Classifications

• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2869—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against hormone receptors
• • 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
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

• the present invention relates to methods for identifying agents that modulate the effect of cytokine class I receptor binding compounds, said agents being useful for decreasing IGF-1 levels in a cell, and for the treatment of medical disorders caused by hormone dysregulation, such as growth hormone or prolactin dysregulation.
• GH BACKGROUND Growth hormone
• IGF-1 GH-induced insulin-like growth factor-1
• GHR growth hormone receptor
• receptors lack intrinsic catalytic activity but are associated to cytosolic proteins with tyrosine-kinase activity.
• the receptors possess a single membrane-spanning domain and they exist as monomers that dimerize and become activated upon ligand binding.
• Several intracellular second messengers have been implicated in the signal transduction of GH, including calcium ions, phospholipase C, phospholipase A2, G-proteins, protein kinase C (PKC), Janus kinase 2 (JAK2) and signal transducer and activator of transcription (STAT) 1, 3 and 5 (Wood, 1996).
• the signal transduction of GH has been investigated in the serine protease inhibitor (SPI) 2.1 gene, where activation is mediated through phosphorylation of JAK2 and STAT5 (Wood, 1996).
• SPI serine protease inhibitor
• the GHR is internalized in the cell by endocytosis and transported to lysosomal vesicles for destruction.
• the GHR has also been reported to get internalized and translocate to the nucleus upon GH-stimulation (Lobie, Wood 1994). It has been suggested that GHR itself might be involved in gene regulation.
• the nuclear translocation of both GH and the GHR is independent of JAK2 (Graichen et al., 2003), which suggests that this nuclear translocation might be an alternative signal transduction pathway independent of the JAK-STAT pathway.
• the transcription factor AP2 belongs to a family with four members, which all have been implicated as tissue- specific effectors of proliferation and differentiation during embryogenesis (Pfisterer et al., 2002; Werling and Schorle 2002).
• OCTl is a ubiquitous transcription factor found in most mammalian cell types, where it activates transcription of a variety of genes.
• Fig. IA depicts the results of electrophoresis mobility shift assays (EMS A) in nuclear extracts from growth hormone receptor (GHR) transfected and non-transfected WRL-98 cells incubated with an anti-GHR antibody and OCTl DNA probes (SEQ ID NOS:3 and 4).
• Fig. IB depicts the results of EMS A in nuclear extracts from GHR transfected and non-transfected WRL-98 cells incubated with an anti-GHR antibody and AP2 DNA probes (SEQ ID NOS:5 and 6).
• FIG. 1C depicts the results of EMS A in nuclear extracts from rat liver cells incubated with an anti-GHR antibody and AP2 DNA probes (SEQ ID NOS:5 and 6).
• Fig. ID depicts the results of EMS A in nuclear extracts from GHR transfected and non-transfected WRL-98 cells incubated with an anti-GHR antibody and Pr2F DNA probes (SEQ ID NOS:9 and 10).
• Fig. IE depicts the results of EMS A in nuclear extracts from prolactin receptor transfected WRL-98 cells incubated with Pr2F DNA probes (SEQ ID NOS: 9 and 10).
• IF depicts the results of EMSA in nuclear extracts from WRL-98 cells incubated with an anti-GHR antibody and growth hormone response element DNA probes (SEQ ID NOS: 1 and 2).
• Free probe (P) was used as a negative control.
• 400x excess of un-labeled specific (+) or non-specific (-) probe was added to the reaction.
• Investigation of possible supershifts was made by incubating the extracts with an anti-GHR antibody (MAb 263) prior to the addition of the labeled DNA-probe. Non-specific competition was not performed; instead antibody only was incubated with the DNA-probe (Ab).
• FIG. 1G depicts the results of EMSA in nuclear extracts from GHR transfected WRL-98 cells incubated with an anti-GHR antibody and growth hormone response element DNA probes (SEQ ID NOS: 1 and 2). Free probe (P) was used as a negative control. To confirm specific protein-DNA interaction, 400x excess of un-labeled specific (+) or non-specific (-) probe was added to the reaction. Investigation of possible supershifts was made by incubating the extracts with an anti-GHR antibody (MAb 263) prior to the addition of the labeled DNA-probe.
• Non-specific competition was not performed; instead antibody only was incubated with the DNA-probe (Ab).
• S-BVTA S-BVTA.
