EP1592391A2 - Tra16, represseur de tr4/tr2 - Google Patents

Tra16, represseur de tr4/tr2

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Publication number
EP1592391A2
EP1592391A2 EP04711255A EP04711255A EP1592391A2 EP 1592391 A2 EP1592391 A2 EP 1592391A2 EP 04711255 A EP04711255 A EP 04711255A EP 04711255 A EP04711255 A EP 04711255A EP 1592391 A2 EP1592391 A2 EP 1592391A2
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Prior art keywords
fragment
composition
variant
combination
seq
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Chawnshang Chang
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University of Rochester
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University of Rochester
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Priority claimed from US10/366,811 external-priority patent/US20030235860A1/en
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Publication of EP1592391A2 publication Critical patent/EP1592391A2/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/743—Steroid hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/721—Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor

Definitions

  • FIG. 29 Figure 22 shows interaction between TR4 and TRA16.
  • TRA16 can interact with TR4 in mammalian two-hybrid system.
  • COS-1 cells in 60-mm dishes were transiently co-transfected with 3 ⁇ g of reporter plasmid pG5-LUC and 4 ⁇ g each of GAL4DBD, VP16, VP16-TR4, and GAL4-TRA16 in various combinations as indicated. Luciferase assay was performed 24 hours after transfection. All values represent the mean +/-SD of three independent experiments.
  • Figure 23 shows that TRA16 can suppress TR4-target gene expression in a dose- dependent manner.
  • A COS-1 cells were transiently co-transfected with 3 ⁇ g of reporter plasmid HCR-1LUC, 1 ⁇ g ofTR4, and increasing amounts ofTRA16 expression plasmid for 24 hours as indicated.
  • B HI 299 cells were transiently co-transfected with 3 ⁇ g of reporter plasmid HCR- ILUC, and increasing amounts ofTRAl ⁇ expression plasmid for 24 h. All values represent the mean +/-SD of three independent experiments.
  • COS-1 (pBig-TRA16) and COS-1 (pBig) stably transfected cells were transiently co-transfected with 3 ⁇ g of HCR-ILUC reporter plasmid, 10 ng of SV40-pRL internal control plasmid, and 1 ⁇ g of TR4 for 16 h.
  • the cells were then treated with 6 ⁇ l DMSO (as negative control) or 6 ⁇ g/ml doxycycline for another 24 h, and then harvested for Luciferase assay. All values represent the mean +/-SD of three independent experiments.
  • Ranges can be expressed herein as from “about” one particular value, and/or to "about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10" is also disclosed.
  • Primers are a subset of probes which are capable of supporting some type of enzymatic manipulation and which can hybridize with a target nucleic acid such that the enzymatic manipulation can occur.
  • a primer can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art which do not interfere with the enzymatic manipulation.
  • TR4 or TR2 or AR or ER or TRA16 For example, if a particular TR4 or TR2 or AR or ER or TRA16 is disclosed and discussed and a number of modifications that can be made to a number of molecules including the TR4 or TR2 or AR or ER or TRA16 are discussed, specifically contemplated is each and every combination and permutation of TR4 or TR2 or AR or ER or TRA16 and the modifications that are possible unless specifically indicated to the contrary.
  • compositions wherein the composition reduces the transcription activity of ER, for example, wherein the composition reduces the transcription activity of ERby 10%, 25%, 50%, or 90%.
  • compositions wherein the composition reduces the Gl/S transition of the cell cycle, for example, wherein the composition reduces the Gl/S transition of the cell cycle by 10%, 25%, 50%, or 90%.
  • methods of inhibiting transcription activity of ER comprising administering any of the disclosed compositions related to ER, AR, TR2, or TR4, as well as molecules that interact with these.
  • Disclosed are methods of identifying inhibitors of ER transcription activity comprising mixing a compound with an ER, ER fragment, ER variant, or combination, and identifying compounds which compete with TR2 binding with the ER, ER fragment, or ER variant.
  • the ER comprises a polypeptide having at least 80%, 85%, 90%, or 95% identity to the sequence set forth in SEQ ID NO:32, and/or wherein the fragment comprises a polypeptide having at least 80%, 85%, 90%, or 95% identity to amino acids 312-340, set forth in SEQ ID NO:32, the fragment comprises a polypeptide having at least 80%, 85%, 90%, or 95% identity to amino acids 123-340, set forth in SEQ ID NO:32, or the fragment comprises a polypeptide having at least 80%, 85%, 90%, or 95% identity to amino acids 312-595, set forth in SEQ ID NO:32, and for example, wherein any variation between the ER and the sequence set forth in SEQ ID NO: 32 is a conserved variation.
  • Disclosed are methods of inhibiting AR transcription activity comprising administering a composition that binds AR, wherein the composition is TR4 or fragment thereof, or a molecule that competitively competes with AR for TR4 binding.
  • Also disclosed are methods of identifying an inhibitor of AR transcription activity comprising mixing a compound with AR and identifying compounds which compete with TR4 interaction with AR. 79.
  • methods of identifying inhibitors of AR transcription activity comprising mixing a set of compounds with AR, AR fragment, AR variant, or combination and identifying compounds which compete with the TR4 interaction with AR ⁇ SONS.
  • a method of identifying an inhibitor of an interaction between AR and TR4 comprising incubating a library of molecules with AR, AR fragment, AR variant, or combination forming a mixture, and identifying the molecules that disrupt the interaction between the AR, AR fragment, AR variant, or combination and TR4, wherein the interaction disrupted comprises an interaction between the AR, AR fragment, AR variant, or combination and TR4 binding site.
  • Disclosed are methods of identifying an inhibitor of an interaction between AR and TR4 comprising incubating a library of molecules with TR4, TR4 fragment, TR4 variant, or combination forming a mixture, and identifying the molecules that disrupt the interaction between AR and the TR4, TR4 fragment, TR4 variant, or combination, wherein the interaction disrupted comprises an interaction between the AR and the TR4, TR4 fragment, TR4 variant, or combination binding site.
  • step of isolating comprises incubating the mixture with molecule comprising AR, AR fragment, AR variant or combination.
  • TR4, TR4 fragment, TR4 variant or combination comprises a polypeptide having at least 80%, 85%, 90%, or 95% identity to SEQ ID NO: 16, as well as methods wherein any variation between the TR4 and the sequence set forth in SEQ ID NO: 16 is a conserved variation, or wherein the TR4 comprises the sequence set forth in SEQ ID NO: 16, or is a conserved variant thereof, or is a fragment thereof.
  • Disclosed are methods of identifying an inhibitor of ER transcription activity comprising mixing a compound with ER and identifying compounds which compete with TR4 interaction with ER.
  • methods of identifying inhibitors of ER transcription activity comprising mixing a set of compounds with ER and identifying compounds which compete with TR4 interaction with ER.
  • step of isolating comprises incubating the mixture with molecule comprising TR4, TR4 fragment, TR4 variant or combination.
  • the step of isolating comprises incubating the mixture with a molecule comprising an ER, ER fragment, ER variant or combination.
  • any variation between the ER and the sequence set forth in SEQ ID NO: 32 is a conserved variation as well as methods wherein the ER comprises the sequence set forth in SEQ ID NO:32, or is a conserved variant thereof, or is a fragment thereof.
  • TR4, TR4 fragment, TR4 variant or combination comprises a polypeptide having at least 80%, 85%, 90%, or 95% identity to SEQ ID NO: 16, as well as methods wherein any variation between the TR4 and the sequence set forth in SEQ ID NO: 16, as well as methods wherein any variation between the TR4 and the sequence set forth in SEQ ID NO: 16, as well as methods wherein any variation between the TR4 and the sequence set forth in SEQ ID NO: 16, as well as methods wherein any variation between the TR4 and the sequence set forth in SEQ ID NO:
  • a composition that binds AR comprising administering a composition that binds AR, wherein the composition is TR2 or fragment thereof, or a molecule that competitively competes with AR for TR2 binding.
  • methods of identifying an inhibitor of AR transcription activity comprising mixing a compound with AR, AR fragment, AR variant, or combination and identifying compounds which compete with TR2 interaction with the AR, AR fragment, AR variant, or combination.
  • Also disclosed are methods of identifying an inhibitor of an interaction between AR and TR2 comprising incubating a library of molecules with TR2, TR2 fragment, TR2 variant or combination forming a mixture, and identifying the molecules that disrupt the interaction between AR and the TR2, TR2 fragment, TR2 variant or combination, wherein the interaction disrupted comprises an interaction between the AR and the TR2, TR2 fragment, TR2 variant or combination binding site.
  • the AR, AR fragment, AR variant or combination comprises a polypeptide having at least 80%, 85%, 90%, or 95% identity to SEQ ID NO:31, as well as methods wherein any variation between the AR and the sequence set forth in SEQ ID NO: 31 is a conserved variation, and methods wherein the AR comprises the sequence set forth in SEQ ID NO:31, or is a conserved variant thereof, or is a fragment thereof.
  • compositions that comprise combinations of TR2 and ER or TR2 and AR or TR4 and ER or TR4 and AR, as well as any other combination of TR2, TR4, ER, and AR.
  • TR4 o ⁇ han receptor and the AR are capable of mutual co-suppression of each other by the formation of a heterodimer.
  • TR2 o ⁇ han receptor represses AR by the formation of a heterodimer.
  • TR4 o ⁇ han receptor and the estrogen receptor are capable of mutual co-suppression by the formation of a heterodimer.
  • TRA16 a protein called TRA16 which is capable of binding the ligand binding domain of TR4 and suppressing the transactivation activity of TR4.
  • TR2 o ⁇ han receptor TR2 o ⁇ han receptor
  • ER-#6 an interaction blocker, ER-#6 (aa 312-340), can mediate the TR2 interaction.
  • Receptor DNA recognition 116 Differential recognition of target genes by the steroid/thyroid hormone members is determined by at least three properties: protein-DNA interactions, protein-protein interactions, and protein environment. For the protein-DNA interaction, DNA-binding domains of family members selectively interact with HREs, which are structurally related but functionally distinct. Based on the zinc finger model, the proximal box in the DNA-binding domain of receptor proteins can determine target HRE specificity (Umesono, K. et al., (1989) Cell 57, 1139-1146).
  • the TR4 can be grouped into members of the estrogen receptor subfamily, recognizing direct repeats of the hexameric consensus motif AGGTCA (Umesono, K. et al., (1989) Cell 57, 1139-1146).
  • AGGTCA hexameric consensus motif
  • TR2 o ⁇ han receptor a member of the nuclear hormone receptor superfamily
  • TR2 TR2 o ⁇ han receptor
  • cDNA encodes a protein of 603 amino acids with a calculated molecular mass of 67 kilo-daltons [Chang C. et al, (1988) Biochem Biophys Res Commun 155:971-977; Chang C.
  • TR2 is evolutionarily conserved among species from primitive creatures to mammalians, including sea urchin, rainbow trout, axolotl, xenopus, drosophila, mouse, and human (Chang, C. et al., (1989) Biochem. Biophys. Res. Commun. 165, 735-41, Chang, C. et al., (1988) Biochem. Biophys. Res. Commun. 155, 971-7; Kontrogianni-Konstantopoulos, A. et al., (1996) Dev. Biol. Ill, 371-82; Le Jossic, C. et al., (1998) Biochem Biophys Res Commun 245, 64-9).
  • TR2 is primarily expressed in the mouse testis, particularly in the developing germ cells, indicating a role of TR2 in spermatogenesis (Lee, C. et al., (1996) Mol. Reprod. Dev. 44, 305-14; Lee, C. et al, (1995) Genomics 30, 46-52).
  • TR2 has been detected widely in the male reproductive system including testis, prostate, and seminal vesicle [Chang C. et al., (1988) Biochem Biophys Res Commun 155:971-977; Chang C. et al., (1988) Biochem Biophys Res Commun 165:735-741; Lee C. et al., (1996) Mol Reprod Dev 44:305-314].
  • TR2 is also relatively highly expressed in prostate cancer tissue and cell lines [Chang C. et al., (1988) Biochem Biophys Res Commun 155:971-977; Chang C. et al, (1988) Biochem Biophys Res Commun 165:735-741; Lee C. et al, (1996) Mol ReprodDev 44:305-314; Hu Y-C. et al., (2002) JBiol Chem 277:33571-33579].
  • TR2 has a broad range of biological functions, h terms of the regulation of TR2 expression, TR2 can be induced during neuronal differentiation in P19 embryonic carcinoma cells stimulated by ciliary neurotrophic factor (CNTF). hi return, TR2 activates its target gene, CNTFR, expression which mediates CNTF signaling and is required for the motor neuron development (Young, W. et al., (1998) J.
  • TR4 functions as a transcriptional activator when bound to the DR separated by four nucleotides (a DR-4 element) (Lee, Y.
  • TR4 also induces the transcription of the cytokine receptor, which is a ciliary neurotrophic factor receptor (Young, W. et al., (1997) J. Biol. Chem. 272(5) 3109-16).
  • TR4 can also modulate other nuclear receptors' transactivation. Previous studies have indicated that TR4 can compete for binding to the hormone response elements of retinoic acid receptor (RAR), retinoid X receptor (RXR) (Lee, Y. et al., (1998) J. Biol. Chem. 273(22) 13437-43) and vitamin D receptor (VDR) (Lee, Y. et al., (1999) J. Biol. Chem. 274(23) 16198-205) to suppress RAR/RXR-or VDR- mediated transcription.
  • RAR retinoic acid receptor
  • RXR retinoid X receptor
  • VDR vitamin D receptor
  • TR4 has been demonstrated to suppress many other receptors' transactivation, such as VDR, RAR, RXR, and PPAR (Lee, Y. et al., (1998) J. Biol. Chem. 273(22) 13437-43; Lee, Y. et al., (1999) J. Biol. Chem. 274(23) 16198-205; Yan, Z. et al., (1998) J. Biol. Chem. 273(18) 10948-57.
  • TR4 The suppression mechanism for these receptors' transactivation has been demonstrated through the competition of TR4 with those receptors' ability to bind their hormone response elements.
  • TR4 cDNA shares structural homology with members of the steroid hormone receptor superfamily (Chang, C. et al., (1994) Proc. Natl. Acad. Sci. USA. 91, 6040- 6044).
  • the TR4 also named as TAKl (Hirose, T. et al, (1994) Mol. Endocrinol. 8, 1667-1680), is most closely related to the previously identified TR2 o ⁇ han receptor (Chang, C. et al., (1988) Biochem. Biophys. Res. Commun.
  • TR4 was designated as the TR2 ⁇ , while the TR2 o ⁇ han receptor was referred to as TR2 ⁇ (Mangelsdorf, D J. et al., (1995) Cell 83, 841-850).
  • TR4 cDNA has been cloned from mouse testis by reverse transcription-PCR (Young, W. et al, (1997) J. Biol. Chem. 272 3109-3116). Subsequently, the human TR4 gene has been mapped to chromosome 3p24.3 (Lin, D. et al., (1998) Endocrine 8 123-134).
  • the P-box sequence of the DNA binding domain (DBD), TR4 is classified as a member of the estrogen receptor and thyroid hormone receptor subfamily, which can recognize the hormone response elements (HREs) composed of the AGGTCA motif.
  • HREs hormone response elements
  • Examples of HREs with this motif include those of the retinoic acid receptor (RARE), retinoid X receptor (RXRE) (Lee, Y.-F., et al., (1998) J. Biol. Chem.
  • TR4 is highly expressed in adult mouse brain especially in the regions in which cells undergo active proliferation and in the granule cells of the hippocampus and cerebellum (Chang, C. et al, (1994) Proc. Natl. Acad. Sci. USA. 91(13), 6040-4). It has been demonstrated that TR4 inhibits the retinoic acid (RA) pathway that is highly involved in the development of the nervous system (Young, W. et al., (1998) J. Biol. Chem. 273, 20877-20885).
  • RA retinoic acid
  • TR4 can be involved in the regulation of differentiation of neuron cells.
  • In situ hybridization analysis has demonstrated that TR4 is expressed in a complex spatiotemporal pattern, including, the central neural system (assia, hippocampal pyramidal cell, and granule cells of both hippocampus and cerebellum) and peripheral organs (most abundantly expressed in spermatocytes of testis, with lower amounts in adrenal cortex, spleen, thyroid, prostate, and pituitary gland) (Chang, C. et al., (1994) Proc. Natl. A cad. Sci. USA 91 :6040-6044; Hirose, T. et al., (1994) Mol. Endocrinol.
  • TR4 transcripts occurs widely in many mouse tissues, including the central nervous system and peripheral organs such as the adrenal gland, spleen, thyroid gland, and prostate (Yoshikawa, T. et al., (1996) Endocrinol. 137, 1562-1571; Young, W. et al., (1997) J. Biol. Chem. 272, 3109-3116). These data are consistent with TR4 playing a role in neurogenesis and neuronal maturation.
  • TR4 also can function as enhancer to induce the ciliary neurotrophic factor receptor-a and thyroid hormone receptor (TR) (Young, WJ. et al., (1997) J: Biol Chem. 272:3109-3116; Lee, Y.F. et al., (1997) J. Biol. Chem. 272:12215-12220).
  • TR4 ciliary neurotrophic factor receptor-a and thyroid hormone receptor
