WO1999000488A1 - Methods for screening nuclear transcription factors for the ability to modulate an estrogen response - Google Patents
Methods for screening nuclear transcription factors for the ability to modulate an estrogen response Download PDFInfo
- Publication number
- WO1999000488A1 WO1999000488A1 PCT/US1998/013089 US9813089W WO9900488A1 WO 1999000488 A1 WO1999000488 A1 WO 1999000488A1 US 9813089 W US9813089 W US 9813089W WO 9900488 A1 WO9900488 A1 WO 9900488A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- receptor
- cell
- transcription factor
- estrogen
- reporter gene
- Prior art date
Links
- 102000040945 Transcription factor Human genes 0.000 title claims abstract description 108
- 108091023040 Transcription factor Proteins 0.000 title claims abstract description 108
- 239000000262 estrogen Substances 0.000 title claims abstract description 95
- 229940011871 estrogen Drugs 0.000 title claims abstract description 95
- 238000000034 method Methods 0.000 title claims abstract description 75
- 238000012216 screening Methods 0.000 title claims abstract description 14
- 230000004044 response Effects 0.000 title description 36
- 108010018242 Transcription Factor AP-1 Proteins 0.000 claims abstract description 139
- 102000015694 estrogen receptors Human genes 0.000 claims abstract description 115
- 108010038795 estrogen receptors Proteins 0.000 claims abstract description 115
- 108700008625 Reporter Genes Proteins 0.000 claims abstract description 88
- 102000005962 receptors Human genes 0.000 claims abstract description 84
- 108020003175 receptors Proteins 0.000 claims abstract description 84
- 239000003446 ligand Substances 0.000 claims abstract description 77
- 230000014509 gene expression Effects 0.000 claims abstract description 66
- 230000004913 activation Effects 0.000 claims abstract description 43
- 230000001404 mediated effect Effects 0.000 claims abstract description 34
- 150000001875 compounds Chemical class 0.000 claims abstract description 26
- 230000001076 estrogenic effect Effects 0.000 claims abstract description 23
- 102100023132 Transcription factor Jun Human genes 0.000 claims description 166
- 108091027981 Response element Proteins 0.000 claims description 59
- 102000003676 Glucocorticoid Receptors Human genes 0.000 claims description 57
- 108090000079 Glucocorticoid Receptors Proteins 0.000 claims description 57
- 238000003556 assay Methods 0.000 claims description 39
- 239000003862 glucocorticoid Substances 0.000 claims description 39
- 101001050288 Homo sapiens Transcription factor Jun Proteins 0.000 claims description 30
- 239000000583 progesterone congener Substances 0.000 claims description 22
- RJKFOVLPORLFTN-LEKSSAKUSA-N Progesterone Chemical class C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 RJKFOVLPORLFTN-LEKSSAKUSA-N 0.000 claims description 21
- 102000003998 progesterone receptors Human genes 0.000 claims description 21
- 108090000468 progesterone receptors Proteins 0.000 claims description 21
- 239000003795 chemical substances by application Substances 0.000 claims description 12
- 108020004414 DNA Proteins 0.000 claims description 9
- 239000000463 material Substances 0.000 claims description 9
- 102000016978 Orphan receptors Human genes 0.000 claims description 7
- 108070000031 Orphan receptors Proteins 0.000 claims description 7
- 239000002395 mineralocorticoid Substances 0.000 claims description 7
- 229930003316 Vitamin D Natural products 0.000 claims description 5
- QYSXJUFSXHHAJI-XFEUOLMDSA-N Vitamin D3 Natural products C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C/C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-XFEUOLMDSA-N 0.000 claims description 5
- SHGAZHPCJJPHSC-YCNIQYBTSA-N all-trans-retinoic acid Chemical compound OC(=O)\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C SHGAZHPCJJPHSC-YCNIQYBTSA-N 0.000 claims description 5
- 229930002330 retinoic acid Natural products 0.000 claims description 5
- 229960001727 tretinoin Drugs 0.000 claims description 5
- 235000019166 vitamin D Nutrition 0.000 claims description 5
- 239000011710 vitamin D Substances 0.000 claims description 5
- 150000003710 vitamin D derivatives Chemical class 0.000 claims description 5
- 229940046008 vitamin d Drugs 0.000 claims description 5
- 239000003098 androgen Substances 0.000 claims description 4
- 102000001307 androgen receptors Human genes 0.000 claims description 4
- 108010080146 androgen receptors Proteins 0.000 claims description 4
- 150000003180 prostaglandins Chemical class 0.000 claims description 3