• Fig. IH depicts the results of EMSA in nuclear extracts from HX rat liver cells incubated with an anti-GHR antibody and growth hormone response element DNA probes (SEQ ID NOS: 1 and 2). Free probe (P) was used as a negative control.
• P free probe
• 400x excess of un-labeled specific (+) or non-specific (-) probe was added to the reaction. Investigation of possible supershifts was made by incubating the extracts with an anti-GHR antibody (MAb 263) prior to the addition of the labeled DNA-probe.
• Fig. 2A depicts the total protein on filter as visualized by Ponceau staining.
• Fig. 2B depicts a Western blot with an anti-GHR antibody.
• Fig. 2C depicts an EMSA gel, showing that the GHR is present in the shifted band.
• Fig. 3 A depicts a Western blot of nuclear extracts from GHR-transfected WRL-68 cells, using a rabbit anti-GHR antibody as a primary antibody, visualized by a pig anti-rabbit secondary antibody coupled to HRP.
• FIG. 3B depicts a control Western blot of nuclear extracts from GHR-transfected WRL-68 cells, using the pig anti-rabbit secondary antibody alone.
• Fig. 3C depicts a Western blot of nuclear extracts from WRL-68 cells, using a rabbit anti-GHR antibody as a primary antibody, visualized by a pig anti-rabbit secondary antibody coupled to HRP.
• GHR is present in nuclear extracts from both transfected and untransfected cells, regardless of treatment, the amount of the receptor is higher in transfected cells.
• 3D depicts a Western blot of nuclear extracts from HX rat liver cells, using a rabbit anti-GHR antibody as a primary antibody, visualized by a pig anti-rabbit secondary antibody coupled to HRP.
• the amount of GHR in HX rat liver cells is as high as in transfected WRL-68 cells.
• GH growth hormone
• this invention provides a method for identifying an agent that modulates an interaction between a cytokine class I receptor and a nuclear factor, the method comprising: (i) contacting a cell with a candidate agent; and (ii) determining whether the candidate agent modulates an interaction between a cytokine class I receptor and a nuclear factor that interacts with the receptor; with the proviso that the nuclear factor is other than STAT5.
• An agent identified by such a method can be used, for example, for the treatment or prevention of a medical disorder caused by dysregulation of a cytokine class I receptor binding compound.
• the method can optionally include the following steps: (i) contacting the cell with the candidate agent, wherein the candidate agent modulates the interaction between the cytokine class I receptor and the nuclear factor; (ii) measuring, in the presence of the candidate agent, a biological effect of a cytokine class I receptor binding compound in the cell; and (iii) determining whether the candidate agent modulates the biological effect of the cytokine class I receptor binding compound in the cell.
• the candidate agent inhibits the interaction between growth hormone receptor and the nuclear factor.
• the candidate agent stimulates the interaction between growth hormone receptor and the nuclear factor.
• the method can optionally include a step of determining the expression of a reporter gene coupled to a promoter comprising a response element for a nuclear factor selected from the group consisting of AP2, OCTl, and Pr2F.
• Candidate agents that can be used in the methods described herein include, for example, polypeptides, peptides, antibodies or antibody fragments, non-peptide compounds, carbohydrates, small molecules, lipids, single or double stranded DNA, single or double stranded RNA, antisense nucleic acid molecules, and ribozymes.
• the identification methods described herein can be carried out in vitro or in vivo. For in vitro methods, the identification can be made using a cell based system or a cell free system. .
• the invention features a method for identifying a nuclear factor that interacts with a cytokine class I receptor, the method comprising: (i) transfecting a cell with a nucleic acid encoding a cytokine class I receptor; (ii) preparing a nuclear extract from the cell; (iii) incubating the nuclear extract with a labeled oligonucleotide probe that binds to a candidate nuclear factor; (iv) separating the reaction mixture in a polyacrylamide gel; and (v) detecting bands corresponding to protein-DNA complexes.
• the method can optionally include a step of, prior to preparing the nuclear extract, stimulating the cell with a cytokine class I receptor binding compound.
• nuclear factors include transfecting cells with a reporter construct wherein the nuclear factor oligonucleotide is part of the promoter regulating transcription of the reporter gene.
• the cells are stimulated with a cytokine class I receptor binding compound and the reporter gene activity is measured.
• the nuclear factor is optionally a DNA binding protein, such as AP2, OCTl, or Pr2F.