  • a recent report has shown that the TR4 is an important regulator of myeloid progenitor cell proliferation and development.
  • the hormone response element for the TR4 is AGGTCA with a variety of direct repeats (DRs) (Lee, Y.F. et al, (1999) Proc. Natl. Aca ⁇ . Sci. USA 96:14724-14729; Young, WJ. et al, (1998) J. Biol. Chem. 273:20877-20885).
  • DRs direct repeats
  • TR4 associated protein TRA 16
  • TR4RE TR4 response element
  • TRA16 was isolated using a two-hybrid system and is shown to bind to TR4.
  • TRA16 comprises a peptide of 139 amino acids having the sequence set forth in SEQ ID NO:34. It is understood that as disclosed herein variants of TRA16, for example, functional variants, are also disclosed. For example, conserved variants of TRA16 where conservative substitutions or deletions have been made or where fragments of TRA16 have been made.
  • TRA16 interacts with TR4 to repress TR4's transcription activity, it interacts with TR2, to repress TR2's transcription activity, and it interacts with ER to enhance ER's transcription activity.
  • Androgen is the most conspicuous amount of steroid hormone in ovary (Risch HA. (1998) JNatl Cancer Inst. 90(23): 1774-86).
  • concentrations of testosterone and esfradiol in the late-follicular phase when estrogens are at their peak are 0.06-O.lOmg/ day and 0.04-0.08mg.day respectively (Risch HA. (1998) JNatl Cancer Inst. 90(23): 1774-86).
  • the ratio of androgens versus estrogens in the ovarian veins of postmenopausal women is 15 to 1 (Risch HA. (1998) JNatl Cancer Inst. 90(23):1774-86; Doldi N, 1998).
  • Estrogen receptors include ER ⁇ and ER ⁇ , belong to nuclear hormone receptor superfamily and mediate estrogen actions in regulation of cell growth and differentiation, particularly in mammary glands and uterus in females (see reviews in (Nilsson, S. et al, (2001) PhysiolRev 81, 1535-65; Couse, J. et al., (1999) Endocr Rev 20, 358-417)).
  • the proliferation of mammary glands is mainly dependent on estrogen stimulation; however, the proliferating epithelial cells detected in terminal end buds (TEBs) at the tip of elongating ducts in mammary glands are usually ER-negative (Zeps, N.
  • TR4 can suppress ER function via protein-protein interaction that results in the interruption of ER-ER homodimerization and in preventing ER binding to its estrogen response element (ERE).
  • ER ⁇ KO mice The analysis of ER ⁇ KO mice indicated that ER ⁇ can play important in vivo functions, such as the growth of the adult female reproductive tract and mammary gland, the regulation of gonadotropin gene transcription, mammary neoplasia induction, and sexual behaviors. Su ⁇ risingly, ER ⁇ also play important roles in spermatogenesis and sperm function (see review (Couse, J. et al., (1999) Endocr. Rev. 20(3), 358-417)).
  • Protein domains 140 A variety of proteins are discussed herein, including TR2, TR4, AR, and ER. It is understood that these proteins have a variety of functional domains which are herein disclosed (Table 3) Table 3
  • Table 4 shows the various interactions which have been tested. Furthermore, the domains are interchangeable. For example, it is understood what the F domain interacts with, and it can be predicted, for example that if it binds TR2 in a particular spot, it would be expected that it could bind TR4 in a homologous domain. Table 4
  • Table 5 shows the effect of the various interactions on the transcription activity of a particular transactivator. These relationships shown have been tested and it is understood that these relationships and effects indicate other effects. For example, as TR4 down regulates AR and AR down regulates TR4 it can be indicated since TR2 down regulates AR that AR also down regulates TR2. Table 5.
  • TR2 can interact with a number of proteins. These interactions can alter TR2 transcriptional activation activity as well as altering the transcriptional activation activity of the proteins TR2 interacts with. Disclosed herein TR2 interacts with ER. a) Interaction between ER and TR2
  • TR2/ER dimerization lies first in the fact that it prevented ER from binding to its target DNA and thus repressed ER transactivation.
  • TR2 LBD was sufficient to prevent ER from binding to its target DNA.
  • ER was induced to transactivate several downstream genes. Overexpression of TR2 in the same cells could block the ER-induced expression of these downstream genes in a dose-dependent manner. Therefore, the present invention is also directed at new treatment strategies for ER-related diseases such as breast cancer by modulating TR2 levels to enhance or repress ER' s transactivation activity. If a disease or clinical condition is associated with an increase of ER- mediated transactivation, the disease or clinical condition can be cured or controlled by increasing the TR2 level.
  • a disease or clinical condition is associated with a decrease of ER- mediated transactivation
  • the disease or clinical condition can be cured or controlled by decreasing the TR2 level.
  • the full length TR2 could repress AR' s transactivation activity.
  • Any method or agent that can increase or decrease TR2 levels in human bodies are candidates for treating ER-related diseases. Since the LBD of TR2 could prevent ER from binding to its target DNA as well, any truncated form ofTR2 that retains the LBD or any chimeric protein that contains the TR2 LBD can also be used when treatment requires repressing ER' s transactivation activity.
  • TR2 transactivation induced in any manner can be repressed by TR2.
  • the compositions and methods make it possible to screen for drugs for ER- related diseases by testing a compound's effect on TR2 level. If a compound can increase or decrease the level of TR2 in a cell, then it can be selected for further testing for treatment of ER- related diseases.
  • the screening method can measure TR2 level directly. It can also measure 1X2 level indirectly, for example, through any reporter system that measures the increase or decrease of TR2 transactivation. Examples of such reporter systems are described below.
  • TR2/ER heterodimerization lies second in the fact that it repressed TR2 transactivation activity in an ER dose dependent manner. Therefore, human clinical conditions such as hair loss that can relate to TR2 transactivation activity can be controlled through modulating ER levels in human. If a disease or clinical condition is associated with an increase of TR2-mediated transactivation, the disease or clinical condition can be cured or controlled by increasing the ER level. If a disease or clinical condition is associated with a decrease of TR2-mediated transactivation, the disease or clinical condition can be cured or controlled by decreasing the ER level. Any method or agent that can change ER levels in human bodies are treatment candidates.
  • compositions and methods also makes it possible to screen for drugs for TR2-related diseases by testing a compound's effect on ER level. If a compound can increase or decrease the level of ER in a cell, then it can be selected for further testing for treatment of TR2-related diseases.
  • the screening method can measure ER level directly. It can also measure ER level indirectly, for example, through any reporter system that measures the increase or decrease of ER transactivation.
  • TR2 can form heterodimers with either TR4 or AR as well. Co- repression exists in these two dimers as well. b) TR2-ER interaction
  • TR2 interacts with ER. This interaction causes a decrease in TR2 activated transcription and a decrease in ER activated transcription.
  • a disease or clinical condition is associated with an increase of AR-mediated transactivation, the disease or clinical condition can be cured or controlled by increasing the TR2 level. If a disease or clinical condition is associated with a decrease of AR-mediated transactivation, the disease or clinical condition can be cured or controlled by decreasing the TR2 level. The full length TR2 could repress AR' s transactivation activity. Any method or agent that can increase or decrease TR2 levels in human bodies are candidates for treating ER-related diseases.
  • AR transactivation induced in any manner, including that by constitutively active AR, can be repressed by TR2.
  • a disease or clinical condition is associated with an increase of TR2 -mediated transactivation, the disease or clinical condition can be cured or controlled by increasing the AR level. If a disease or clinical condition is associated with a decrease of TR2 -mediated transactivation, the disease or clinical condition can be cured or controlled by decreasing the AR level. Any method or agent that can change AR levels in human bodies are treatment candidates. 161.
  • the disclosed compositions and methods also make it possible to screen for drugs for TR2-related diseases by testing a compound's effect on AR level. If a compound can increase or decrease the level of AR in a cell, then it can be selected for further testing for treatment of TR2-related diseases.
  • the screening method can measure AR level directly.
  • TR4 and AR The interaction between TR4 and AR can repress TR4 mediated transcription activation at TR4 induced promoters as well as AR mediated transcription activation at AR induced promoters.
  • Transfected TR4 was mainly located in the nucleus while transfected AR was mainly located in the cytoplasm, in the absence of its cognate ligand DHT. However, when TR4 and AR were co-transfected, the majority of the AR was transported to the nucleus by TR4 even in the absence of DHT.
  • the ligand binding domain of TR4 was sufficient for binding to a near full- length AR (amino acids 33-918).
  • the binding was DHT-dependent.
  • the difference between DHT-dependent interaction detected in the mammalian one-or two-hybrid systems and DHT- independent interaction detected in the GST pull-down and the immunocytofluorescence assays could be due to the involvement of AF-1 ligand-independent interaction in the GST pull-down and the immunocytofluorescence assays, which used the full length of TR4 containing AF-1, vs.
  • TR4/AR heterodimerization lies first in the fact that such dimerization prevents TR4 from binding to its target DNA and thus repressed TR4-mediated transactivation. This repression was observed in mammalian cells in a reporter assay, as described below. The repression is AR dose-dependent. Therefore, human diseases or clinical conditions such as hepatitis, hepatoma and hair loss that can relate to TR4 transactivation activity can be controlled or cured through modulating AR levels in the subject, such as a human.
  • a disease or clinical condition is associated with an increase of TR4 transactivation, the disease or clinical condition can be affected by modulating the AR level. If a disease or clinical condition is associated with a decrease of TR4 transactivation, the disease or clinical condition can be controlled by decreasing the AR level. Any method or agent that can change the AR level in human bodies is a candidate to treat TR4-related diseases.
  • the present invention makes it possible to screen for drugs for TR4-related diseases by testing a compound's effect on AR level. If a compound can increase or decrease the level of AR in a cell, it can be selected for further testing for treatment of TR4-related diseases.
  • the screening method can measure the AR level directly. It can also measure the AR level indirectly, for example, through any reporter system that measures A transactivation. Examples of such reporter systems are described below.
  • TR4/AR heterodimerization lies secondly in the fact that it also represses AR-mediated transactivation.
  • AR was induced to transactivate several downstream genes. Overexpression of TR4 in the same cells could block the AR-induced expression of these downstream genes. Therefore, the present invention is also directed at new treatment strategies for AR-related diseases such as prostate cancer by modulating TR4 levels to enhance or repress AR' s transactivation activity. If a disease or clinical condition is associated with an increase of AR-mediated transactivation, the disease or clinical condition can be cured or controlled by increasing the TR4 level.
  • a disease or clinical condition is associated with a decrease of AR-mediated transactivation
  • the disease or clinical condition can be cured or controlled by decreasing the TR4 level.
  • the full length TR4 can repress AR' s transactivation activity. Any method or agent that can change TR4 levels in human bodies are candidates for treating AR-related diseases. Since the LBD of TR4 could bind to AR as well, any truncated form of TR4 that retains the LBD or any chimeric protein that contains the TR4 LBD can also be used when treatment requires a repression of AR transactivation.
  • the present invention makes it possible to screen for drugs for AR-related diseases by testing a compound's effect on TR4 level. If a compound can increase or decrease the level of TR4 in a cell, then it can be selected for further testing for treatment of AR-related diseases.
  • the screening method can measure TR4 level directly. It can also measure TR4 level indirectly, for example, through any reporter system that measures TR4 transactivation. b) TR4-ER interactions
  • TR4 interacts with ER to repress ER transactivation.
  • the LBD of ER interacts well with TR4 in the presence or absence of E2.
  • the TR4-ER interaction occurs in the LBD of TR4.
  • a further deletion of the C-terminal of LBD (pCMV ⁇ C-TR4) could not repress ER activity and this indicates that the LBD of TR4 is required for TR mediated ER effects.
  • a compound that is identified or designed as a result of any of the disclosed methods can be obtained (or synthesized) and tested for its biological activity, e.g., inhibition of TR2, TR4, AR, ER, or TRA16 transcription activity.
  • Disclosed are methods of treating a subject comprising administering to the subj ect an inhibitor of transcription activity of AR and TR4, ER and TR4, TRAl 6, wherein the inhibitor reduces the heterodimerzation between AR and TR4, ER and TR4, AR and TR2, TR4 and TRAl 6, and/or ER and TR2, for example, and wherein the subject is in need of such treatment.
  • TR4-TRA16 interactions 178.
  • compositions comprising TRA16, wherein the composition interacts with TR4, such that TR4 transcription activity is decreased relative to transcription activity in the absence of the composition.
  • compositions comprising a fragment of TRAl 6, wherein the composition interacts with TR4, such that TR4 transcription activity is decreased relative to transcription activity in the absence of the composition, wherein the fragment of TRAl 6 has at least 80%, 85%, 90%, or 95% identity to SEQ ID NO:34.
  • compositions wherein any variation between the TRAl 6 and the sequence set forth in SEQ ID NO: 34 is a considered a conserved variation.
  • composlions wherein the composition reduces the transcription activity of TR4, wherein the composition reduces the transcription activity of TR4 by 10%, 25%, 50%, or 90%.
  • Disclosed are methods of inliibiting transcription activity of TR4 comprising administering the TRAl 6 compositions, or any of the other compositions.
  • composition reduces the transcription activity of TR4 by 10%, 25%, 50%, or 90%.
  • TRAl 6 and TR4 comprising incubating a library of molecules with an TRAl 6, TRAl 6 fragment, TRAl 6 variant or combination, forming a mixture, and identifying the molecules that disrupt the interaction between the TRAl 6, TRAl 6 fragment, TRAl 6 variant, or combination and TR4, wherein the interaction disrupted comprises an interaction between the TRAl 6, TRAl 6 fragment, TRAl 6 variant, or combination and TR4 binding site.
  • TRAl 6 and TR4 comprising incubating a library of molecules with TR4, TR4 fragment, TR4 variant or combination forming a mixture, and identifying the molecules that disrupt the interaction between TRAl 6 and the TR4, TR4 fragment, TR4 variant or combination, wherein the interaction disrupted comprises an interaction between the TRAl 6 and the TR4, TR4 fragment, TR4 variant or combination binding site.
  • Disclosed are methods of identifying inhibitors of TR4 transcription activity comprising mixing a set of compounds with TRAl 6, TRAl 6 fragment, TRAl 6 variant, or combination and identifying compounds which compete with ER binding with TRAl 6, TRAl 6 fragment, TRAl 6 variant, or combination.
  • TR4 has a sequence of at least 80%, 85%, 90%, or 95% identity to SEQ ID NO: 16, methods wherein any variation between the TR4 and the sequence set forth in SEQ ID NO: 16 is considered a conserved variation. 192. Disclosed are methods, wherein the TR4 comprises SEQ ID NO:16.
  • TR4 comprises a polypeptide having at least 80%, 85%, 90%, or 95% identity to the sequence set forth in SEQ ID NO:16.
  • isolated compositions comprising TRAl 6 and TR4 or variants or fragments of either, TRAl 6 and AR or variants or fragments of either, or TRAl 6 and ER or variants or fragments of either.
  • TRA16 also interacts with TR2 and ER. TRA 16 inhibits TR2 transcription activity and it enhances ER's transcription activity.
  • TR4 and (AR or ER, or TRAl 6) or TR2 and (AR or ER or TRAl 6) can be performed using high through put means.
  • putative inhibitors can be identified using Fluorescence Resonance Energy Transfer (FRET) to quickly identify interactions.
  • FRET Fluorescence Resonance Energy Transfer
