cells NCI H1299 were transfected with penicillin (25 U / ml), streptomycin (25 μg / ml) and 5% fetal calf serum (FCS).
Were grown in Dulbecco's Minimum Essential Medium (DMEM) containing One hour prior to transfection, the medium was changed to DMEM with charcoal stripped FCS. Phenol red and serum free media were used for experiments with E2 or TGFβ, respectively. The β-galactosidase expression plasmid pCMV-β-gal was used as an internal standard for transfection efficiency. Cells were transfected using the calcium phosphate method (Yeh, et al. Mol
ec. Endocrinol. 8: 77-88, 1994). The medium was changed 24 hours after transfection,
Cells were treated with either steroid hormones or hydroxyflutamide and cultured for an additional 24 hours. Cells were harvested and cell lysates were assayed for CAT activity after normalization using β-galactosidase as an internal standard. Chloramphenicol acetyltransferase (CAT) activity was visualized using a phosphoimager (Molecular Dynamics), and image quantification (Ima
geQuant) software (Molecular Dynamics).

Mammalian Protein Complex Detection Assay The mammalian protein complex detection system used was essentially a Clontech (California) protocol with the following modifications. To obtain good expression, GAL4DBD (amino acids 1-147) was fused to pSG5 under the control of the SV40 promoter and named pGALO. The hinge and LBD of the wild type AR were inserted into pGALO. Similarly, V
The P16 activation domain is fused to pCMX under the control of the CMV promoter, resulting in pCMX VP16
(Provided by Dr. RM Evan). DHT-dependent interaction between AR and ARA54 was confirmed in prostate DU145 cells using a protein complex detection system using a CAT reporter gene assay. ARA54
Alternatively, transient transfection of wild type AR alone showed negligible transcriptional activity. However, co-expression of AR and ARA54 in the presence of ^10-8 M DHT
Activity was significantly induced. ARA54 functions as a relatively specific coactivator for AR-mediated transcription. ARA
54 induces AR and PR transcriptional activity up to 6-fold and 3- to 5-fold, respectively. In contrast, ARA
54 showed marginal effects (less than 2-fold) on GR and ER in DU145 cells.
These data indicate that ARA54 is less specific for AR as compared to ARA70, which shows higher specificity for AR. However, we could not rule out the possibility that ARA54 was more common for other steroid receptors in other cell types under different conditions. Co-expression of ARA54 with SRC-1 or ARA70 is additive rather than synergistic.
It has been found to enhance R transcription activity. These results indicate that these cofactors independently contribute to adequate or maximal AR-mediated transcriptional activity.

The C-terminal region of ARA54 (amino acids 361-471 of SEQ ID NO: 2) isolated from a prostate cDNA library was shown to be sufficient for interaction with AR in a yeast protein complex detection assay. We have found that the region contains endogenous ARA54 H1229.
Whether to suppress the effects of ARA54 on AR-activated transcription in cells was further investigated. The C-terminal region of ARA54 inhibited AR-mediated transcription by about 70%, and co-expression of exogenous full-length ARA54 reversed the pressure-reducing effect in a dose-dependent manner. These results indicate that A
The C-terminal domain of RA54 can serve as a dominant negative inhibitor,
Furthermore, it has been demonstrated that ARA54 is required for proper or maximal AR transactivation in human H1229 cells. ARA54 for transcriptional activity of wild type AR and mtARs in the presence of DHT, E2 and HF
Studies of the effects of have revealed differential ligand specificity. Transactivation of wild-type AR occurred in the presence of DHT (10 ^-10 to 10 ^-8 M), and co-expression of ARA54 enhanced transactivation a further 3-5 fold. However, wild-type AR
It responded only marginally to E2 (10 ^-9 -10 ^-7 M) or HF (10 ^-7 -10 ^-5 M) in the presence or absence of ARA54. As expected, the positive control ARA70 is 10 ^- could be enhanced 10 ^-5 AR transcriptional activity in the presence of HF - ¹ -10 ^-7 M E2 and ^10-7. This result is in good agreement with previous reports (Yeh, PNAS, Miyamoto, PNAS). The AR mutant Art877a (23) found in many prostate tumors and the AR mutant Are708k (24) found in yeast gene screens and partially androgen-insensitive patients showed differential specificity for ligands . ARA
In the absence of 54, Art877a responds to E2 (10 ^-9 -10 ^-7 M) and HF (10 ^-7 -10 ^-5 M), and AR
A54 further enhanced E2- or HF-mediated AR transactivation. These results
mtARs have also been suggested to require cofactors for proper or maximal DHT-, E2-, or HF-mediated AR transcriptional activity. mARe708k DHT response is wild-type A
It was slightly less sensitive than the response of R or mARt877s. On the other hand, E2 and HF
Showed no agonist activity against ARe708k. These results show that
The modification of 708 is critical for the interaction of the anti-androgen-ARe708k-ARA54 complex and suggests that both AR structure and coactivator play a role in determining ligand specificity.