• a method according to the invention can comprise the determination whether the candidate agent modulates the effect of growth hormone in the cell. In one embodiment of the invention, such determination comprises determining the expression of a reporter gene coupled to a promoter, e.g., the SPI 2.1, AP2, OCT-1 or Pr2F promoter.
• Reporter genes such as, for example, luciferase, ⁇ -galactosidase, alkaline phosphatase, chloramphenicol acetyl transferase (CAT), Green Fluorescent Protein and other members of the Reef Coral Fluorescent Protein (RCFP) family, can be used to determine transcriptional activity in screening assays according to the invention (see, for example, Goeddel (ed.), Methods Enzymol., Vol. 185, San Diego: Academic Press, Inc. (1990)).
• CAT chloramphenicol acetyl transferase
• RCFP Reef Coral Fluorescent Protein
• Such disorders include, e.g., acromegaly, growth hormone deficiency, growth retardation associated with the Prader-Willi syndrome and Turner's syndrome, growth hormone insensitivity, wasting disorders associated with Acquired Immunodeficiency Syndrome (AIDS), and osteoporosis.
• the agents can be used for decreasing or inhibiting the IGF-1 levels or IGF-1 production in a cell. For the treatment of acromegaly, it is expected that the identified agent will inhibit or decrease the interaction between GHR and the nuclear factor.
• prolactinemia is a disease caused by excess production and secretion of prolactin, and results in clinical symptoms such as suppression of reproductive function and galactorrhea.
• prolactin-secreting pituitary tumor prolactinoma
• a subject in need of prolactin is, e.g., a person in need of stimulation of lactation, e.g., a mother; a person in need of stimulation of the immune system, e.g., a person at risk for an immune disorder, e.g., a person at risk of AIDS, or a person infected with a human immunodeficiency virus (HIN), or a person having a nutritional deficiency (see, e.g., US Patent No. 6,545,198).
• the invention features a method for treating or preventing a medical disorder caused by dysregulation of a cytokine class I receptor binding compound, the method comprising administering to a subject in need thereof an effective amount of an agent that modulates an interaction between a cytokine class I receptor and a nuclear factor.
• the nuclear factor is other than STAT5.
• the method can optionally include a step of identifying a subject as having or being at risk of having a medical disorder described herein prior to the administration of the agent.
• the method can include a step of, following the administration of the agent, evaluating the subj ect for the presence of severity of one or more symptoms of the medical disorder.
• the amount of the agent administered to the subject can optionally be selected based upon the results of such an evaluation.
• the agent inhibits the interaction between the cytokine class I receptor and the nuclear factor.
• the agent stimulates the interaction between the cytokine class I receptor and the nuclear factor.
• the invention features a method for modulating IGF-1 transcription in a cell, the method comprising contacting a cell with an effective amount of an agent that modulates an interaction between growth hormone receptor and a nuclear factor, thereby modulating IGF-1 transcription in the cell.
• the agent inhibits the interaction between growth hormone receptor and the nuclear factor, thereby decreasing IGF-1 transcription in the cell.
• the agent stimulates the interaction between growth hormone receptor and the nuclear factor, thereby increasing IGF-1 transcription in the cell.
• the invention features a method for modulating transcription in a cell, the method comprising contacting a cell with an effective amount of an agent that modulates an interaction between a cytokine class I receptor and a nuclear factor, thereby modulating transcription induced by the cytokine class I receptor in the cell, with the proviso that the nuclear factor is other than STAT5.
• the agent can be, for example, a compound that binds to the cytokine class I receptor and prevents or reduces the ability of the cytokine class I receptor to bind to the nuclear factor.
• the agent inhibits the interaction between the cytokine class I receptor and the nuclear factor, thereby decreasing transcription induced by the cytokine class I receptor in the cell.
• the agent stimulates the interaction between the cytokine class I receptor and the nuclear factor, thereby increasing transcription induced by the cytokine class I receptor in the cell.
• the cytokine class I receptor can be, for example growth hormone receptor or prolactin receptor.
• the cytokine class I receptor binding compound is growth hormone and the cytokine class I receptor is growth hormone receptor.
• the cytokine class I receptor binding compound is prolactin and the cytokine class I receptor is prolactin receptor.
• the nuclear factor used in the methods and compositions described herein can be, for example, AP2, OCTl , or Pr2F.
• the agent used in the methods described herein can be, for example, a polypeptide, peptide, antibodiy or antibody fragment, non-peptide compound, carbohydrate, small molecule, lipid, single or double stranded DNA, single or double stranded RNA, antisense nucleic acid molecule, or ribozyme.