  • the underlying theory of the techniques is that when two molecules are close in space, ie, interacting at a level beyond background, a signal is produced or a signal can be quenched. Then, a variety of experiments can be performed, including, for example, adding in a putative inhibitor.
  • the inhibitor competes with the interaction between the two signaling molecules, the signals will be removed from each other in space, and this will cause a decrease or an increase in the signal, depending on the type of signal used. This decrease or increasing signal can be correlated to the presence or absence of the putative inhibitor. Any signaling means can be used.
  • disclosed are methods of identifying an inhibitor of the interaction between any two of the disclosed molecules comprising, contacting a first molecule and a second molecule together in the presence of a putative inhibitor, wherein the first molecule or second molecule comprises a fluorescence donor, wherein the first or second molecule, typically the molecule not comprising the donor, comprises a fluorescence acceptor; and measuring Fluorescence Resonance Energy Transfer (FRET), in the presence of the putative inhibitor and the in absence of the putative inhibitor, wherein a decrease in FRET in the presence of the putative inhibitor as compared to FRET measurement in its absence indicates the putative inhibitor inhibits binding between the two molecules.
  • FRET Fluorescence Resonance Energy Transfer
  • compositions prevents AR transcription activity
  • composition is defined as a composition capable of being identified by administering the composition to a system, wherein the system supports AR transcription activity, assaying the effect of the composition on the amount of transcription activity in the system, and selecting a composition which causes a decrease in the amount of AR transcription activity present in the system relative to the system without the addition of the composition.
  • Also disclosed are methods of inhibiting AR transcription activity comprising administering a composition that binds AR, wherein the composition is TR4 or fragment thereof, or a molecule that competitively competes with TR4 for AR binding.
  • Disclosed are methods of manufacturing an inhibitor to AR transcription activity comprising, a) administering a composition to a system, wherein the system supports AR transcription activity, b) assaying the effect of the composition on the amount of AR transcription activity in the system, c) selecting a composition which cause a decrease in the amount of AR transcription activity present in the system relative to the system with the addition of the composition, and d) synthesizing the composition.
  • Also disclosed are methods comprising the step of admixing the composition with a pharmaceutical carrier.
  • Also disclosed are methods of identifying inhibitors of TR4 transcription activity comprising, a) administering a composition to a system, wherein the system supports TR4 transcription activity, b) assaying the effect of the composition on the amount of TR4 transcription activity in the system, and c) selecting a composition which causes a decrease in the amount of TR4 transcription activity present in the system relative to the system without the addition of the composition. 207.
  • Disclosed are methods of inhibiting TR4 transcription activity comprising administering a composition, wherein the composition prevents TR4 transcription activity, wherein the composition is defined as a composition capable of being identified by administering the composition to a system, wherein the system supports TR4 transcription activity, assaying the effect of the composition on the amount of transcription activity in the system, and selecting a composition which causes a decrease in the amount of TR4 transcription activity present in the system relative to the system without the addition of the composition.
  • Also disclosed are methods of inhibiting TR4 transcription activity comprising administering a composition that binds TR4, wherein the composition is AR, AR fragment, AR variant, or a molecule that competitively competes with TR4 for AR binding, or a combination thereof.
  • Also disclosed are methods of inhibiting TR4 transcription activity comprising administering a composition that binds TR4, wherein the composition is ER, ER fragment, ER variant, or a molecule that competitively competes with TR4 for ER binding, or a combination thereof.
  • Also disclosed are methods of inhibiting TR4 transcription activity comprising administering a composition that binds TR4, wherein the composition is TRAl 6, TRAl 6 fragment, TRAl 6 variant, or a molecule that competitively competes with TR4 for TRAl 6 binding, or a combination thereof.
  • Disclosed are methods of manufacturing an inhibitor to TR4 transcription activity comprising, a) administering a composition to a system, wherein the system supports TR4 transcription activity, b) assaying the effect of the composition on the amount of TR4 transcription activity in the system, c) selecting a composition which cause a decrease in the amount of TR4 transcription activity present in the system relative to the system with the addition of the composition, and d) synthesizing the composition.
  • Disclosed are methods of inhibiting TR4 transcription activity comprising administering a composition, wherein the composition prevents TR4 transcription activity, wherein the composition is defined as a composition capable of being identified by administering the composition to a system, wherein the system supports TR4 transcription activity, assaying the effect of the composition on the amount of transcription activity in the system, and selecting a composition which causes a decrease in the amount of TR4 transcription activity present in the system relative to the system without the addition of the composition.
  • Also disclosed are methods of inhibiting TR4 transcription activity comprising administering a composition that binds TR4, wherein the composition is ER, AR, TRAl 6, or fragment or variant thereof, or a molecule that competitively competes with ER, AR, or TRAl 6 for TR4 binding.
  • Disclosed are methods of making a composition capable of inhibiting TR4 transcription activity comprising admixing a compound with a pharmaceutically acceptable carrier, wherein the compound is identified by administering the compound to a system, wherein the system supports TR4 transcription activity, assaying the effect of the compound on the amount of TR4 transcription activity in the system, and selecting a compound which causes a decrease in the amount of TR4 transcription activity in the system relative to the system without the addition of the compound.
  • an inhibitor to TR4 transcription activity comprising, a) administering a composition to a system, wherein the system supports TR4 transcription activity, b) assaying the effect of the composition on the amount of TR4 transcription activity in the system, c) selecting a composition which cause a decrease in the amount of TR4 transcription activity present in the system relative to the system with the addition of the composition, and d) synthesizing the composition.
  • methods comprising the step of admixing the composition with a pharmaceutical carrier.
  • Disclosed are methods of inhibiting ER transcription activity comprising administering a composition, wherein the composition prevents ER transcription activity, wherein the composition is defined as a composition capable of being identified by administering the composition to a system, wherein the system supports ER transcription activity, assaying the effect of the composition on the amount of transcription activity in the system, and selecting a composition which causes a decrease in the amount of ER transcription activity present in the system relative to the system without the addition of the composition. 221. Also disclosed are methods of inhibiting ER transcription activity comprising administering a composition that binds ER, wherein the composition is TR4 or fragment thereof, or a molecule that competitively competes with TR4 for ER binding.
  • Disclosed are methods of manufacturing an inhibitor to ER transcription activity comprising, a) administering a composition to a system, wherein the system supports ER transcription activity, b) assaying the effect of the composition on the amount of ER franscription activity in the system, c) selecting a composition which cause a decrease in the amount of ER transcription activity present in the system relative to the system with the addition of the composition, and d) synthesizing the composition.
  • Also disclosed are methods comprising the step of admixing the composition with a pharmaceutical carrier.
  • Disclosed are methods of inhibiting TR2 transcription activity comprising administering a composition, wherein the composition prevents TR2 transcription activity, wherein the composition is defined as a composition capable of being identified by administering the composition to a system, wherein the system supports TR2 transcription activity, assaying the effect of the composition on the amount of transcription activity in the system, and selecting a composition which causes a decrease in the amount of TR2 transcription activity present in the system relative to the system without the addition of the composition.
  • Also disclosed are methods of inhibiting ER transcription activity comprising administering a composition that binds ER, wherein the composition is TR2 or fragment thereof, or a molecule that competitively competes with TR2 for ER binding.
  • Disclosed are methods of making a composition capable of inhibiting TR2 transcription activity comprising admixing a compound with a pharmaceutically acceptable earner, wherein the compound is identified by administering the compound to a system, wherein the system supports TR2 transcription activity, assaying the effect of the compound on the amount of TR2 transcription activity in the system, and selecting a compound which causes a decrease in the amount of TR2 transcription activity in the system relative to the system without the addition of the compound.
  • Disclosed are methods of manufacturing an inhibitor to TR2 transcription activity comprising, a) administering a composition to a system, wherein the system supports TR2 transcription activity, b) assaying the effect of the composition on the amount of TR2 transcription activity in the system, c) selecting a composition which cause a decrease in the amount of TR2 transcription activity present in the system relative to the system with the addition of the composition, and d) synthesizing the composition.
  • Also disclosed are methods comprising the step of admixing the composition with a pharmaceutical carrier.
  • Disclosed are methods of inhibiting TR2 transcription activity comprising administering a composition, wherein the composition prevents TR2 transcription activity, wherein the composition is defined as a composition capable of being identified by administering the composition to a system, wherein the system supports TR2 transcription activity, assaying the effect of the composition on the amount of transcription activity in the system, and selecting a composition which causes a decrease in the amount of TR2 transcription activity present in the system relative to the system without the addition of the composition.
  • Also disclosed are methods of inhibiting TR2 transcription activity comprising administering a composition that binds TR2, wherein the composition is AR or fragment thereof, or a molecule that competitively competes with TR2 for AR binding.
  • Disclosed are methods of making a composition capable of inhibiting TR2 transcription activity comprising admixing a compound with a pharmaceutically acceptable earner, wherein the compound is identified by administering the compound to a system, wherein the system supports TR2 transcription activity, assaying the effect of the compound on the amount of TR2 transcription activity in the system, and selecting a compound which causes a decrease in the amount of TR2 transcription activity in the system relative to the system without the addition of the compound.
  • Disclosed are methods for screening a compound for use in treatment of androgen related diseases comprising the steps of testing the compound to determine the effect of the compound on nuclear receptor mediated transcriptional activity, the activity being mediated by a nuclear receptor selected from the group consisting of the TR2, the TR4, and the RXR, and observing the effect of such compound on the level of AR initiated transcription in the test.
  • Also disclosed are methods for modulating androgen receptor-mediated transactivation activity in a cell comprising the step of: treating the cell with a compound that can modulate TR2 level or TR4 level in the cell.
  • compositions that interact with ER, wherein the interaction occurs within the region defined by amino acids 312 to 340 of ER and methods of inhibiting transcription activity of ER comprising administering a composition, wherein the composition interacts within amino acids 312 to 3 0 of ER.
  • Steroid/thyroid honnones function through the action of specific receptor proteins (Mangelsdorf, D.J. et al., (1995) Cell 83:835-839; Tingley, D.W. (1996) J. NIHRes. 8:81-88).
  • Steroid/thyroid hormone receptors comprise a huge family of transcriptional factors that regulate complex gene networks in a wide variety of biological processes, such as growth, development, and differentiation (Evans, R.M. (1988) Science 240, 889-895), and include more than 150 proteins so far identified.
  • steroid hormone receptors Members of this superfamily include receptors for steroid hormones, thyroid hormones, vitamin A and D derivatives, as well as a large group of o ⁇ han receptors whose cognate ligands remain to be identified.
  • the steroid hormone receptors can undergo an activation or transformation step (O'Malley, B.W. et al., (1992) Biol. Reprod. 46, 163-1675; Truss, M. et al, (1993) Endocrine Rev. 14, 459-479). Regardless of whether transcriptional activity is controlled by the binding of a ligand or not, each of these proteins must be capable of binding to a specific DNA sequence that identifies particular genes as targets for regulation.
  • DBD DNA binding domains
  • Homodimeric receptors in this family include the receptors for androgen (AR), glucocorticoid, estrogen (ER), and mineralocorticoid and a large diverse subfamily of non-steroid receptors including receptors for thyroid hormone, retinoids and vitamin D, as well as many o ⁇ han receptors, for which the majority will heterodimerize with retinoid X receptor (RXR).
  • RXR heterodimers function as dynamic transcription factors in which one subunit influences the capacity of the other subunit to interact with the ligand and with other co-factors.
  • the hormone-receptor complex serving as a tr ⁇ ws-regulator, can specifically bind to a cw-acting DNA sequence, known as a hormone response element (HRE), and thereafter regulate the transcription of target genes (Mangelsdorf, D.J. et al., (1995) Cell 83, 841-850).
  • HRE hormone response element
  • the o ⁇ han receptors belong to the nuclear receptor superfamily which mediates extracellular hormonal signals to transcriptional response. The roles of o ⁇ han receptors have been linked to development, homeostasis, and diseases. (Mangelsdorf, D. et al., (1995) Cell 83(6) 841-50-3; Enmark, E., et al., (1996) Mol. Endocrinol.
  • EGSs External guide sequences
  • RNAse P RNAse P
  • EGSs can be designed to specifically target a RNA molecule of choice.
  • RNAse P aids m processing transfer RNA (tRNA) within a cell.
  • Bacterial RNAse P can be recruited to cleave virtually any RNA sequence by using an EGS that causes the target RNA:EGS complex to mimic the natural tRNA substrate. (WO 92/03566 by Yale, and Forster and Altaian, Science 238:407-409 (1990)).
  • compositions can be used as targets for any combinatorial technique to identify molecules or macromolecular molecules that interact with the disclosed compositions in a desired way.
  • the nucleic acids, peptides, and related molecules disclosed herein, such as TR2, TR4, AR, or ER, or fragments thereof can be used as targets for the combinatorial approaches.
  • Molecules isolated which bind TR2, TR4, AR, or ER, or fragments thereof, are typically competitive inhibitors so that the heterodimerzation properties, such as inhibition of TR2, TR4, AR, or ER, transactivation activity, possessed between TR2 and ER as well as TR4 and ER and TR4 and AR are present.
  • the inhibitors are non-competitive inhibitors, which, for example, cause allosteric reanangements which prevent TR2, TR4, AR, or ER, activity such as the heterodimers disclosed herein.
  • combinatorial methods and libraries included traditional screening methods and libraries as well as methods and libraries used in interactive processes.
  • Computer modeling technology allows visualization of the three-dimensional atomic structure of a selected molecule and the rational design of new compounds that will interact with the molecule.
  • the three-dimensional construct typically depends on data from x-ray crystallo graphic analyses or NMR imaging of the selected molecule.
  • the molecular dynamics require force field data.