DU145 cells cotransfected with a plasmid encoding the hormone binding domain of wild type AR fused to GAL4 DBD (GALOAR) and a plasmid encoding full length ARA55 fused to the activation domain of VP16 (VP16-ARA55) CAT activity in
Significantly induced by co-transfection of VP16 ARA55 and GALOAR in the presence of nM DHT, but not E2 or HF. GALO empty vector and VP
The combination with 16-ARA55 did not show any CAT activity. The combination of GALOAR and VP16 vector showed negligible CAT activity. These results show that ARA55
And R interact in an androgen-dependent manner. Transient transfection assays were performed to investigate the role of ARA55 in AR transactivation activity. DU145 cells were co-transfected with an increased amount of ARA55 and wild-type AR under control of a eukaryotic promoter and an MMTV CAT reporter. The AR without ligand has minimal MMTV-in the presence and absence of ARA55.
It had CAT reporter activity. ARA55 alone also had minimal reporter activity. Addition of 10 nM DHT increased the AR transcriptional activity by 4.3-fold, and ARA55 increased this induction by a further 5.3-fold (from 4.3 to 22.8-fold) in a dose-dependent manner. The induced activity is
The AR: ARA55 ratio reached a stable state at 1: 4.5. Similar results were obtained with PC3 with DU145 cells.
Obtained using the CAT reporter gene under control of the 2.8 kb promoter region of the cell or PSA gene. C-terminal region of ARA55 (ARA _251-444 ) (amino acids 251-
444) did not enhance CAT activity. Of PC3 cells containing ARA55 endogenous, ARA55 _{251- 444,} co-transfection with AR and MMTV-CAT reporter, 10 nM
In the presence of DHT, it showed dramatically reduced AR transcriptional activity compared to cells transfected with AR and MMTV-CAT alone. These results demonstrate that ARA55 is required for adequate or maximal AR transcriptional activity in PC3 cells and that the C-terminus of ARA55 serves as a dominant negative inhibitor.

Effect of ARA55 on mARt877s and mARe708k in the presence of DHT and its antagonists E2 and HF. The mARt877s receptor is found in LNCaP cells and / or advanced prostate cancer and has a point mutation at codon 877 (Thr to Ser) (G
addipati et al., Cancer Res. 54: 2861-2864 (1994); Veldscholte et al., Bi
ochem. Biophys. Commun. 173: 534-540 (1990)). mARe708k receptor is codon 7
08 has a point mutation (Glu to Lys) and is isolated by yeast genetic screening and shows reduced susceptibility to HF and E2 compared to wild-type AR (Wang, C., University of Wisconsin-Madison) PhD Dissertation (1997)). Wild-type AR, the transcriptional activity of mARt877s and MARe708k, was induced by ^{DHT (10 -11 ~ 10 -8 M} ). ARA55 enhanced transactivation of all three receptors by a factor of 4-8. In the presence of E2 or HF, wild type AR responded marginally only at high concentrations (10 ^-1 M for E2 and 10 ^-5 M for HF). However, co-transfection of wild-type AR with ARA55 at a ratio of 1: 4.5 enhanced AR transcriptional activity at 10 ^-8 -10 ^-7 M for E2 or 10 ^-6 to 10 ^-5 M for HF. Compared to wild type AR, LNCaP mAR responded better to E2 and HF, and ARA55 significantly enhanced its transcriptional activity. ARA55 is suitable or maximal DHT-
, E2- or HF-mediated AR transcriptional activity. The effect of ARA55 on transcriptional activation by GR, PR and ER was tested in DU145 cells. ARA55 is fairly specific for AR but enhances GR and PR to a small extent and has a marginal effect on ER. ARA70 shows much higher specificity for AR than ARA55 compared to other tested steroid receptors. ARA55 is GR-
It enhances AR-mediated transcription to a greater degree than PR- or ER-mediated transcription, but appears to be less specific than ARA70. The amino acid sequence of ARA55 has very high homology to mouse hic5,
Since early studies on ic5 suggested that expression of this mouse gene could be induced by negative TGFβ (Shibanuma et al., J. Biol. Ch.
em. 269: 26767-26774 (1994)), we were interested in examining whether ARA55 serves as a bridge between TGFβ and the AR steroid hormone system. Northern blot analysis showed that TGFβ treatment (5 ng / ml)
Was induced about 2-fold. In the same cell, TGFβ treatment reduced AR transcriptional activity by 7
Increased to 0%. This induction is weak (70% vs. 4-fold) compared to the effect achieved in transfecting PC3 cells with exogenous ARA55. This is AR and ARA55
May be related to the difference in AR: A for maximum transcription activity
The optimal ratio of RA55 is 1: 4.5. Whether other mechanisms are involved in this TGFβ-induced AR transcriptional activity is an interesting question to investigate. TGFβ is ARA in prostate
The unexpected finding of enhancing AR transcriptional activity through induction of 55 links negative regulatory protein function in a positive manner by inducing the transcriptional activity of AR, a major promoter of prostate tumor growth May show first proof.