• all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Suitable methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.
• WRL-68 cells a human fetal hepatic cell-line, were cultured in EMEM medium with NaHCO3 (Statens Neterinarmedicinska -Anstalt), supplemented with 10% fetal bovine serum (FBS), 2% L-Glutamine, 1% Pyruvate and 1% ⁇ on essential amino acids ( ⁇ EA), all from GIBCO. The cells were sub-cultivated twice weekly by trypsination to maintain a cell density of approximately 80%.
• Transfection Cells were transfected with human full-length GH-receptor
• Nuclear extracts were prepared according to the methods described in Dignam et al. (1983) and Ausubel et al. (1993), with the following modifications: WRL-68 cells were scraped into ice cold PBS, pooled in 50 ml Falcon tubes and centrifuged 3000 rpm for 5 minutes.
• the cells were allowed to swell on ice for ten minutes and then homogenized in a Dounce homogenizer with a Teflon pestle type C. Cell lysis was confirmed in a microscope by adding trypan blue (0.4%, Sigma) to an aliquot of cells, since nuclei will stain blue in broken cells. Approximately 40 strokes were necessary to get 70-80% clean nuclear fractions. The nuclei were collected by centrifugation at 4000 ⁇ m for 15 minutes and immediately frozen in -70°C. The supernatant was also saved in -70°C as the cytosolic fraction. Nuclear and cytosolic fractions were prepared from hypophysectomized
• Sprague Dawley rat livers (ethical license Nl 76/02).
• the rats had minipumps implanted at 5 weeks of age for administration of hGH.
• Control animals were not stimulated and thus completely GH-deficient while GH-animals were stimulated continuously with hGH 0.12 mg/kg/day for five days.
• Animals were anaesthetized and livers were cut out and placed in ice-cold PBS. Livers were then cut into smaller pieces and transferred to 8 ml ice-cold hypotonic buffer with protease and phosphatase inhibitors and were allowed to swell on ice for 10 minutes.
• the small pieces of liver were homogenized in a Dounce homogenizer with Teflon pestle type C and the suspension was filtered through a sterile compress to get a cell-suspension.
• the cell- suspension was further homogenized in a Dounce homogenizer with a glass pestle.
• Cell lysis was confirmed in a microscope by adding trypan blue (0.4%), Sigma) to an aliquot of cells, since nuclei will stain blue in broken cells. Approximately 10-20 strokes were necessary to achieve 70-80%> clean nuclear fractions.
• the nuclei were collected by centrifugation at 4000 rpm and 4°C for 15 minutes. The supernatant was removed and saved as the cytosolic fraction. Pelleted nuclei and cytosol were immediately frozen in - 70°C.
• Protein concentrations were determined by using a BCA Protein Assay Reagent Kit (Pierce), which uses the Biuret reaction (reduction of Cu 2+ to Cu 1+ by proteins in an alkaline medium) and a reagent containing bicinchoninic acid (BCA). Two molecules of BCA form a complex with one Cu 1+ ion, which gives a purple color and has a strong absorbance at 562 nm. Bovine serum albumin (BSA) was used as standard.
• BSA Bovine serum albumin
• Protein G Sepharose (Amersham) was used to immunoprecipitate GHR and phospho-STAT5b from the total nuclear extracts. Protein G is immobilized on sepharose beads and binds to the F c region of IgG, leaving the F a region available for binding the antigen.
• 60 ⁇ l protein G Sepharose slurry was used (approximately 30 ⁇ l clean sepharose). The sepharose was pooled in one 2 ml Eppendorf tube and washed in 3x1 ml of phosphate buffer saline (PBS) by centrifuging 3000 rpm for 2 minutes.
• PBS phosphate buffer saline
• the antibody was added, 5 ⁇ l/sample of either anti-GHR (MAb 263, AGEN) or anti phos ⁇ hoSTAT5a/b, Y694/Y699 (Upstate Biotechnology).
• the sepharose with antibody was diluted with PBS and mixed end over end at room temperature for 2 hours. After that the sepharose slurry was divided in clean 2 ml Eppendorf tubes and washed in lxl ml PBS. Nuclear lysates were then thawed and added to the sepharose, 100-200 ⁇ l lysate from each sample.