  • the computer graphics systems enable prediction of how a new compound will link to the target molecule and allow experimental manipulation of the structures of the compound and target molecule to perfect binding specificity. Prediction of what the molecule-compound interaction will be when small changes are made in one or both requires molecular mechanics software and computationally intensive computers, usually coupled with user-friendly, menu-driven interfaces between the molecular design program and the user.
  • CHARMm performs the energy minimization and molecular dynamics functions.
  • QUANTA performs the construction, graphic modeling and analysis of molecular structure. QUANTA allows interactive construction, modification, visualization, and analysis of the behavior of molecules with each other.
  • Also disclosed are methods of identifying inhibitors of ER transcription activity comprising, a) administering a composition to a system, wherein the system supports ER transcription activity, b) assaying the effect of the composition on the amount of ER transcription activity in the system, and c) selecting a composition which causes a decrease in the amount of ER transcription activity present in the system relative to the system without the addition of the composition. f) Methods of identifying inhibitors of T2-ER interactions 312. Disclosed are methods of identifying an inhibitor of an interaction between ER and TR2, comprising incubating a library of molecules with ER forming a mixture, and identifying the molecules that disrupt the interaction between ER and TR2, wherein the interaction disrupted comprises an interaction between the ER and TR2 binding site.
  • step of isolating comprises incubating the mixture with molecule comprising TR2.
  • Disclosed are methods of identifying an inhibitor of an interaction between ER and TR2 comprising incubating a library of molecules with TR2 forming a mixture, and identifying the molecules that disrupt the interaction between ER and TR2, wherein the interaction disrupted comprises an interaction between the ER and TR2 binding site. 315. Also disclosed are the methods, wherein the step of isolating comprises incubating the mixture with molecule comprising ER.
  • methods of identifying inhibitors of ER and TR2 interaction comprising, a) administering a composition to a system, wherein the system supports ER and TR2 interaction, b) assaying the effect of the composition on the amount of ER-TR2 in the system, and c) selecting a composition which causes a decrease in the amount of ER-TR2 present in the system relative to the system without the addition of the composition.
  • variants of genes and proteins herein disclosed typically have at least, about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% percent homology to the stated sequence or the native sequence.
  • the homology can be calculated after aligning the two sequences so that the homology is at its highest level.
  • Another way of calculating homology can be performed by published algorithms. Optimal alignment of sequences for comparison can be conducted by the local homology algonthm of Smith and Watennan Adv. Appl Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. Mol Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection.
  • a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using both the Zuker calculation method and the Pearson and Lipman calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by the Smith and Watennan calculation method, the Needleman and Wunsch calculation method, the Jaeger calculation methods, or any of the other calculation methods.
  • a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using each of calculation methods (although, in practice, the different calculation methods will often result in different calculated homology percentages).
  • hybridization typically means a sequence driven interaction between at least two nucleic acid molecules, such as a primer or a probe and a gene.
  • Sequence driven interaction means an interaction that occurs between two nucleotides or nucleotide analogs or nucleotide derivatives in a nucleotide specific manner. For example, G interacting with C or A interacting with T are sequence driven interactions. Typically sequence driven interactions occur on the Watson-Crick face or Hoogsteen face of the nucleotide.
  • the hybridization of two nucleic acids is affected by a number of conditions and parameters known to those of skill in the art. For example, the salt concentrations, pH, and temperature of the reaction all affect whether two nucleic acid molecules will hybridize.
  • the temperature and salt conditions are readily determined empirically in preliminary experiments in which samples of reference DNA immobilized on filters are hybridized to a labeled nucleic acid of interest and then washed under conditions of different stringencies. Hybridization temperatures are typically higher for DNA-RNA and RNA- RNA hybridizations. The conditions can be used as described above to achieve stringency, or as is known in the art. (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989; Kunkel et al., Methods Enzymol. 1987:154 :367, 1987 which is herein inco ⁇ orated by reference for material at least related to hybridization of nucleic acids).
  • a preferable stringent hybridization condition for a DNA:DNA hybridization can be at about 68°C (in aqueous solution) in 6X SSC or 6X SSPE followed by washing at 68°C.
  • Stringency of hybridization and wasliing if desired, can be reduced accordingly as the degree of complementarity desired is decreased, and further, depending upon the G-C or A-T richness of any area wherein variability is searched for.
  • stringency of hybridization and washing if desired, can be increased accordingly as homology desired is increased, and further, depending upon the G-C or A-T richness of any area wherein high homology is desired, all as known in the art. 328.
  • selective hybridization is by looking at the amount (percentage) of one of the nucleic acids bound to the other nucleic acid.
  • selective hybridization conditions would be when at least about, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% percent of the limiting nucleic acid is bound to the non-limiting nucleic acid.
  • the non-limiting primer is in for example, 10 or 100 or 1000 fold excess.
  • This type of assay can be performed at under conditions where both the limiting and non-limiting primer are for example, 10 fold or 100 fold or 1000 fold below their k , or where only one of the nucleic acid molecules is 10 fold or 100 fold or 1000 fold or where one or both nucleic acid molecules are above their k d -
  • selective hybridization conditions would be when at least about, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
  • nucleic acid based there are a variety of molecules disclosed herein that are nucleic acid based, including for example the nucleic acids that encode, for example AR, ER, TR2, TR4, TRAl 6, and/or fragments thereof, as well as various functional nucleic acids.
  • the disclosed nucleic acids are made up of for example, nucleotides, nucleotide analogs, or nucleotide substitutes. Non-limiting examples of these and other molecules are discussed herein. It is understood that for example, when a vector is expressed in a cell, that the expressed mRNA will typically be made up of A, C, G, and U.
  • an antisense molecule is introduced into a cell or cell environment through for example exogenous delivery, it is advantageous that the antisense molecule be made up of nucleotide analogs that reduce the degradation of the antisense molecule in the cellular environment.
  • a nucleotide is a molecule that contains a base moiety, a sugar moiety and a phosphate moiety. Nucleotides can be linked together through their phosphate moieties and sugar moieties creating an internucleoside linkage.
  • the base moiety of a nucleotide can be adenin-9-yl (A), cytosin-1-yl (C), guanin-9-yl (G), uracil-1-yl (U), and thymin-1-yl (T).
  • the sugar moiety of a nucleotide is a ribose or a deoxyribose.
  • the phosphate moiety of a nucleotide is pentavalent phosphate.
  • a non-limiting example of a nucleotide would be 3'- AMP (3'- adenosine monophosphate) or 5'-GMP (5'-guanosine monophosphate).
  • a nucleotide analog is a nucleotide which contains some type of modification to either the base, sugar, or phosphate moieties. Modifications to nucleotides are well known in the art and would include for example, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, and 2-aminoadenine as well as modifications at the sugar or phosphate moieties. 335.
  • conjugates can be link other types of molecules to nucleotides or nucleotide analogs to enhance for example, cellular uptake.
  • Conjugates can be chemically linked to the nucleotide or nucleotide analogs.
  • conjugates include but are not limited to lipid moieties such as a cholesterol moiety.
  • a Hoogsteen interaction is the interaction that takes place on the Hoogsteen face of a nucleotide or nucleotide analog, which is exposed in the major groove of duplex DNA.
  • the Hoogsteen face includes the N7 position and reactive groups (NH2 or O) at the C6 position of purine nucleotides.
  • the primers are used for the DNA amplification reactions, such as PCR or direct sequencing. It is understood that in certain embodiments the primers can also be extended using non-enzymatic techniques, where for example, the nucleotides or oligonucleotides used to extend the primer are modified such that they will chemically react to extend the primer in a sequence specific manner.
  • the disclosed primers hybridize with the nucleic acids of AR, ER, TR2, TR4, TRA16, and/or fragments thereof, or a region of the nucleic acids of AR, ER, TR2, TR4, TRA16, and/or fragments thereof, or they hybridize with the complement of the nucleic acids of AR, ER, TR2, TR4, TRAl 6, and/or fragments thereo or complement of a region of a nucleic acid of AR, ER, TR2, TR4, TRAl 6, and/or fragments thereof. d) Delivery of the compositions to cells
  • compositions and methods which can be used to deliver nucleic acids to cells, either in vitro or in vivo. These methods and compositions can largely be broken down into two classes: viral based delivery systems and non-viral based delivery systems.
  • the nucleic acids can be delivered through a number of direct delivery systems such as, elecfroporalion, lipofection, calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or via transfer of genetic material in cells or carriers such as cationic liposomes.
  • Transfer vectors can be any nucleotide construction used to deliver genes into cells (e.g., a plasmid), or as part of a general strategy to deliver genes, e.g., as part of recombinant retro virus or adenovirus (Ram et al., Cancer Res. 53:83-88, (1993)). 344.
  • plasmid or viral vectors are agents that transport the disclosed nucleic acids, such as nucleic acids encoding AR, ER, TR2, TR4, TRAl 6, and/or fragments thereof into the cell without degradation and include a promoter yielding expression of the gene in the cells into which it is delivered.
  • the vectors are derived from either a virus or a retrovirus.
  • Viral vectors are, for example, Adenovirus, Adeno-associated virus, He ⁇ es vims, Vaccinia virus, Polio virus, AIDS virus, neuronal trophic virus, Sindbis and other RNA viruses, including these viruses with the HIV backbone, as well as lentiviruses. Also prefened are any viral families which share the properties of these viruses which make them suitable for use as vectors. Retroviruses include Murine Maloney Leukemia virus, MMLV, and retroviruses that express the desirable properties of MMLV as a vector. Retroviral vectors are able to carry a larger genetic payload, i.e., a transgene or marker gene, than other viral vectors, and for this reason are a commonly used vector.
  • Adenovirus vectors are relatively stable and easy to work with, have high titers, and can be delivered in aerosol formulation, and can transfect non-dividing cells.
  • Pox viral vectors are large and have several sites for inserting genes, they are thermostable and can be stored at room temperature.
  • a prefened embodiment is a viral vector which has been engineered so as to suppress the immune response of the host organism, elicited by the viral antigens. Prefened vectors of this type will carry coding regions for h terleukin 8 or 10. 345.
  • Viral vectors can have higher transaction (ability to introduce genes) abilities than chemical or physical methods to introduce genes into cells.
  • viral vectors typically contain, nonstructural early genes, structural late genes, an RNA polymerase UJ transcript, inverted terminal repeats necessary for replication and encapsidation, and promoters to control the transcription and replication of the viral genome.
  • viruses When engineered as vectors, viruses typically have one or more of the early genes removed and a gene or gene/promotor cassette is inserted into the viral genome in place of the removed viral DNA. Constructs of this type can carry up to about 8 kb of foreign genetic material.
  • the necessary functions of the removed early genes are typically supplied by cell lines which have been engineered to express the gene products of the early genes in trans.
  • a retrovirus is an animal virus belonging to the virus family of Retro viridae, including any types, subfamilies, genus, or tropisms.
  • Retroviral vectors in general, are described by Verma, I.M., Retroviral vectors for gene transfer. In Microbiology- 1985, American Society for Microbiology, pp. 229-232, Washington, (1985), which is inco ⁇ orated by reference herein. Examples of methods for using retroviral vectors for gene therapy are described in U.S. Patent Nos.
  • Retrovirus vectors typically contain a packaging signal for inco ⁇ oration into the package coat, a sequence which signals the start of the gag transcription unit, elements necessary for reverse transcription, including a primer binding site to bind the tRNA primer of reverse transcription, terminal repeat sequences that guide the switch of RNA strands during DNA synthesis, a purine rich sequence 5' to the 3' LTR that serve as the priming site for the synthesis of the second strand of DNA synthesis, and specific sequences near the ends of the LTRs that enable the insertion of the DNA state of the retrovirus to insert into the host genome.
  • gag, pol, and env genes allow for about 8 kb of foreign sequence to be inserted into the viral genome, become reverse transcribed, and upon replication be packaged into a new retroviral particle. This amount of nucleic acid is sufficient for the delivery of a one to many genes depending on the size of each transcript. It is preferable to include either positive or negative selectable markers along with other genes in the insert. 348. Since the replication machinery and packaging proteins in most retroviral vectors have been removed (gag, pol, and env), the vectors are typically generated by placing them into a packaging cell line.
  • a packaging cell line is a cell line which has been transfected or transformed with a retrovirus that contains the replication and packaging machinery, but lacks any packaging signal.
  • the vector carrying the DNA of choice When the vector carrying the DNA of choice is transfected into these cell lines, the vector containing the gene of interest is replicated and packaged into new retroviral particles, by the machinery provided in cis by the helper cell. The genomes for the machinery are not packaged because they lack the necessary signals.
  • Adenoviral Vectors 349 The construction of replication-defective adeno viruses has been described (Berkner et al., J. Virology 61:1213-1220 (1987); Massie et al., Mol. Cell. Biol. 6:2872-2883 (1986); Haj-Ahmad et al., J. Virology 57:267-274 (1986); Davidson et al, J. Virology 61:1226-1239 (1987); Zhang “Generation and identification of recombinant adenovirus by liposome-mediated transfection and PCR analysis” BioTechniques 15:868-872 (1993)).
  • Recombinant adenoviruses achieve gene transduction by binding to specific cell surface receptors, after which the virus is internalized by receptor-mediated endocytosis, in the same manner as wild type or replication-defective adenovirus (Chardonnet and Dales, Virology 40:462-477 (1970); Brown and Burlingham, J.
  • a viral vector can be one based on an adenovirus which has had the El gene removed and these virions are generated in a cell line such as the human 293 cell line. In another prefened embodiment both the El and E3 genes are removed from the adenovirus genome.
  • AAV adeno-associated virus
  • This defective parvovirus is a prefened vector because it can infect many cell types and is nonpathogenic to humans.
  • AAV type vectors can transport about 4 to 5 kb and wild type AAV is known to stably insert into chromosome 19.
  • Vectors which contain this site specific integration property are prefened.
  • An especially prefened embodiment of this type of vector is the P4.1 C vector produced by Avigen, San Francisco, CA, which can contain the he ⁇ es simplex virus thymidine kinase gene, HS V-tk, and/or a marker gene, such as the gene encoding the green fluorescent protein, GFP.
  • the AAV contains a pair of inverted terminal repeats (ITRs) which flank at least one cassette containing a promoter which directs cell-specific expression operably linked to a heterologous gene.
  • ITRs inverted terminal repeats
  • Heterologous in this context refers to any nucleotide sequence or gene which is not native to the AAV or B19 parvovirus.
  • AAV and B19 coding regions have been deleted, resulting in a safe, noncytotoxic vector.