- 102000015433 Prostaglandin Receptors Human genes 0.000 claims 2
- 108010050183 Prostaglandin Receptors Proteins 0.000 claims 2
- 102100023118 Transcription factor JunD Human genes 0.000 abstract 3
- 210000004027 cell Anatomy 0.000 description 200
- 230000000694 effects Effects 0.000 description 49
- 150000003431 steroids Chemical class 0.000 description 45
- 108090000623 proteins and genes Proteins 0.000 description 42
- VOXZDWNPVJITMN-ZBRFXRBCSA-N 17β-estradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 VOXZDWNPVJITMN-ZBRFXRBCSA-N 0.000 description 36
- 229960005309 estradiol Drugs 0.000 description 35
- 229930182833 estradiol Natural products 0.000 description 34
- 238000011282 treatment Methods 0.000 description 33
- 238000013518 transcription Methods 0.000 description 32
- 230000035897 transcription Effects 0.000 description 32
- 238000002474 experimental method Methods 0.000 description 26
- NKANXQFJJICGDU-QPLCGJKRSA-N Tamoxifen Chemical compound C=1C=CC=CC=1C(/CC)=C(C=1C=CC(OCCN(C)C)=CC=1)/C1=CC=CC=C1 NKANXQFJJICGDU-QPLCGJKRSA-N 0.000 description 20
- 230000001965 increasing effect Effects 0.000 description 19
- 230000003993 interaction Effects 0.000 description 18
- 229940037128 systemic glucocorticoids Drugs 0.000 description 17
- 150000007523 nucleic acids Chemical class 0.000 description 15
- 230000037361 pathway Effects 0.000 description 15
- 230000000638 stimulation Effects 0.000 description 15
- 238000001514 detection method Methods 0.000 description 14
- 102000039446 nucleic acids Human genes 0.000 description 14
- 108020004707 nucleic acids Proteins 0.000 description 14
- PHEDXBVPIONUQT-RGYGYFBISA-N phorbol 13-acetate 12-myristate Chemical compound C([C@]1(O)C(=O)C(C)=C[C@H]1[C@@]1(O)[C@H](C)[C@H]2OC(=O)CCCCCCCCCCCCC)C(CO)=C[C@H]1[C@H]1[C@]2(OC(C)=O)C1(C)C PHEDXBVPIONUQT-RGYGYFBISA-N 0.000 description 14
- 108060005980 Collagenase Proteins 0.000 description 13
- 201000008752 progressive muscular atrophy Diseases 0.000 description 13
- 238000001890 transfection Methods 0.000 description 13
- 102000029816 Collagenase Human genes 0.000 description 12
- 229960002424 collagenase Drugs 0.000 description 12
- 230000003149 estradiol stimulation Effects 0.000 description 12
- 239000013604 expression vector Substances 0.000 description 12
- 102100027584 Protein c-Fos Human genes 0.000 description 10
- 108010071563 Proto-Oncogene Proteins c-fos Proteins 0.000 description 10
- 239000012190 activator Substances 0.000 description 10
- 239000008186 active pharmaceutical agent Substances 0.000 description 10
- 239000000556 agonist Substances 0.000 description 10
- 230000005764 inhibitory process Effects 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 10
- 230000001105 regulatory effect Effects 0.000 description 10
- 229960001603 tamoxifen Drugs 0.000 description 10
- 230000002103 transcriptional effect Effects 0.000 description 10
- 230000029279 positive regulation of transcription, DNA-dependent Effects 0.000 description 9
- 238000012360 testing method Methods 0.000 description 9
- 230000004568 DNA-binding Effects 0.000 description 8
- 108060001084 Luciferase Proteins 0.000 description 8
- 239000005089 Luciferase Substances 0.000 description 8
- 239000005557 antagonist Substances 0.000 description 8
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 8
- 230000006698 induction Effects 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 7
- 238000011002 quantification Methods 0.000 description 7
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 6
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 6
- 229960003957 dexamethasone Drugs 0.000 description 6
- 230000002267 hypothalamic effect Effects 0.000 description 6
- QFFCYTLOTYIJMR-XMGTWHOFSA-N promegestone Chemical compound C1CC2=CC(=O)CCC2=C2[C@@H]1[C@@H]1CC[C@@](C(=O)CC)(C)[C@@]1(C)CC2 QFFCYTLOTYIJMR-XMGTWHOFSA-N 0.000 description 6
- 102000005969 steroid hormone receptors Human genes 0.000 description 6
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 5
- 108010035563 Chloramphenicol O-acetyltransferase Proteins 0.000 description 5
- 206010028980 Neoplasm Diseases 0.000 description 5
- 108010085012 Steroid Receptors Proteins 0.000 description 5
- 102000005936 beta-Galactosidase Human genes 0.000 description 5
- 108010005774 beta-Galactosidase Proteins 0.000 description 5
- 230000027455 binding Effects 0.000 description 5
- 201000011510 cancer Diseases 0.000 description 5
- 210000002569 neuron Anatomy 0.000 description 5