The ability of ARA55 to induce the transcriptional activity of both wild-type Ar and mARt877s in the presence of DHT, E2 and HF suggests an important role for ARA55 in the development of prostate cancer and the development of resistance to hormonal therapy I have. Evaluation of molecules that interfere with the function of ARA55 will help identify potent chemotherapeutic drugs. Human small cell lung cancer H1299 cell line without endogenous AR protein was transformed with AR and ARA2
Transfected with 4 / Ran. Since ARA24 / Ran is one of the most abundant and ubiquitously expressed proteins in various cells, sense and antisense ARA24 / Ran mammalian expression plasmids were tested. Overexpression of sense ARA24 / Ran did not significantly enhance AR transactivation. This result indicates that endogenous ARA24 / Ran
Is not surprising, given the large presence of. However, antisense ARA24 / Ran (ARA24as) significantly reduced DHT-induced CAT activity in a dose-dependent manner. Furthermore, when the DHT concentration was increased from 0.1 nM to 10 nM, DHT strongly induced AR transactivation and reduced the inhibitory effect of ARA24as. This indicates that increasing DHT levels antagonizes the negative effects of ARA24as. The affinity between ARA24 / Ran and AR is inversely related to the length of the AR polyQ stretch. AR transactivation decreases with increasing length of AR polyQ. Exchanged fusion partners Gal4DBD-ARA24 / Ran and VP16AD-ARNs (length 1, 25,
A mutual protein complex detection assay using amino acids 34-555 with 35,49 residues of polyQ was performed using mammalian CHO cells. These results indicate that ARA24 / Ran and AR
It has been consistently shown that affinity between the polyQ region is inversely correlated with the length of AR polyQ in both yeast and mammalian CHO cells. Regulation of AR transactivation by ARA24 / Ran correlates with its affinity. These results demonstrate that different lengths of polyQ can exist in a single cell or cell line.
Using ARs with, it is suggested that ARA24 / Ran can achieve differential transactivation of AR. ARA24as was used again in the ARE-Luc transfection assay to resolve the role of AR poly-Q length in controlling AR by ARA24 / Ran. ARs of polyQ lengths of 1, 25 and 49 residues and increasing amounts (1, 2 and 4 μm)
g) ARA24as expression vector together with an equal amount of reporter plasmid (pMMTV-Luc)
HO cells were co-transfected. Basic reporter activity is antisense AR
Although slightly affected by increased A24 / Ran levels, ARA24as showed a significant decrease in AR transactivation. As the length of AR polyQ increased, the effect of ARA24as on AR transactivation decreased. These results indicate that ARA24 / Ran
Suggests that the effect of reduced ARA24 / Ran on the affinity of AR and AR transactivation decreases with increasing AR polyQ length.