  • the AAV ITRs, or modifications thereof, confer infectivity and site- specific integration, but not cytotoxicity, and the promoter directs cell-specific expression.
  • Patent No. 6,261,834 is herein inco ⁇ orated by reference for material related to the AAV vector.
  • the vectors of the present invention thus provide DNA molecules which are capable of integration into a mammalian chromosome without substantial toxicity.
  • the inserted genes in viral and retroviral usually contain promoters, and/or enhancers to help control the expression of the desired gene product.
  • a promoter is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard to the transcription start site.
  • a promoter contains core elements required for basic interaction of RNA polymerase and transcription factors, and can contain upstream elements and response elements.
  • compositions can be delivered to the target cells in a variety of ways.
  • the compositions can be delivered through electroporation, or through lipofection, or through calcium phosphate precipitation.
  • the delivery mechanism chosen will depend in part on the type of cell targeted and whether the delivery is occurring for example in vivo or in vitro.
  • compositions can comprise, in addition to the disclosed compositions or vectors for example, lipids such as liposomes, such as cationic liposomes (e.g., DOTMA, DOPE, DC-cholesterol) or anionic liposomes.
  • liposomes can further comprise proteins to facilitate targeting a particular cell, if desired.
  • Administration of a composition comprising a compound and a cationic liposome can be administered to the blood afferent to a target organ or inhaled into the respiratory tract to target cells of the respiratory tract.
  • liposomes see, e.g., Brigham et al., Am. J. Resp. Cell. Mol. Biol.
  • the compound can be administered as a component of a microcapsule that can be targeted to specific cell types, such as macrophages, or where the diffusion of the compound or delivery of the compound from the microcapsule is designed for a specific rate or dosage.
  • delivery of the compositions to cells can be via a variety of mechanisms.
  • delivery can be via a liposome, using commercially available liposome preparations such as LIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, MD), SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison, WI), as well as other liposomes developed according to procedures standard in the art.
  • nucleic acid or vector of this invention can be delivered in vivo by electroporation, the technology for which is available from Genetronics, Inc. (San Diego, CA) as well as by means of a SONOPORATION machine (ImaRx Pharmaceutical Co ⁇ ., Arlington, AZ).
  • the materials can be in solution, suspension (for example, inco ⁇ orated into microparticles, liposomes, or cells). These can be targeted to a particular cell type via antibodies, receptors, or receptor ligands.
  • the following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem ⁇ 2:447-451, (1991); Bagshawe, K.D., Br. J Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al, Cancer Immunol. Immunothen, 35:421-425, (1992); Pietersz and McK.enzie,_Immunolog.
  • Vehicles such as "stealth” and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo.
  • the internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).
  • Nucleic acids that are delivered to cells which are to be integrated into the host cell genome typically contain integration sequences. These sequences are often viral related sequences, particularly when viral based systems are used. These viral integration systems can also be inco ⁇ orated into nucleic acids which are to be delivered using a non-nucleic acid based system of deliver, such as a liposome, so that the nucleic acid contained in the delivery system can be come integrated into the host genome.
  • a non-nucleic acid based system of deliver such as a liposome
  • cells or tissues can be removed and maintained outside the body according to standard protocols well known in the art.
  • the compositions can be introduced into the cells via any gene transfer mechanism, such as, for example, calcium phosphate mediated gene delivery, electroporation, microinjection or proteoliposomes.
  • the transduced cells can then be infused (e.g., in a pharmaceutically acceptable carrier) or homotopically transplanted back into the subject per standard methods for the cell or tissue type. Standard methods are known for transplantation or infusion of various cells into a subject.
  • Expression systems are known for transplantation or infusion of various cells into a subject.
  • the nucleic acids that are delivered to cells typically contain expression controlling systems.
  • the inserted genes in viral and retroviral systems usually contain promoters, and/or enhancers to help control the expression of the desired gene product.
  • a promoter is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard to the transcription start site.
  • a promoter contains core elements required for basic interaction of RNA polymerase and franscription factors, and can contain upstream elements and response elements.
  • Prefened promoters controlling transcription from vectors in mammalian host cells can be obtained from various sources, for example, the genomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus and most preferably cytomegalovirus, or from heterologous mammalian promoters, e.g. jS-actin promoter.
  • viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus and most preferably cytomegalovirus, or from heterologous mammalian promoters, e.g. jS-actin promoter.
  • the early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication (Fiers et al., Nature, 273: 113 (1978)).
  • the immediate early promoter of the human cytomegalovirus is conveniently obtained as a Hindffl E restriction fragment (Greenway, P.J. et al., Gene 18: 355-360 (1982)).
  • promoters from the host cell or related species also are useful herein.
  • Enhancer generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5' (Laimins, L. et al., Proc. Natl. Acad. Sci. 8: 993 (1981)) or 3' (Lusky, M.L., et al., Mol. Cell Bio. _3: 1108 (1983)) to the franscription unit. Furthermore, enhancers can be within an intron (Banerji, J.L. et al., Cell 33: 729 (1983)) as well as within the coding sequence itself (Osborne, T.F., et al., Mol. Cell Bio._4: 1293 (1984)).
  • Enhancers function to increase transcription from nearby promoters. Enhancers also often contain response elements that mediate the regulation of transcription. Promoters can also contain response elements that mediate the regulation of transcription. Enhancers often determine the regulation of expression of a gene. While many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, -fetoprotein and insulin), typically one will use an enhancer from a eukaryotic cell virus for general expression.
  • Prefened examples are the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. 369.
  • the promotor and/or enhancer can be specifically activated either by light or specific chemical events which trigger their function.
  • Systems can be regulated by reagents such as tetracycline and dexamethasone.
  • compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.
  • unprotected peptide segments are chemically linked where the bond formed between the peptide segments as a result of the chemical ligation is an unnatural (non-peptide) bond (Schnolzer, M et al., Science, 256:221 (1992)).
  • This technique has been used to synthesize analogs of protein domains as well as large amounts of relatively pure proteins with full biological activity (deLisle Milton RC et al., Techniques in Protein Chemistry TV. Academic Press, New York, pp. 257-267 (1992)).
  • RNA from the DHT-treated transfected LNCaP cells was prepared by the ultracentrifugation method as described. Lee, et al., J: Biol. Chem. 273, 13437-13443 (1998). The probe was obtained from exon 3 of PSA gene and labeled with ⁇ - 32 p dCTP.
  • TR4 Interacts with AR both In vitro and In vivo.
  • DU145 cells were seeded on two-well Lab Tek Chamber slides (Nalge Nunc International) 18 hours before fransfection.
  • AR unliganded or liganded
  • TR4 or ER alone or in combination with the FuGENETM6 transfection reagent (Boehringer-Mannheim). 24 hours after transfection, cells were treated with 100 nM DHT or ethanol.
  • hnmunostaining was performed by incubation with the anti-AR polyclonal antibody, anti-TR4 monoclonal antibody, or anti-ER ⁇ monoclonal antibody, followed by incubation with either fluorescein-conjugated goat anti-rabbit or anti-mouse antibodies (ICN Pharmaceuticals, Inc.). It was found that unliganded AR was located mainly in the cytoplasm. The AR signal moved to the nucleus in the presence of its cognate ligand, DHT. These data agreed with a previous report shown in COS cells. Simental, et al., J: Biol. Chem. 266, 510-518 (1991). In contrast, TR4 was detected as a nuclear protein as was ER even in the absence of exogenous ligand.
  • a modified mammalian one-hybrid system was used to avoid the possibility that the DHT-dependent interaction between AR and TR4 is due to artificial conformational changes created by the VPI6-AR fusion protein in the mammalian two-hybrid system.
  • a full-length AR( ⁇ SG5AR) was co-transfected with GAL4-TR4E and PG5-Luc reporter in HI 299 cells (pSG5AR, ⁇ SG5GR, pSG5PR, disclosed in Yeh et al., Proc Natl. Acad. Sci., 93:5517-5521 (1996), which is herein inco ⁇ orated by reference at least for material related to plasmids and assays, including sequence).
  • reporter plasmids (DR4-CAT and CNTFR-15-LUC) was co-transfected with 200 ng ofpCMX ⁇ TR4 and increasing amounts of pCMV-AR (200,600, and 1,200 ng), pSG5GR (1,200 ng), or pSG5 PR (1,200 ng) using the SuperFect transfection kit (Qiagen) (PCMV AR Mowszowicz et al., (1993) Molecular Endocrinology 7:861:869). As shown in Fig.
  • the CAT activity induced by pCMXTR4 could be repressed significantly, in a dose-dependent manner by co-transfection of pSG5AR in the absence or presence of DHT.
  • This repression of TR4 transactivation is AR specific, as other activated steroid receptors, such as GR or PR, have no suppressive effects (Fig. 3A, lanes 9-10).
  • Similar results were obtained when the DR4-TK-CAT reporter was replaced with DRl -CNTFR- 15-LUC, another TR4 response element (Fig. 3 A). Young, et al., J: Biol. Chem. 272,3109-3116 (1997). 439.
  • Another TR4 potential target gene which is located in the hepatitis B virus
  • HepG2 cells were co-transfected with 1.5 ⁇ g CpFL(4)- LUC reporter and 0.5 ⁇ g pCMX-TR4, with increasing amounts of pCMV-AR (0.5, 2.5, and 5 ⁇ g) by modified calcium phosphate precipitation method.
  • the relative reporter gene activities were compared to the CAT activities (or luciferase activities) with vector alone.
  • the /3-galactosidase expression vector and pRL-TK were co- transfected in the CAT assay and in the Dual-luciferase reporter assay system (Promega), respectively.
  • TR4 can induce CpFL(4)-LUC activity, which is significantly decreased by co-transfection of AR with TR4 in a dose-dependent manner. This rinding indicated that AR could regulate HBV gene expression through protein-protein interaction.
  • EMSA using 32 p labeled DR1-TR4RE as a probe were applied to further dissect the mechanism of how AR repressed the TR4-mediated transactivation.
  • One ⁇ l of in vitro translated TR4 protein was incubated with increasing amounts of in vitro translated AR (1 ⁇ l, 2 ⁇ l, and 4 ⁇ l) in EMSA reaction buffer (10 mM HEPES pH 7.9, 2% (v/v) glycerol, 100 mM KC1, ImM EDTA, 5 mM MgC12, and 1 mM DTT) for 15 min.
  • 32 P-end labeled DRl was added into the protein mixture and incubated for 15 min before loading.
  • AR itself acts as a transcription factor to activate many androgen target genes.
  • 500 ng of MMTV-Luc (Fig. 4A), or PSA-Luc (Fig. 4B) were co-transfected with 40 ng pCMV-AR (lane 2-3) with increasing amounts of pCMX-TR4 (as indicated in Figure 4). After 24 hours transfection, cells were treated with 10 nM of DHT. After 16-18 hours incubation, cells were harvested for Dual-luciferase reporter assay.
  • Fig. 4A MMTV-Luc
  • PSA-Luc Fig. 4B
  • MMTV-luciferase reporter As GR and PR can also induce MMTV-luciferase reporter, Beato, M., Ce//56, 335-344 (1989), determining if TR4 could also repress OR-or PR-mediated transactivation was analyzed. Three ⁇ g of MMTV-Luc was co-transfected with 4 ⁇ g of pCMX-TR4 in the presence of 1 ⁇ g of pSG5AR, pSG5GR, or pSG5PR by modified calcium-phosphate method. After 24 hours transfection, the cells were treated with 10 nM of synthetic steroids (DHT, dexamethasone, and progesterone). Dual-luciferase reporter assays were performed. PRL-TK was used to normalize the transfection efficiency.
  • AGGTCA consensus response element
  • Chloramphenicol was obtained from Amersham Co ⁇ (Arlington Heights, IX 60005). Acetyl coenzyme A was purchased from Phannacia Biotech Inc (Piscataway, NJ 08854). 17
  • Inserts of the ER fragments were released from the pSG5-ER and generated by the following strategies: GST-ER-N-terminal (GST-ER-N), the BamHI-MscI fragment of ER into the pGEX-3x Smal-RcoRI site; GST-ER-DNA Binding Domain (GST-ER-DBD), the Hindlll-Pstl fragment into the pGEX-2T Smal site; GST-ER- Ligand Binding Domain (GST-ER-LBD), the Pstl-EcoRI fragment into the pGEX-2T Smal- EcoRI site; and GST-ER-F Domain (GST-ER-F) the Hhal-EcoRI fragment into the pGEX-3x
  • H1299 human lung cancer cells and PC-3 human prostate cancer cells were maintained in DMEM containing 5% fetal calf serum (FCS), 100 U/mL penicillin and 100 ⁇ g/ml streptomycin sulfate at 5% CO2 at 37°C.
  • T47D human breast cancer cells were maintained in RPMI 1640 medium containing 5% FCS, 100 U/ml penicillin and 1 00 ⁇ g /ml streptomycin sulfate at 5% CO2 at 37°C.
  • the total amount of DNA was adjusted up to 10.5 ⁇ g with parent vectors, pSG5 or pCMV in each transcriptional activity assay. After 24 hour fransfection, the medium was changed again and the cells were treated with 10-8 M E2 for another 24 h. The cells were then harvested and whole cell extracts were used for CAT assay. The CAT activity was quantitated by Phosphorhnager (Molecular Dynamics). Data are presented as means ⁇ S.D. of at least three independent experiments. (d) GST ' Pull-down Assay
  • Fusion proteins of GST-TR2, GST-ER segments and GST were obtained by transforming expressing plasmids into BL21(DE3)pLysS competent bacteria followed with 2- hour IP TG induction.
  • GST-proteins were then purified by mixing Glutathione-SepharoseTM 4B (Pharmacia) into bacteria lysates on a rotating disk at 4°C for 40 mm followed by washing with ImL NENT buffer (20 mM Tris-HCL (pH 8.0), 100 mM NaCI, 1 mM EDTA, 6 mM MgC12, 1 mM Dithiothretiol, 8% Glycerol, 1 mM PMSF and 0.5% (v/v) NP-40).
  • the ER, AR, RXR ⁇ ., and TR2 proteins labeled with [35S] were generated in vitro by using the TNT reticulocyte lysate system (Promega).
  • the glutathione-Sepharose bound GST-proteins were resuspended with 100 ⁇ l of interaction buffer (20 mM HEPES/pH 7.9, 150 mM KC1, 5 mM MgCI2.
  • the bound proteins were separated on an SDS/8% PAGE and visualized by using autoradiography.
  • EMSA was carried out as described previously with some modification.
  • 0.1 ⁇ g of Double-stranded oligonucleotide ERE primers were end-labeled with 5 ⁇ l of [ ⁇ - 32 P] ATP (DuPont NEN) by using T4 polynucleotide kinase. In vitro translated proteins.
  • ⁇ CMV-TR2 was transiently transfected into T47D cells, and lysed in RIP A buffer (lOmM sodium phosphate, pH 7.0. 150 mM NaCI, 2 mM EDT A. 1 % (w/v) Nonidet P-40, 0.1 % (w/v) SDS. l% (w/v) sodium deoxycholate) with freshly adding proteinase inhibitors.
  • the soluble protein was quantified using the Bio-Rad Protein Assay reagent (Bio-Rad Laboratories) and 50 ⁇ g soluble proteins were loaded onto SDS/10% PAGE and then transfened to hnmobion-P transfer membrane (Millipore). After the blocking reaction overnight, the membrane was incubated with rat anti-PR polyclonal antibody (H-190. Santa Cruz) in PBS(-) containing 0.1% Skim milk for 2 hour at room temperature. The membrane was washed and then incubated in 15 ⁇ Ci 1251] of protein-A (DuPont NENV30 ml of PBS(-) containing 0.1% Skim milk for 1 hour at room temperature. The western blots were autoradiographed and quantitated by using Phosphorhnager.
  • Bio-Rad Protein Assay reagent Bio-Rad Laboratories
  • 50 ⁇ g soluble proteins were loaded onto SDS/10% PAGE and then transfened to hnmobion-P transfer
  • vztr ⁇ -translated [35S]-labeled TR2 protein was incubated with GST, GST-ER-N (aa 1-165 of ER), GST-ER-DBD (aa 123-340 of ER), GST-ER- LBD (aa 499-595 of ER), or GST-ER-F (aa 552-595 of ER) in the absence or presence of 1 ⁇ M E2.
  • the partial LBD, (aa 499-595) or the F domain (aa 552-595) of the ER, but not the N- terminal domain (aa 1-165) or DBD (aa 123-340) of the ER can interact with the [35SJ-TR2 in the presence of 1 ⁇ M E2.
  • TR2 The in vivo mammalian two-hybrid system was applied to further confirm the interaction between the ER and TR2.
  • a full-length TR2 was fused to the transcriptional activator W ⁇ 6 (VPI6-TR2) and then co-transfected with GAL4-DBD fused with ER-LBD (GAL4-ER, aa 282 to 595) and a GAL4-responsive CAT reporter (pG5-CAT) in H1299 cells.
  • H1299 cells and PC-3 cells were transiently co-transfected with 3 g of pSG5- CA T reporter plasmid, 3 ⁇ g of GAL4 or GAL4-ER expression plasmid, and 3 ⁇ g of VPI6 or VP16-TR2 expression-plasmid. Interaction was estimated by determining, CAT activity levels in the presence or absence of 108 M E2. Cells were also transfected with a /3-galactosidase expression plasmid, pCMV-/3-gal, as an internal control for transfection efficiency. As shown in Fig.