- NMJREATYWWNIKX-UHFFFAOYSA-N GnRH Chemical compound C1CCC(C(=O)NCC(N)=O)N1C(=O)C(CC(C)C)NC(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)CNC(=O)C(NC(=O)C(CO)NC(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)C(CC=1NC=NC=1)NC(=O)C1NC(=O)CC1)CC1=CC=C(O)C=C1 NMJREATYWWNIKX-UHFFFAOYSA-N 0.000 description 4
- 101000857870 Squalus acanthias Gonadoliberin Proteins 0.000 description 4
- 230000001270 agonistic effect Effects 0.000 description 4
- 230000033228 biological regulation Effects 0.000 description 4
- 238000012761 co-transfection Methods 0.000 description 4
- 238000013537 high throughput screening Methods 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 210000002997 osteoclast Anatomy 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000000186 progesterone Substances 0.000 description 4
- 229960003387 progesterone Drugs 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- 206010006187 Breast cancer Diseases 0.000 description 3
- 208000026310 Breast neoplasm Diseases 0.000 description 3
- 108010040163 CREB-Binding Protein Proteins 0.000 description 3
- 102100021975 CREB-binding protein Human genes 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 3
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 3
- 102000007399 Nuclear hormone receptor Human genes 0.000 description 3
- 108020005497 Nuclear hormone receptor Proteins 0.000 description 3
- 208000001132 Osteoporosis Diseases 0.000 description 3
- 230000010632 Transcription Factor Activity Effects 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 229940046836 anti-estrogen Drugs 0.000 description 3
- 230000001833 anti-estrogenic effect Effects 0.000 description 3
- 230000002238 attenuated effect Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 230000002860 competitive effect Effects 0.000 description 3
- 230000037011 constitutive activity Effects 0.000 description 3
- 239000000328 estrogen antagonist Substances 0.000 description 3
- 230000035558 fertility Effects 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 239000005090 green fluorescent protein Substances 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 108020004999 messenger RNA Proteins 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 210000004940 nucleus Anatomy 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 238000003259 recombinant expression Methods 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 210000004291 uterus Anatomy 0.000 description 3
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 2
- 206010001197 Adenocarcinoma of the cervix Diseases 0.000 description 2
- 208000034246 Adenocarcinoma of the cervix uteri Diseases 0.000 description 2
- 102100038415 ELKS/Rab6-interacting/CAST family member 1 Human genes 0.000 description 2
- 102100035183 ERC protein 2 Human genes 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 101001100208 Homo sapiens ELKS/Rab6-interacting/CAST family member 1 Proteins 0.000 description 2
- 101000876444 Homo sapiens ERC protein 2 Proteins 0.000 description 2
- 101000904173 Homo sapiens Progonadoliberin-1 Proteins 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 102100024028 Progonadoliberin-1 Human genes 0.000 description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 description 2
- 101710159283 Vitellogenin-A2 Proteins 0.000 description 2
- VREFGVBLTWBCJP-UHFFFAOYSA-N alprazolam Chemical compound C12=CC(Cl)=CC=C2N2C(C)=NN=C2CN=C1C1=CC=CC=C1 VREFGVBLTWBCJP-UHFFFAOYSA-N 0.000 description 2
- 230000004075 alteration Effects 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 208000007502 anemia Diseases 0.000 description 2
- 230000003042 antagnostic effect Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000002981 blocking agent Substances 0.000 description 2
- 210000000988 bone and bone Anatomy 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 201000006662 cervical adenocarcinoma Diseases 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 108700010039 chimeric receptor Proteins 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000003292 diminished effect Effects 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- -1 mineralcorticoids Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 230000001537 neural effect Effects 0.000 description 2
- 108020004017 nuclear receptors Proteins 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 229940094443 oxytocics prostaglandins Drugs 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 230000001991 pathophysiological effect Effects 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 238000012827 research and development Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000012289 standard assay Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 230000007704 transition Effects 0.000 description 2