Co-expression of the Rb and AR expression plasmids in DU145 cells using the mammalian protein complex detection system was achieved by co-transfection of almost full-length AR (nAR, amino acids 36-918) and Rb in CAT activity. Doubled. nAR and PR-
Cells co-transfected with LBD or Rb and ARA70 did not show increased CAT activity. Surprisingly, the addition of 10 nM DHT made very little difference in the interaction between Rb and nAR. The inability of Rb to interact with AR-LBD suggests that the interaction site of AR is located in the N-terminal domain (amino acids 36-590). Our data suggest that the interaction between Rb and AR is unique in that: First, the interaction is androgen-independent, binding is specific, but significantly weaker than other AR-related proteins, such as ARA70 (3-fold vs. 12-fold; in mammalian protein complex detection assays). Induced CAT activity, data not shown). Second, the steroid receptor
Unlike most identified steroid receptor-related proteins that bind to the C-terminal domain, Rb binds to the N-terminal domain of AR. Third, Rb and ARA7
No interaction between the two AR-related proteins, 0, in DU145 cells. DU145 cells containing the mutant Rb (Singh et al., Nature 374: 562-565 (1995)) were cultured with charcoal-stripped FCS in the presence or absence of 1 nM DHT. DHT
Transiently transfected DU145 with a 1: 3 ratio of wild type AR and Rb in the absence of
No AR transcription activity was observed in the cells. However, wild type AR is 1n
When expressed in the presence of MDHT, AR transcriptional activity could be induced 5-fold. Rb and
Co-transfection with AR increases AR transcriptional activity from 5-fold in the presence of 1 nM DHT
It can be further increased by a factor of 21. As a control, co-transfection of ARA70, the first identified AR coactivator, was able to enhance DU145 cell transcriptional activity 5- to 36-fold. In DU145 cells co-transfected with Rb, ARA70 and AR, the induction of AR transcriptional activity increased synergistically from 5-fold to 64-fold.
When transfecting wild-type AR without Rb or ARA70, 10 nM E2 or 1 μM H
A slightly marginal induction (less than 2-fold) was detected in the presence of F. In contrast, wild type
Co-transfection of AR with Rb or ARA70 increases AR transcriptional activity by a factor of 12 (E2) or 3
AR transactivation with Rb and ARA70 can enhance AR transcriptional activity 36-fold (E2) or 12-fold (HF). We extended these findings to two different AR variants: mAt877s from prostate cancer patients and mARe708k from partially androgen-insensitive patients. MARt wild type AR
Similar induction was obtained when replacing with 877s. In contrast, when the wild type AR was replaced with mARe708k, a similar induction was detected in the presence of 1 nM DHT, but 10 nM E2 or 1
There was little induction by co-transfection of meAR708k with Rb and / or ARA70 in the presence of μHF. These results indicate that Rb and ARA70 were wild-type AR and m
It has been shown that the transcriptional activity of AR877 can be synergistically induced in the presence of 1 nM DHT, 10 nM E2 or 1 μM HF. However, Rb and ARA70 synergistically induced the transcriptional activity of mAR708 only in the presence of 1 nM DHT, but not 10 nM E2 or 1 μM HF. The fact that Rb and ARA70 can induce the transcriptional activity of both wild-type AR and mutant AR that occurs in many prostate tumors strongly emphasizes the importance of Rb and ARA70 in normal prostate and prostate tumors. Would argue. Furthermore, the differential induction of DHT versus E2 / HF would suggest that site 708 of AR plays an important role in androgen: antiandrogen recognition for AR.