  • ER rabbit polyclonal (H- 184), ER mouse monoclonal (C314), and progesterone receptor (PR) rabbit polyclonal (HI 90) were obtained from Santa Cruz Biotechnology.
  • TR2 rabbit polyclonal (#1132) and mouse monoclonal anti-TR2 IgM antibody (G204) were described previously (Lee, S. et al. (1998) Mol. Endocrinol. 12(8): 1184-92).
  • Monoclonal anti-FLAG antibody (M2) was purchased from Sigma.
  • Biotinylated secondary antibodies (goat-anti rabbit IgG and goat-anti mouse IgM) were from Vector Laboratories, h e. (Burlingame, CA).
  • AP- conjugated secondary antibodies (goat anti-rabbit IgG and goat anti-mouse IgM) were from Santa Cruz Biotechnology.
  • the pCMV-TR2, pGEX-3x-TR2, and pCMX-VP 16-TR2 were constructed by insertion of full-length TR2 cDNA (Chang, C. et al., (1989) Biochem. Biophys. Res. Commun. 165, 735-41; Chang, C. et al., (1988) Biochem. Biophys. Res. Commun. 155, 971-7) into individual vectors.
  • the doxycycline-inducible expression vector pBIG2i bearing hygromycin B resistance gene was a gift from Dr. Jay Reeder (University of Rochester, NY) (Sfrathdee, C. et al, (1999) Gene 229, 21-9).
  • pBIG2i and pBIG2i-FLAG-TR2 were used for generating MCF7- pBIG and MCF7-TR2 stable clones, respectively.
  • the GAL4-ER (aa 282-595) and pCMV- mER ⁇ were gifts from Dr. Hinrich Gronemeyer (Strasbourg, France) and Vincent Giguere (McGill University, Quebec, Canada), respectively.
  • To construct GST-ER fragments ER cDNA fragments were released from pSG5-ER (Green, S. et al., (1988) Nucleic Acids Res.
  • pGEX-3X-ER-#l (aa 1 to 165), pGEX-2T-ER-#2 (aa 123-340), pGEX- 2T-ER-#3 (aa 312-595), pGEX-3X-ER-#4 (aa 552-595), pGEX-2T-ER-#5 (aa 123-312), and pGEX-2T-ER-#6 (aa 312-3 0).
  • the pGEX-KG-TR2-#l, #2, and #3 plasmids were constructed by insertion of PCR-generated cDNA fragments conesponding to aa 1-112, aa 88-196, and aa 179-603, respectively, into pGEX-KG vector (Guan et al., (1991), Analytical Biochemistry 192:362-4, which is herein inco ⁇ orated by reference al least for material related to plasmids and assays, including sequence).
  • pCDNA3-TR2-fl AS and pIRES-TR2-N AS were constructed by insertion of opposite orientation of cDNAs encoding full length and N terminal (aa 1-112) into pCDNA3 (Invitrogen) and pIRES (Clontech), respectively.
  • Co-im ⁇ ranoprecipitation 469 MCF7 cells plated on 100-mm dishes were solubilized in 1 ml RIPA buffer containing 0.5% NP-40 and protease inhibitors. Immunoprecipitation was performed using rabbit anti-ER antibody (1:1000) (H- 184) and then analyzed by Western blotting with anti-ER (1 : 1000) (H-184) or anti-TR2 (1:1000) (G204) antibodies, followed by incubation with AP conjugate goat anti-rabbit or rabbit anti-mouse IgM antibodies, and visualized with AP conjugate kit (Bio-Rad).
  • EMSA was carried out as described previously (Lee, Y. et al., (1999) Proc. Natl. Acad. Sci. USA 96, 14724-9) with some modifications.
  • Human complement C3 ERE (containing one imperfect palindromic inverted repeat: S'-AGGTGGCCCTGACCC-S') end- labeled with ⁇ -[ PJ-ATP was used as probe.
  • ER and TR2 were in vitro translated by TNT system as instructed by manufacturer (Promega).
  • TR2 is known to be an important regulator for breast cancer development as two thirds of breast tumors contain a functional ER that mediates estrogen responsiveness for cell growth.
  • Northern blotting was employed to determine whether TR2 is expressed in ER-positive breast cancer cells. TR2 transcripts around 2.5 kb were expressed at different levels in three breast cancer cell lines (MCF7, T47D, and ZR-75-1), which were well documented as containing both ER ⁇ and ER ⁇ . TR2 could also be detected in prostate cancer PC-3 and LNCaP cells.
  • TR2 specifically suppresses ER-mediated transcription
  • TR2 did not affect TR2's influence on ER function.
  • TR2 consistently suppressed either exogenous (in PC-3 and hl299 cells) or endogenous (in MCF7 and T47D cells) ER in a dose-dependent manner (Fig. 11A).
  • MCF7-pBIG and MCF7-TR2 cells were stably transfected with doxycycline-inducible pBIG2i and pBIG2I-FLAG-TR2 plasmids, respectively.
  • ER was not suppressed by doxycycline treatment in the MCF7-pBIG cells which were stably transfected with the pBIG2i parent vector.
  • PR expression an endogenous target gene of ER, was examined.
  • T-47D cells seeded in 60-mm dishes were transfected with 10 mg of pCMV or pCMV-TR2 for 16 h, followed by treatment with 10 nM E2 for another 16 h.
  • Cell extracts (50 mg) and mRNA (15 mg) were used for Western blotting with anti-PR antibody (H-190) and Northern Blotting, respectively.
  • TR2 Relative mRNA expression amounts were normalized by 28S expression and quantitated by ImageQuant V.1.2 (Molecular Dynamics). It was observed that TR2 could repress E2-induced PR expression at mRNA and protein levels in T47D cells and MCF7 cells. TR2 could also suppress the basal level of ER franscription in the absence of E2. For examining specificity, the effect of TR2 on other classical steroid receptors was analyzed. As shown in Fig. 1 IB, while TR2 could also suppress ER ⁇ -and AR-mediated transcription in
  • TR2 has marginal effect on the PR-or glucocorticoid receptor (GR)-mediated transcription in T47D cells.
  • GR glucocorticoid receptor
  • GST pull-down assay the interaction of GST-TR2 fusion protein with AR, ER, and RxR ⁇ was shown. Briefly, the GST and GST-TR2 fusion proteins were purified as instructed by the manufacturer (Amersham Pharmacia). 5 ml of in vz ' tr ⁇ -franslated [35S]-labeled AR, ER, and RXR were incubated with the GST or GST-TR2 bound to glutathione-Sepharose beads in a pulldown assay. After extensive washing, bead-bound protein complexes were loaded onto 8% SDS-PAGE and analyzed by Phosphorhnager. Typically, the input represented 20% amount of [35S]-labeled proteins used in each pull-down assay.
  • GST-ER-#2 (aa 123-340) and GST-ER-#3 (aa 312-595), but not GST-ER-#1 (aa 1-165) and GST-ER-#4 (aa 552-595) can interact with TR2 in the presence or absence of E2 (see Fig. 12A for construct illustrations).
  • GST-ER-#6 (aa 312-340), the overlapping region between GST-ER-#2 and-#3, but not GST-ER-#5 (aa 123-312), showed positive interaction with TR2, indicating that the ER-#6 domain is responsible for this interaction.
  • three GST-fused TR2 fragments, GST-TR2-#l,-#2, and-#3 (see Fig. 12B for construct illustrations), conesponding to N-terminus (aa 1-112), DBD (aa 88-196), and LBD (aa 179-603), respectively, were also examined to locate the ER-binding site.
  • GST or three GST-TR2 fusion proteins were purified and incubated with 5 ml of [35S]-ER in the pull-down assay.
  • the input represents 10% amount of [35S]-labeled proteins used in each pull-down assay.
  • DBD DNA binding domain
  • LBD ligand binding domain.
  • GST-TR2-#2 but not GST-TR2-#1 or-#3, was responsible for binding to ER.
  • Glutathione-Sepharose beads bound GST-TR2 were then incubated with 5 ml of [35S]-ER with increasing amounts of HA-ER-#6, which was in vitro translated from pCDNA3-HA-ER-#6 plasmids, for 2 hour at 4°C in the absence of E2. After extensive washing, bead-bound protein complexes were loaded onto 8% SDS-PAGE and analyzed by Phosphorhnager. Typically, the input represented 10% amount of [35SJ-ER used in each pull-down assay. First, the interaction of GST-TR2 with [ 35 S]-labeled ER was inhibited by increasing amounts of HA-ER-#6 peptide.
  • TR2 The biological significance of TR2 on ER activity 478.
  • SuperFect transfection kit (Qiagen) was used for the transfections. The total amount of plasmids in each dish was made up to 5 mg by adding pCDNA3 parent vector.
  • ER DNA-binding and homodimeric formation are disrupted by associating with TR2 479.
  • TR2 ER expression, stability, nuclear translocation, DNA binding, and interaction with coregulators was tested.
  • [ 32 P] -end-labeled ERE probe (4 x 10 8 dpm/mg) was incubated with in vitro translated TR2 and ER proteins (ratios from 1:1 to 1:4) in EMSA binding buffer and analyzed on a 5% acrylamide native gel containing 2.5% glycerol.
  • TR2 can further suppress the growth of all-trans retinoic acid treated cells to 29-45%.
  • Fig. 15 demonstrates that TR2 could suppress E2/ER-induced cell growth and Gl/S transition via suppression of ER signaling.
  • TR2 staining was observed to be heterogeneous in the ductal epithelia and TEBs as well as the stroma cells within the connective tissues, h contrast, the highly proliferating cap cells comprising a monolayer of stem cells forming the leading edge of TEBs were likely all TR2-negative.
  • the expression pattern of TR2 in mammary glands observed is highly conelated to that of ER described previously (Zeps, N. et al., (1998) Differentiation 62, 221-6; Haslam, S. et al., (1992) J Steroid Biochem Mol Biol 42, 589-95).
  • TR2 a suppressor of ER, can play such a role as disclosed herein TR2 expression was increased in mammary glands at lactation stage and that TR2 inhibited estrogen-dependent PR mRNA expression in breast cancer cells.
  • TR2 was originally identified as a transcriptional factor that can modulate many target genes' expression via binding to the TR2 response elements (Chang, C. et al., (1989) Biochem. Biophys. Res. Commun. 165, 735-41; Chang, C. et al., (1988) Biochem. Biophys. Res. Commun. 155, 971-7; Lin, T. et al, (1995) J. Biol. Chem. 270, 30121-8). It is disclosed herein that TR2 functions as a repressor of ER-mediated transcription via direct protein-protein interaction (Fig.
  • TR2 since the interaction blocker, ER-#6, an ER fragment (aa 312-340) responsible for TR2 binding, was able to rescue ER from suppression by TR2 (Fig. 13 and 14).
  • Adminisfration of antisense TR2 that enhanced ER transcription in MCF7 cells implies that endogenous TR2 normally down-regulates ER signaling (Fig. 14).
  • TR2 neither affects ER expression levels or the nuclear translocation nor the interaction with some coactivators, such as SRC- la, TIF- ⁇ , and ARA70.
  • TR2 and ligand-dependent coactivators on ER are consistent as an explanation for the disclosed results showing that the ER-TR2 interaction was ligand-independent, as demonstrated by co- immunoprecipitation and GST pull-down assay, and that TR2 did not interfere with ER interacting with those coactivators. Consistent with this phenomena, an antiestrogen, tamoxifen, did not affect their interaction. It is also consistent with the finding that a signature motif, LXXLL, located on the TR2 LBD (aa 547-551) is not required for ER interaction since the ER binding site is located on the TR2 DBD (Fig. 135).
  • Fig. 15 demonstrates that TR2 can suppress E2/ER-induced Gl/S transition and cause cell growth inhibition in MCF7-TR2 cells where TR2 could be induced by treatment with doxycycline. This growth suppression is indicated to mainly go through suppression of ER signal since TR2 lost its suppressive effect on cell growth in the presence of tamoxifen (Fig. 15C). TR2 could additionally mediate growth inhibition through pathways independent of ER. Earlier studies have shown that TR2 induction is involved in neuronal differentiation in mouse P19 stem cells stimulated by either retinoic acid or CNTF (Young, W. et al., (1998) J. Biol. Chem. 273, 20877-85; Lee, C.
  • pBIG-2i and pCMV-mER ⁇ (pBIG-2i, Sfrathdee et al, (1999), Gene 229:21-29) and pCMVmER, Tremblay et al, (1999) Molecular Cell, 3:513-19, both of which are herein inco ⁇ orated by reference at least for material related to plasmids and assays, including sequence.
  • pGEX-3X-TR4-N was made by cloning the Spel/Aaffl fragment from pGEM-T EASY-TR4 into the Smal site of pGEX-3X.
  • the pBIG-2i-TR4 was made by inserting the Spel/Notl fragment of pGEM-T EASY-TR4 into SpelTNotl site of pBIG-2i vector. All plasmids were verified by restriction enzyme analysis and DNA sequencing.
  • FBS calcium phosphate precipitation method
  • H1299 cells were transfected with an ER expression plasmid (pSG5-ER or pCMV-mER ⁇ ), ERE-chloramphenicol acetyltransferase (ERE-CAT) or pSG5-PR and MMTV-Luciferase reporter plasmid, and a TR4 expression plasmid (pCMV-TR4) ERZ-CAT Bradshaw et al., (1991) JBiol Chem.
  • ER expression plasmid pSG5-ER or pCMV-mER ⁇
  • ERE-chloramphenicol acetyltransferase pSG5-PR and MMTV-Luciferase reporter plasmid
  • TR4 expression plasmid pCMV-TR4 expression plasmid
  • the fransfection plasmid was created by inserting the Spel-Notl fragment of pGEM-T EASY-TR4 containing the full length TR4, into the multiple cloning site of autoregulated bi-directional tefracycline-responsive pBIG2i expression vector (Sfrathdee, C. et al., (1999) Gene 229(1-2) 21-9).
  • MCF-7 cells were fransfected with ⁇ BIG-2i-TR4 (MCF-7- TR4) or vector (MCF-7-pBIG) using the Superfect reagent (Qiagen) and the cells were maintained in DMEM- 10% FBS selective media containing 200 ⁇ g/ml hygromycin (GTBCO) for two weeks.
  • GST Glutathione-Sepharose-transferase
  • MCF-7-TR4 or MCF-7-pBIG cells were cultured in DMEM containing 10% charcoal dextran-freated FBS for 3 days and then 3 x 10 6 cells were seeded on 100-mm dishes.
  • RNA from cells was prepared by the ultracentrifugation method as described previously (Lee, Y. et al., (1998) J. Biol. Chem. 273(22) 13437-43).
  • MCF-7-TR4 or MCF-7-pBIG cells were cultured in DMEM containing 10% charcoal dextran-freated FBS for 3 days and then 1 x 10 6 cells were seeded on 100-mm dishes.
  • the cells were treated with 2 ⁇ g/ml Dox for 24 hours and then treated with 100 nM E2 or vehicle.
  • Cell lysates were collected after 12 hours with or without E2 treatment by using RIPA buffer. The lysate protein amount was quantitated by Bradford assay (Bio-Rad, Hercules, CA) with bovine serum albumin as a reference standard.
  • Bradford assay Bio-Rad, Hercules, CA
  • bovine serum albumin bovine serum albumin as a reference standard.
  • One hundred ⁇ g protein was loaded and after electrophoresis on 10% SDS-PAGE was fransfened onto Immobulin (Milipore,
  • Electrophoretic Mobility Shift Assay was carried out as described previously (Lee, Y. et al., (1998) J. Biol Chem. 273(22) 13437-43). Briefly, 2.5 ⁇ l of TNT-expressed ER with different amounts of TR4 was included in each reaction and TNT lysate was used to make up equal amounts of lysate. The reaction was preincubated for 15 min at room temperature in 20 ⁇ l of binding buffer (10 mM HEPES/pH 7.4, 50 mM KC1, 1 mM MgCl 2 , 1 mM mercaptoethanol, 0.1 mM ZnCl , and 20% glycerol) containing 1 ⁇ g poly(dI-dC).
  • binding buffer (10 mM HEPES/pH 7.4, 50 mM KC1, 1 mM MgCl 2 , 1 mM mercaptoethanol, 0.1 mM ZnCl , and 20% glycerol
  • [ 32 P]ATP end labeled consensus ERE probes were added to the samples, incubated for 30 min at room temperature, and followed by another 30 min at 4 °C.
  • the reactions were incubated with 2 ⁇ l of a monoclonal anti-ER antibody (C-314) (Santa Cruz) for 15 min at room temperature prior to the addition of probe.
  • C-314 monoclonal anti-ER antibody
  • Protein-DNA complexes were resolved on a 5% native polyacrylamide gel and analyzed by autoradiography.
  • MCF-7-TR4 and MCF-7-pBIG cells which were deprived of estrogen by culturing in phenol-free DMEM medium supplement with 10% charcoal-stripped FBS for 4 days, were plated at 10 4 cells/well into 24-well plate. After 24 hours Dox treatment, 10 nM E2 was added to cells. Cells were trypsinized at the specified time points and counted with a hemocytometer to determine cell density per sample. b) Results
  • progesterone receptor As a confrol, progesterone receptor (PR), which like ER also plays important roles in the mammary gland development (Lydon, J. et al., (1995) Genes Dev. 9(18) 2266-78), was applied to demonstrate that TR4 has selective suppression with these two closely related nuclear receptors. As shown in Fig. 16,4, while 10 nM progesterone can induce PR-mediated MMTV-LUC reporter activity, TR4 failed to suppress the transactivation of PR. The distinct difference in suppression of ER- mediated and PR-mediated transactivation indicates that these events are rather selective. This difference is also not an artifact due to a large amount of exogenously transfected TR4 plasmid, which can result in suppression of general gene transactivation.
  • PR progesterone receptor
  • TR4's ability to repress the MCF-7 endogenous ER-mediated transactivation was assayed. As shown in Fig. 165, increasing the TR4 led to a gradual decrease in the endogenous ER-mediated CAT reporter activity. Together, data in Fig. 16 indicates TR4 is able to repress ER-, but not PR-mediated transactivation.
  • TR4 can repress ER transactivation
  • a GST pull-down assay was used.
  • In vitro interactions of TR4 with ER, [ S] labeled AR, ER, and RXR ⁇ were incubated with GST-TR4 or GST bound glutathione-sepharose beads in a pull-down assay.
  • [ 35 S]-methionine labeled ER was able to interact with the GST-TR4 fusion protein but not with GST alone. This interaction is relatively specific for ER, as TR4 was unable to interact with RXR ⁇ , a common nuclear receptor that binds to many other nuclear receptors (Mangelsdorf, D.
  • TR4-LBD Domain Is Essential for TR4 Suppression Effect on ER Transactivation
  • GST-TR4 N-terminal, GST- ⁇ C-TR4, and GST-TR4-LBD fusion proteins were generated (Fig. 11 A) and their interaction with ER was tested.
  • the GST pull-down assay used [35S] labeled ER and purified GST-fusion protein or GST bound glutathione-Sepharose beads. The input represented 20% of the amount of labeled protein used in the pull-down assay. Only GST-TR4-LBD was able to interact with [ 35 S]-ER, but not GST-TR4 N-terminal and GST- ⁇ C-TR4.