• HRP Horse Radish Peroxidase
• Membranes were stripped from antibodies by incubation in stripping buffer (62.5 mM Tris pH 6.5, 2% SDS, 100 mM ⁇ - Mercaptoethanol) at 50°C for 30 minutes. They were washed for 2x10 minutes in large volumes of TBST and then blocked in blocking buffer (1% milk in TBST) at 4°C over night. Re-blotting could then be performed with another antibody as described above.
• stripping buffer 62.5 mM Tris pH 6.5, 2% SDS, 100 mM ⁇ - Mercaptoethanol
• Electrophoretic Mobility Shift Assay is a method used to investigate protein-DNA interactions.
• a DNA probe with a known sequence is end-labeled with 33 P-ATP and then incubated with nuclear extracts. Proteins that bind specifically to the DNA-probe will reduce the mobility of the complex when separated on a non-denaturing poly acryl-amid gel and a shifted band can be seen.
• an antibody can be added to the nuclear extract before incubation with the DNA-probe. If the antibody recognizes a protein that binds to the DNA, the mobility of the complex will be reduced even further and cause a supershift.
• Oligonucleotides were end-labeled with [ ⁇ - 33 P] ATP (2500 Ci/mmol, -Amersham or 3000 Ci/mmol, Perkin Elmer) by mixing 2 ⁇ l consensus oligonucleotide (1.75 pmol ⁇ l, Promega or SGS DNA), 1 ⁇ l T4 Polynucleotide Kinase lOx Buffer (Promega), 1 ⁇ l T4 Polynucleotide Kinase (Promega), 1 ⁇ l [ ⁇ - 33 P] ATP (Amersham or Perkin Elmer) and 5 ⁇ l nuclease free water (DEPC medium).
• [ ⁇ - 33 P] ATP 2500 Ci/mmol, -Amersham or 3000 Ci/mmol, Perkin Elmer
• the mixture was incubated at 37°C for 10 minutes and the reaction was stopped by adding 1 ⁇ l 0.5 M EDTA. The volume was adjusted to 100 ⁇ l by adding 89 ⁇ l 0.05 M EDTA. To remove unincorporated [ ⁇ - 33 P] ATP, the 100 ⁇ l aliquot was loaded on a NICKTM Column (Amersham Pharmacia Biotech AB) and labeled DNA was eluted with 2x400 ⁇ l 0.05 M EDTA. Specific activity was measured by mixing 2 ⁇ l 33 P-labeled sample with 200 ⁇ l scintillation fluid and counting was performed in a Beta counter (Triluxl450).
• GHRE 5'-TAC GCT TCT ACT AAT CCA TGT TCT GAG AAA TCA T-3' (SEQ ID NO:l) 3'-ATG CGA AGA TGA TTA GGT ACA AGA CTC TTT AGT A-5' (SEQ ID NO: 2)
• OCTl 5'-TGT CGAATG CAAATC ACT AGAA-3' (SEQIDNO: 3) 3'-ACA GCT TAC GTT TAG TGATCT T-5' (SEQ IDNO: 4)
• AP2 5'-GAT CGAACT GAC CGC CCG CGG CCC GT-3' (SEQIDNO: 5) 3'-CTA GCT TGA CTG GCG GGC GCC GGGCA-5' (SEQ ID NO: 6)
• API 5'-CGC TTGATGAGT CAGCCGGAA-3' (SEQIDNO: 7) 3'-GCG AAC TAC TCAGTC GGC CTT-5' (SEQ IDNO: 8)
• Pr2F linker with Rgfll/H dlll overhangs used in EMSA: 5'-GATCTAGATGCTTTCACAAACCCCACCCACAAA-3' (SEQ ID NO: 9) 5'-AGCTTTTGTGGGTGGGGTTTGTGAAAGCATCTA-3' (SEQ ID NO: 10)
• Linker containing two "Promega" AP2 sites, with KpnUmXhol overhangs for cloning to Luc- and SEAP -reporter vectors 5'-CGATCGAACTGACCGCCCGCGGCCCGTGATCGAACTGACCGCCCG CGGCCCGTC-3' (SEQ ID NO: 13) 5'-TCGAGACGGGCCGCGGGCGGTCAGTTCGATCACGGGCCGCGGGCG GTCAGTTCGATCGGTAC-3' (SEQ ID NO: 14)
• OCTl consensus oligonucleotides (Promega) for 20 minutes at room temperature.