The present inventors have determined the effect of Rb and ARA70 on the transcriptional activity of other steroid receptors by examining the cognate DNA response elements [AR, glucocorticoid receptor (GR) and progesterone receptor (PR) MMTV-CAT. Estrogen receptor (ER) was further investigated by ERE-CAT]. While Rb and ARA70 can synergistically induce AR transcriptional activity up to 64 fold, Rb and ARA70 can only marginally induce GR, PR and ER transcriptional activity in DU145 cells. These results indicate that Rb and
ARA70 has been suggested to be a more specific coactivator for AR in prostate DU145 cells. However, the assay conditions in prostate DU145 cells are particularly preferred conditions for Rb and ARA70 to function as an AR-only coactivator, and Rb and ARA70 are E. coli in other cells or conditions.
The possibility of acting as a strong coactivator for R, PR and GR cannot be ruled out. Failure to induce transactivation by Rb mutant AR888,
That is, the inability to bind to androgens means that the interaction between Rb and AR is androgen-independent, but the AR-Rb (and AR-ARA70) complex requires a ligand for transactivation activity Suggest that. Rb activity in the control of the cell cycle is essentially associated with the ability to bind to several proteins, ie the regulation of activity. To date, a number of cellular proteins that bind to Rb have been reported (Weinberg, RA, Cell 81: 323-330 (1995)). These include a number of transcription factors, putative regulators of ras, nuclear structural proteins, protein phosphatases and some protein kinases. Whether all of these proteins are actually complex in cells and are regulated by Rb is under investigation. Much attention has been paid to functional interactions between Rb and transcription factors. To date, some of these factors have been shown to form complexes with Rb in cells. Studies of this complex formation and subsequent function indicate that the regulatory activity of Rb is
Take the form of transcriptional repression as in E2F (Weintraub et al., Nature 375: 812).
-815 (1995)) or NF-IL6 (Chen et al., Proc. Natl. Acad. Sci. USA 93: 465-
469 (1996)) and the hBrm / BRG1 complex (Singh et al., (1995)). Herein, the present inventors have shown that Rb can bind to AR and induce AR transcription activity. To our knowledge, this fact is the first demonstration of the function of a negative growth control protein in a positive manner by initiating transcription via a signaling mechanism associated with binding to a nuclear receptor. In connection with the cell cycle and differentiation, these data suggest a previously unreported function of Rb that highlights the importance of this protein in controlling transcription by direct binding to transcription factors. However, this alleged Kushizu can control transcription by stimulating at least one type of signaling mechanism.

A relationship between Rb expression and response to hormonal treatment of human breast cancer has been suggested (And
erson et al., J. Pathology 180: 65-70 (1996)). Other studies have shown that Rb gene alterations occur in all degrees and stages of prostate cancer, not only in metastatic disease but also in localized disease (Brooks et al., Prostate 26:35). -39
(1995)). It remains unclear how Rb function links to androgen-dependent status in prostate tumor progression. One possible explanation is that Rb alterations are necessary events in prostate carcinogenesis for a subset of prostate tumors. This would also be true for AR expression in prostate tumors. All publications cited herein are incorporated herein by reference. The present invention is not limited to the illustrated embodiment. The present invention is intended to include all modifications and variations that fall within the scope of the appended claims.

[Sequence list]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12Q 1/02 G01N 33/50 Z G01N 33/15 33/74 33/50 C12N 15/00 ZNAA 33/74 5/00 A (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ , UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, C , CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL , TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZW

Claims (12)

[Claims] 1. An isolated polynucleotide comprising a sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 3. 2. A genetic construct comprising a promoter capable of causing expression of a protein coding region in a cell, wherein the promoter is ARA54 or ARA54.
A genetic construct, operably linked to a protein coding region encoding expression of a polypeptide from the 55 coding regions. 3. The polypeptide encoded by the protein coding region comprises a sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4.
The described genetic construct. 4. A eukaryotic host cell comprising the gene construct according to claim 2. 5. A method for testing a compound for androgen or antiandrogen activity, comprising: (a) producing a host cell in the host cell of a polypeptide selected from the group consisting of ARA54, ARA55, ARA24 and Rb. Transfecting with at least one genetic construct, wherein the host cell also produces a human androgen receptor protein, (b) exposing the cell to a compound, and (c) reacting with the androgen receptor. Measuring the level of transcriptional activity caused. 6. The method of claim 5, wherein said host cell is a prostate cell. 7. The method of claim 5, wherein said cells are eukaryotic cells lacking a native endogenous androgen receptor, and wherein said cells further comprise an introduced genetic construct that produces an androgen receptor protein. 8. The method according to claim 5, wherein the gene construct comprises a DNA sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7. 9. The method of claim 5, wherein the cells are transfected with a gene construct that includes a reporter gene that can be expressed in the cell, and the expression of the reporter gene is sensitive to detection and quantification. . 10. The method according to claim 9, wherein the reporter gene is selected from the group consisting of a chloramphenicol acetyltransferase gene and a luciferase gene. 11. A method for testing a compound for androgen or antiandrogen activity, comprising: (a) transforming a host cell with a human androgen receptor protein and ARA54, ARA55;
Transfecting a polypeptide selected from the group consisting of ARA24 and Rb with at least one gene construct capable of producing in the host cell; (b) exposing the cell to a compound; and (c) Measuring the interaction between AR and AR coactivator. 12. The method of claim 11, wherein said activator is selected from the group consisting of ARA54, ARA55, ARA24 and Rb.