  • TR4 is identified as one of the ER-interacting proteins to modulate ER functions.
  • TR4 Both ER and TR4 can use their LBDs to interact with other receptors.
  • the disclosed data show that TR4 did not influence the binding between RLP140 and ER. Instead, TR4 modulates ER functions via interruption of ER homodimerization and ER binding to EREs.
  • TR4 could suppress ER function not only occurring in cell line transient fransfection experiments (Fig. 16A, B), but also in ER target gene expression. TR4 is also able to suppress estrogen-induced cell proliferation in MCF-7 cells stably fransfected with TR4 induced by Dox (Fig. 18 ⁇ 4). This result extends our in vitro results and demonstrates that the consequence of the protein-protein inhibition can result in the suppression of E2/ER-induced cell growth.
  • ER ⁇ is expressed well in testes and epididymis (Couse, j. et al., (1997) Endocrinology 138(11), 4613-21). ER ⁇ KO mice are infertile and produce lower numbers of epididymal spenn, compared to wild-type mice at 12 weeks. Furthermore, the sperms produced in the ER ⁇ KO mice have obvious defects and are unable to fertilize wild-type oocytes (Eddy, E. et al., (1996) Endocrinology 137(11), 4796-805). These studies indicated that ER ⁇ plays important roles in the testes function and spermatogenesis.
  • TR4 is highly expressed in testes, and is also strongly linked to the defective spermatogenesis found in rhesus monkey in either surgery-induced cryptorchid testis (Mu, X. et al, (2000) J. Biol. Chem. 275(31), 23877-83). 4.
  • the plasmid MMTV-CAT, PSA-CAT, pCMV-AR, pCMV-TR2, pSG5-PR, pSG5-GR, pSG5-SRC-l, pSG5-TMI, pSG5-ARA55, pSG5-ARA54, GST-TR2, and pCMV- ⁇ - Gel were reported previously [5-13F, 16F, and their sequences and structures are herein inco ⁇ orated by reference].
  • Human prostate cancer PC-3 cells were maintained in DMEM containing penicillin (25 units/ml), streptomycin (25 g/ml), and 5% FCS.
  • Human prostate cancer LNCaP cells were maintained in RPMI 1640 containing penicillin (25 units/ml), streptomycin (25 g/ml), and 10% FCS.
  • PC-3 cell were transfected using the calcium phosphate precipitation method as described previously [Mu X.M. et al., (1998) Endocrine 9:27-32], and LNCaP cells were transfected by using SuperfectTM according to the manufacturer's procedures (Qiagen, Chatsworth, CA). CAT assay was performed as described previously [Lee Y.
  • GST control protein and GST-TR2 fusion protein were purified by glutathione- sepharose 4B beads as described by manufacture (Amersham. Biosciences). Five microliters of in vz ' tro-translated 35S-methionine-labeled proteins was used to perform the Pull-down assay as described previously.
  • RNA from transfected LNCaP cells was prepared using TRIZOL reagents (Life Technology) as instructed by manufacturer. The probe was obtained from the PSA gene by PCR and labeled with 32 P dCTP. Northern hybridization was performed as described previously [Mu X.M. et al, (2002) JBiol Chem 275:23877-23883]. b) Results
  • TR2 suppresses AR mediated transactivation in prostate cancer PC-3 cells
  • AR is known to be highly involved in prostate cancer progression, and both TR2 and AR are expressed in prostate cancer, therefore the TR2 effect on AR fransactivation activity was analyzed.
  • Fig. 20 addition of AR induced both MMTV-CAT (A) and PSA- CAT (B), two common AR target gene reporters in prostate cancer PC-3 cells, activity in the presence of 10 nM DHT.
  • TR2 expression plasmid strongly suppresses MMTV-CAT (Fig. 20A) and PSA-CAT (Fig. 20B) activity (Lanes 3-5 vs 2) in a dosage-dependent manner.
  • Glucocorticoid receptor can also induce MMTV reporter gene activity in the presence of 10 nM dexamethasone (Lane 7 vs 6), but transfection of TR2 has less suppression effect on the GR-mediated transactivation (Lanes 8-10 vs 7).
  • AR transactivation activity is highly regulated by coregulators, and the amount of co-regulator is relatively limited, therefore nuclear receptors can be able to suppress each other via competing for shared coregulators.
  • MMTV-CAT, pCMV-AR, and increasing amount of pCMV-TR2 were transfected into human prostate PC-3 cells, and one or more of the AR coregulators SRC-1 (Lane 6-10), CBP (Lane 11-15), ARA70 (Lane 16-20), TIF TL (Lane 21-25), ARA54 (Lane 26-30), and ARA55 (Lane 31-35) were also transfected.
  • PSA is an androgen target gene, which is widely used as a marker for prostate cancer progression.
  • TR2 suppresses AR target gene PSA expression.
  • Prostate cancer LNCaP cells were transfected with either vector confrol or TR2 expression plasmid pCMV-TR2, and cells were treated with or without DHT. Total RNA was extracted and PSA expression was measured by Northern blot. Addition of 10 nM DHT induces PSA mRNA expression. Addition of TR2 expression plasmid suppresses endogenous PSA expression level in LNCaP cells.
  • a GST Pull-down assay was applied.
  • the GST-TR2 fusion protein and GST confrol were purified according to manufacturer's protocol. 5 ⁇ l in vz ' tro-franslated 35S methionine-labeled AR (Lane 4, 6, and 8) and RXR ⁇ (Lane 3, 5, and 7) were incubated with the GST-TR2 (Lane 4-7) and GST (Lane 3 and 8) bound glutathione-sepharose beads.
  • the pulldown complex was loaded on 10% SDS-PAGE and visualized by autoradiography.
  • Methionine- labeled AR was able to interact with GST-TR2 fusion protein (Lane 4 and 6), but not GST alone (Lane 3 and 8). This interaction was relatively specific for AR, as TR2 was not able to interact with RXR ⁇ (Lane 5 and 7), a common nuclear receptor that binds to many other nuclear receptors.
  • TR2 is a transcription factor that can regulate several target genes expression [Lin T-M. et al. (1995) JBiol Chem 270:30121-30128; Lin T-M, et al. (1995) JBiol Chem 270:30121-30128; Lee H.J. et al. (1995) JBiol Chem; 270:5434-5440; Lee H.J. et al. (1996) JBiol Chem 271, 10405-10412; Lee H.J. et al. (1999) Mol Cell Biochem 194:199-207; Chang C. et al. (1997) Biochem Biophys Res Commun 235:205-211; Young W.J. et al.
  • TR4 Testicular Orphan Receptor-4 (TR4) Associated Protein (TRAl 6) as Repressor for the Selective Suppression of TR4-Mediated Transactivation 525. While many coactivators have been identified for various nuclear receptors, relatively fewer corepressors have been isolated and characterized.
  • TR4-mediated fransactivation a testicular o ⁇ han nuclear receptor-4 (TR4) associated protein (TRAl 6), which is mainly localized in the nucleus of cells as repressor to suppress TR4-mediated fransactivation.
  • TR4-mediated trans activation is selective as TRAl 6 shows only slight influence on the trans activation of AR, glucocorticoid receptor, and progesterone receptor.
  • Sequence analysis shows that TRAl 6 is a gene with 139 amino acids in an open reading frame with a 16-kDa molecular weight, which did not match any published gene sequences. Mammalian two-hybrid system and co-immuno-precipitation assays both demonstrated that TRAl 6 can interact strongly with TR4.
  • the electrophoretic mobility shift assay indicates that TRAl 6 can suppress TR4-mediated transactivation via decreased binding between TR4 protein and TR4-response-element on the target gene(s). Furthermore, TRAl 6 can also block the interaction between TR4 and the TR4 ligand binding domain (TR4-LBD) through interaction with the TR4-DNA binding and ligand binding domains (TR4-DL). These unique suppression mechanisms indicate TRAl 6 can function as a repressor to selectively suppress the TR4-mediated transactivation.
  • the library was screened by transformation with the pSos-TR4 bait constructor, and then the transformed yeast cells were selected for growth on both synthetic dropout / glucose minus uronolactone (-UL) agar and synthetic dropout / galactose (- UL) agar plates, and cultured at 25°C and 37°C.
  • the positive clones grow at 25°C both on synthetic dropout / glucose (-UL) and synthetic dropout / galactose (-UL) plates, but at 37°C grow on synthetic dropout / galactose (-UL) plates and not on synthetic dropout / glucose (-UL) plates.
  • TR4 associated protein (TRAl 6) (SEQ JJD NO:34).
  • PMyr- TRA16 fragment and pSos-TR4 were co-transformed into the yeast to verify the interaction between these two proteins. All the positive and negative controls were set up following the system's instruction manual.
  • the missing 5'-coding region was isolated by RACE-PCR technology (Chang, C. et al., (1994) Proc. Nail A cad. Sci. USA 91:6040-6044).
  • the gene-specific antisense primer used for 5* RACE-PCR was 5'-TCACACCTTCTCCCCAAGCACCCG CAGGTGGTA-3'.
  • the PCR reaction condition was 95°C for 2 min, 30 cycles of 95°C for 20 sec to 58°C for 30 sec to 72°C for 30 sec, then 72°C for 7 min.
  • the PCR product was sequenced for confirmation.
  • TR4-DL containing DBD and LBD, or full length of TRAl 6 cDNAs were constructed into pET expression system, and then large-scale expression and purification were carried out from transformed E. coli BL21 (DE3) bacteria following the manufacturer's procedures (Qiagen, Chatsworth, CA). Both proteins of TR4-DL and TRA16 were then confirmed directly on 15% SDS-PAGE followed by Coomassie Blue staining.
  • TRA16 using pET expression system was purified from transformed E. coli BL21 (DE3) bacteria and used directly for immunizing rabbits.
  • the polyclonal TRAl 6 antibody was obtained from Cocalico Biologicals, Inc. Reamstown, P A. It was diluted I: 100 in PBS for immunofluorescence assay and I: 1000 for Western blot assay.
  • Immunostaining was performed by incubating with anti-TRA16 polyclonal antibody, or with anti-TR4 monoclonal antibody (#17), then followed by incubating with either fluorescein- conjugated goat anti-rabbit or anti-mouse antibodies. Coverslips were fixed on the glass slides with a drop of DAPI to stain the nucleus. The slides were observed under 40-fold magnification using a fluorescence microscope or confocal fluorescence microscope. Typically, a green signal represents the localization of TRAl 6, a red signal represents the localization of TR4, and a blue signal represents the DAPI-stained nucleus.
  • HI 299, DU145, and CaS-I were cultured with Dulbecco's minimal essential medium containing penicillin (25 units/ml), streptomycin (25 ⁇ g/ml) and 10% fetal calf serum. Transfection was perfo ⁇ ned by modified calcium phosphate precipitation as previously described or using SuperFect according to the manufacturer's procedures (Qiagen, Chatsworth, CA). The dual-Luciferase (LUC) reporter assay system was conducted according to the manufacturer's instructions (Promega). The Renilla Luciferase reporter plasmid-simian virus 40 (pRL-SV 40) was used as an internal control.
  • pRL-SV 40 Renilla Luciferase reporter plasmid-simian virus 40
  • the chloramphenicol acetyltransferase (CAT) assay was performed as described previously.
  • the (3-galactosidase expression gene was co- transfected to normalize the fransfection efficiency.
  • a Phosphohnager visualized the CAT activity.
  • the total amount of plasmids in each fransfection was adjusted to be equal by addition of backbone vectors.
  • COS-1 cells were transiently co-transfected with 3 ⁇ g reporter plasmid pG5-LUC and 4 ⁇ g of both the GAL4DBD and VP16-hybrid expression plasmids as described previously.
  • the pRL-SV40 plasmid (10 ng) was co-transfected for normalization of transfection efficiency.
  • the Luciferase (Luc) assay was performed 24 hours after transfection.
  • DL containing DBD and LBD
  • TNT-TRA16 containing DBD and LBD
  • TNT-pcDNA4c-His control proteins were purified as instructed by the manufacturer (Promega).
  • IP immunoprecipitation
  • 5 ⁇ l of each in vz ' tro-translated [ 35 S]-labeled proteins were incubated 1 hours at 30°C in various combinations as indicated, then incubated with an antibody bound to protein-G Agarose beads (Santa Cruz Biotechnology) at 4°C for another 2 h. After washing each mixture 4 times, each complex was loaded onto 15% SDS-PAGE gel and visualized by autoradiography.
  • COS-1 cells which are TR4-negative and show little expression of TRA16, were transfected with pBig or pBig-TRA16 using SuperFect (Qiagen, Chatsworth, CA). The cells were then selected using 100 ⁇ g/ml hygromycin B (Sfrathdee, CA. et al., (1999) Gene 229, 21- 29), a single colony was chosen, amplified, and confirmed by reporter gene assay.
  • Nuclear Extract Preparation 537 The nuclear protein extraction was prepared as described previously (Andrews,
  • the EMSA was performed as described previously (Lee, Y.F. et al., (1997) J. Biol. Chem. 272:12215-12220; Young, W.J. et al.,, (1998) J. Biol. Chem. 273:20877-20885). Briefly, the reaction was performed by incubating the [ 32 P]-radiolabeled DRI-TR4RE probe with 10 ⁇ g nuclear extracts of different cells, or only with the purified proteins of TR4-DL and TRA16 from bacteria.
  • TR4 human TR4 associated protein
  • SEQ ID NO. 34 and SEQ ID NO. 35 show cDNA sequences and deduced amino acid sequences ofhuman o ⁇ han receptor TR4 associated protein (TRAl 6). Sequence data have been deposited into GenBank (GenBank accession number: AY1O1377).
  • TRAl 6 protein was made.
  • HI 299 cells were transiently fransfected either with 10 ⁇ g of pcDNA4c-His or pcDNA4c-His-TRA16 for 24 h. After harvesting, 50 ⁇ g of whole cell lysates were run in the 15% SDS PAGE gel. The anti-TRA16 antibody or anti- His-tag antibody was used to immunoblot the membrane to detect the expression of TRAl 6.
  • ⁇ - Actin expression level was used as a loading confrol. Both the endogenous TRAl 6 and transiently transfected pcDNA4c-TRA16 expressed TRAl 6 are recognized by anti-TRA16 antibody.
  • TR4 HCR-1-LUC reporter was replaced with other reporters containing different TR4REs, such as CpFL4-LUC and DR4-TK-CAT (Lee, Y.F. et al., (1999) Proc. Natl. Aca ⁇ . Sci. USA 96:14724-14729). As shown in Fig.
  • [ 32 P]-radiolabeled DR1-TR4RE oligonucleotides probe only was used as a loading confrol.
  • two proteins, TR4-DL (containing DBD and LBD ofTR4) and TRA 16 from E. coli strain DE3, were purified and then checked with EMSA.
  • EMSA was performed using the [ P] -radiolabeled DRI-TR4RE oligonucleotides with both TR4-DL containing DBD and LBD of TR4, and TRA16 purified from the E. coli strain DE3 bacteria (1 :5).
  • TR4 (#C-16) polyclonal antibody produced a TR4-DNA supershift band and separated TR4 from the rest of DR1-TR4RE- binding proteins.
  • TRAl 6 definitely can decrease TR4 binding to its target gene, even with addition of anti-TR4 antibody.
  • the results clearly demonstrate that TRA 16 can suppress TR4-mediated transactivation via the interruption of the binding between TR4 and TR4RE on its target gene. 549. The dimerization of TR4 was assayed to see if it might play any roles in the
  • TR4 can form dimers via the interaction between TR4 and TR4-LBD (amino acids 224-615) in a mammalian two-hybrid assay as shown in Fig. 27 (lane 4).
  • TR4-LBD amino acids 224-615
  • TRAl 6 could then suppress the interaction between TR4 and TR4-LBD significantly (Fig. 27, lane 5).
  • AR showed little influence on the interaction between TR4 and TR4-LBD (Fig. 27A, lane 6).
  • AR can also function as repressor to suppress TR4 transactivation (Lee, Y.F. et al., (1999) Proc. Natl. Aca ⁇ . Sci.
  • TR4-N terminus aa 1-125
  • TR4-DL aa 125-615
  • DBD DBD
  • LBD LBD
  • nuclear receptor coactivators such as pI60/SRC, p300/CBP, and P/CAF act through an inherent histone acetyltransferase activity to increase the level of histone acetylation and enhance the transcriptional activity of ligand bound receptors (Chen, H. et al., (1997) Cell 90:569-580; Spencer, T.E. et al, (1997) Nature 389:194-198; Bannister, AJ. et al., (1996) Nature 384:641-643; Ogryzko, V.V. et al., (1996) Cell 87:953-959; Yang, X.J.
  • nuclear receptor coactivators such as pI60/SRC, p300/CBP, and P/CAF
  • the nuclear receptor corepressor (N-CoR) and the silencing mediator for retinoid and thyroid hormone receptors (SMR T) also contain three repeated receptor interaction domains that include the conserved hydrophobic core motif I/LXXIJ, which can interact with unliganded nuclear receptors, such as retinoic acid receptor ⁇ , thyroid hormone receptor and the o ⁇ han receptor, chicken ovalbumin upstream promoter-transcription factor I (Perissi, V. et al., (1999) Genes & Dev. 13:3198-3208; Nagy, L. et al., (1999) Genes & Dev. 13:3209-3216; Webb, P. et al., (2000) Mol. Endocrinol.
  • N-CoR and SMRT proteins function as corepressors with HDACs activity that can recruit a complex containing Sin3, HDACs, and several additional proteins (Heinzel, T. et al., (1997) Nature 387:43-48; Alland, L.
  • Cyclin Dl a cell cycle regulating protein that functions as an AR corepressor may rely on its cell cycle regulating function to suppress AR (Knudsen, K.E. et al, (1999) Cancer Res. 59:2297-2301).
  • proteins such as a small ubiquitous nuclear corepressor that may function as nuclear receptor corepressor via forming complexes with N-CoR (Zamir I. et al., (1997) Proc. Natl. Acad. Sci. USA 94:14400-14405).
  • Mathur Mathur (Mathur, M.
  • SEQ ID NO:4 Genbank Accession No. A31521. orphan receptor TR2, splice form TR2-5-human.
  • SEQ ID NO:5 Genbank Accession No. 154075. gene mTR2Rl protein- mouse
  • TR4 orphan receptor protein 17. SEQ ID NO:17 TR4 nucleic acid Genbank Accession No: L27586. Human TR4 orphan receptor mRNA, complete eds encoding TR4 protein
  • SEQ ID NO:34 TRA16 Protein, (see figure 28),