- 102000009310 vitamin D receptors Human genes 0.000 description 2
- 108050000156 vitamin D receptors Proteins 0.000 description 2
- 101150116358 API gene Proteins 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 208000009304 Acute Kidney Injury Diseases 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 206010003694 Atrophy Diseases 0.000 description 1
- 108090000363 Bacterial Luciferases Proteins 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- OMFXVFTZEKFJBZ-UHFFFAOYSA-N Corticosterone Natural products O=C1CCC2(C)C3C(O)CC(C)(C(CC4)C(=O)CO)C4C3CCC2=C1 OMFXVFTZEKFJBZ-UHFFFAOYSA-N 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 108700010895 Drosophila ADH Proteins 0.000 description 1
- 206010014733 Endometrial cancer Diseases 0.000 description 1
- 206010014759 Endometrial neoplasm Diseases 0.000 description 1
- 102220572626 Estrogen receptor_G400V_mutation Human genes 0.000 description 1
- 108090000331 Firefly luciferases Proteins 0.000 description 1
- 101000609762 Gallus gallus Ovalbumin Proteins 0.000 description 1
- 108010026132 Gelatinases Proteins 0.000 description 1
- 102000013382 Gelatinases Human genes 0.000 description 1
- 206010018366 Glomerulonephritis acute Diseases 0.000 description 1
- 229940123127 Glucocorticoid agonist Drugs 0.000 description 1
- 108010068250 Herpes Simplex Virus Protein Vmw65 Proteins 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 101000882584 Homo sapiens Estrogen receptor Proteins 0.000 description 1
- 101000926939 Homo sapiens Glucocorticoid receptor Proteins 0.000 description 1
- 101001071608 Homo sapiens Glutathione reductase, mitochondrial Proteins 0.000 description 1
- 101150088952 IGF1 gene Proteins 0.000 description 1
- 101150021395 JUND gene Proteins 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 102000004318 Matrilysin Human genes 0.000 description 1
- 108090000855 Matrilysin Proteins 0.000 description 1
- 102000000422 Matrix Metalloproteinase 3 Human genes 0.000 description 1
- 108010006035 Metalloproteases Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108010085220 Multiprotein Complexes Proteins 0.000 description 1
- 102000007474 Multiprotein Complexes Human genes 0.000 description 1
- 108090000189 Neuropeptides Proteins 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 101150023107 PS2 gene Proteins 0.000 description 1
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 description 1
- 229920012196 Polyoxymethylene Copolymer Polymers 0.000 description 1
- 108010069820 Pro-Opiomelanocortin Proteins 0.000 description 1
- 102100027467 Pro-opiomelanocortin Human genes 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 208000033626 Renal failure acute Diseases 0.000 description 1
- 241000714474 Rous sarcoma virus Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 102220497176 Small vasohibin-binding protein_T47D_mutation Human genes 0.000 description 1
- 206010047115 Vasculitis Diseases 0.000 description 1
- 101710159308 Vitellogenin-B2 Proteins 0.000 description 1
- 241000269370 Xenopus <genus> Species 0.000 description 1
- VRGWBRLULZUWAJ-XFFXIZSCSA-N [(2s)-2-[(1r,3z,5s,8z,12z,15s)-5,17-dihydroxy-4,8,12,15-tetramethyl-16-oxo-18-bicyclo[13.3.0]octadeca-3,8,12,17-tetraenyl]propyl] acetate Chemical compound C1\C=C(C)/CC\C=C(C)/CC[C@H](O)\C(C)=C/C[C@@H]2C([C@@H](COC(C)=O)C)=C(O)C(=O)[C@]21C VRGWBRLULZUWAJ-XFFXIZSCSA-N 0.000 description 1
- 231100000851 acute glomerulonephritis Toxicity 0.000 description 1
- 201000011040 acute kidney failure Diseases 0.000 description 1
- 208000012998 acute renal failure Diseases 0.000 description 1
- 210000004727 amygdala Anatomy 0.000 description 1
- 229940030486 androgens Drugs 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 210000003295 arcuate nucleus Anatomy 0.000 description 1
- 238000003491 array Methods 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 230000037444 atrophy Effects 0.000 description 1
- 235000021028 berry Nutrition 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 201000008275 breast carcinoma Diseases 0.000 description 1
- 238000000423 cell based assay Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
- 239000003610 charcoal Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 1