• EMSA - Western Blot EMSA was performed as described above, but instead of drying the gel and expose it to a phosphor imager screen, the proteins in the gel were transferred to a nitrocellulose membrane and blotted with an anti-GHR antibody as in a regular Western Blot, as described. To confirm that the proteins were transferred to the membrane, the membrane was immersed in Ponceau S Solution (Sigma), which stains all proteins red.
• GHR and STAT5 are present in nuclear extracts Cultured cell lines usually exhibit low amounts of endogenously produced GHR
• Fig. 3C liver tissue contains high amounts of receptors
• Fig. 3D liver tissue contains high amounts of receptors
• Fig. 3C nuclear proteins were separated with gel electrophoresis and visualized in Western Blot.
• WRL-68 GHR-transfected WRL-68
• HX rat livers None of the bands appeared as a result of unspecific binding of the secondary antibody (pig anti-rabbit, Fig. 3B).
• the Western Blot membrane with non-transfected WRL-68 total nuclear extracts was re-blotted with a polyclonal antibody against STAT5, and two bands of 60 and 100 kDa, respectively, could be seen.
• the 100-kDa band probably represented intact STAT5 monomers, while the 60-kDa band could be a cleaved form of STAT5
• EXAMPLE 2 Transfection of WRL-68 cells with full-lenfith GH-receptor increase protein interactions with GHRE, AP2, OCTl and Pr2F DNA probes GH-receptor transfected and non-transfected WRL-68 nuclear extracts were incubated with anti-GHR antibody and 33 P-GHRE (SEQ ID NOS: 1 and 2), 33 P-AP2 (SEQ ID NOS: 5 and 6) or 33 P-OCTl (SEQ ID NOS: 3 and 4) or 33 P-Pr2F (SEQ ID NOS: 9 and 10) consensus oligo-nucleotides and analyzed in EMSA to elucidate any possible interactions of the GHR with proteins binding to these DNA probes.
• 33 P-GHRE SEQ ID NOS: 1 and 2
• 33 P-AP2 SEQ ID NOS: 5 and 6
• 33 P-OCTl SEQ ID NOS: 3 and 4
• 33 P-Pr2F SEQ ID NOS: 9 and
• the intensity of the top band was increased in extracts from GHR-transfected WRL-68 cells (Fig. IA, arrow), but in contrast to AP2, incubation of extracts with an anti-GHR antibody did not affect the binding of nuclear proteins to the OCTl D ⁇ A probe. No effects could be seen in nuclear extracts from hGH stimulated cells compared to un- stimulated control cells. Also in this case BNTA broke some protein-D ⁇ A interactions; the intensity of the top specific band was decreased in extracts from BVTA treated cells. As with GHRE and AP2, the antibody itself showed no interaction with the D ⁇ A probe.
• ⁇ on-transfected WRL-68 nuclear extracts were analyzed with API D ⁇ A probe (SEQ ID ⁇ OS: 7 and 8) as a control since API is activated via GH-activated MAP kinase.
• API D ⁇ A probe SEQ ID ⁇ OS: 7 and 8
• One shifted band could be seen in all fractions showing an activated API transcription factor, and no effects could be seen from either hGH or BNTA.
• Incubation with a GHR-antibody resulted in somewhat fainter bands.
• EMSA was performed with AP2 and OCTl probes together with nuclear extract from GHR-transfected WRL-68 cells. The separated complexes were transferred to a nitrocellulose membrane.
• EXAMPLE 3 Incubation of rat liver nuclear extracts with anti-GHR antibodies enhances protein binding to an AP2 DNA probe HX rat liver nuclear extracts were incubated with anti-GHR and 33 P-GHRE, 33 P-
• EMSA AP2 or 33 P-OCTl consensus oligonucleotides and analyzed in EMSA.
• nuclear extracts not incubated with anti-GHR were also analyzed. Extracts from two control animals and two hGH stimulated animals were used in the assays.
• EMSA with GHRE and OCTl DNA probes did not show any differences in protein-DNA binding either with hGH stimulation or with pre-incubation of nuclear extracts with an anti-GHR antibody.
• EMSA with an AP2 DNA probe showed an enhanced protein-DNA interaction when nuclear extracts were incubated with an anti-GHR antibody (Fig. 1C). As mentioned above, this effect was also seen with GHR-transfected WRL-68 cells and AP2 probe (Fig. IB).
• Hepatocyte nuclear factor l ⁇ activates promoter 1 of the human insulin-like growth factor 1 gene via two distinct binding sites.

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