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  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne des compositions et des techniques relatives au récepteur d'androgène et au récepteur d'oestrogène TR2, TR4 et à TRA16 et aux interactions entre ces protéines.
EP04711255A 2003-02-13 2004-02-13 Tra16, represseur de tr4/tr2 Withdrawn EP1592391A2 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US44772803P 2003-02-13 2003-02-13
US10/366,811 US20030235860A1 (en) 1999-11-12 2003-02-13 Interactions between AR, ER, TR2, and TR4
US366811 2003-02-13
US447728P 2003-02-13
PCT/US2004/004445 WO2004071461A2 (fr) 2003-02-13 2004-02-13 Tra16, represseur de tr4/tr2

Publications (1)

Publication Number Publication Date
EP1592391A2 true EP1592391A2 (fr) 2005-11-09

Family

ID=32871615

Family Applications (1)

Application Number Title Priority Date Filing Date
EP04711255A Withdrawn EP1592391A2 (fr) 2003-02-13 2004-02-13 Tra16, represseur de tr4/tr2

Country Status (4)

Country Link
EP (1) EP1592391A2 (fr)
AU (1) AU2004212015A1 (fr)
CA (1) CA2516344A1 (fr)
WO (1) WO2004071461A2 (fr)

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5614620A (en) * 1988-03-30 1997-03-25 Arch Development Corporation DNA binding proteins including androgen receptor

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2004071461A3 *

Also Published As

Publication number Publication date
AU2004212015A1 (en) 2004-08-26
WO2004071461A2 (fr) 2004-08-26
CA2516344A1 (fr) 2004-08-26
WO2004071461A3 (fr) 2007-08-16

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