- 230000008045 co-localization Effects 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- OMFXVFTZEKFJBZ-HJTSIMOOSA-N corticosterone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@H](CC4)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 OMFXVFTZEKFJBZ-HJTSIMOOSA-N 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 201000003914 endometrial carcinoma Diseases 0.000 description 1
- 230000002357 endometrial effect Effects 0.000 description 1
- 210000004696 endometrium Anatomy 0.000 description 1
- 208000023965 endometrium neoplasm Diseases 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 239000003687 estradiol congener Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- VRGWBRLULZUWAJ-UHFFFAOYSA-N fusaproliferin Natural products C1C=C(C)CCC=C(C)CCC(O)C(C)=CCC2C(C(COC(C)=O)C)=C(O)C(=O)C21C VRGWBRLULZUWAJ-UHFFFAOYSA-N 0.000 description 1
- 230000002518 glial effect Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000012203 high throughput assay Methods 0.000 description 1
- 210000001320 hippocampus Anatomy 0.000 description 1
- 230000004179 hypothalamic–pituitary–adrenal axis Effects 0.000 description 1
- 210000003016 hypothalamus Anatomy 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000013383 initial experiment Methods 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 108020001756 ligand binding domains Proteins 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- FRQMUZJSZHZSGN-HBNHAYAOSA-N medroxyprogesterone Chemical compound C([C@@]12C)CC(=O)C=C1[C@@H](C)C[C@@H]1[C@@H]2CC[C@]2(C)[C@@](O)(C(C)=O)CC[C@H]21 FRQMUZJSZHZSGN-HBNHAYAOSA-N 0.000 description 1
- 229960002985 medroxyprogesterone acetate Drugs 0.000 description 1
- 229960004296 megestrol acetate Drugs 0.000 description 1
- RQZAXGRLVPAYTJ-GQFGMJRRSA-N megestrol acetate Chemical compound C1=C(C)C2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@@](C(C)=O)(OC(=O)C)[C@@]1(C)CC2 RQZAXGRLVPAYTJ-GQFGMJRRSA-N 0.000 description 1
- 230000009245 menopause Effects 0.000 description 1
- 230000002175 menstrual effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 230000014511 neuron projection development Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000011599 ovarian follicle development Effects 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 210000002963 paraventricular hypothalamic nucleus Anatomy 0.000 description 1
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 1
- 239000002644 phorbol ester Substances 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 210000001176 projection neuron Anatomy 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 229930185346 proliferin Natural products 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000026267 regulation of growth Effects 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 238000003571 reporter gene assay Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000003938 response to stress Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 102000003702 retinoic acid receptors Human genes 0.000 description 1
- 108090000064 retinoic acid receptors Proteins 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 108020003113 steroid hormone receptors Proteins 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 108091007196 stromelysin Proteins 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 102000004217 thyroid hormone receptors Human genes 0.000 description 1
- 108090000721 thyroid hormone receptors Proteins 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 230000002463 transducing effect Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000011830 transgenic mouse model Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 230000005751 tumor progression Effects 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 230000001836 utereotrophic effect Effects 0.000 description 1
- 230000001457 vasomotor Effects 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
-
- 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
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
-
- 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
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
-
- 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
- G01N33/6875—Nucleoproteins
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
- G01N2333/723—Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
-
- 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
- This invention relates to the field of signaling and induction of transcription by nuclear transcription factor ligands.
- this invention pertains to the modulation of estrogen activity at the API site by nuclear transcription factor ligands.
- agonistic activity may have beneficial effects, such as preventing osteoporosis and reducing serum cholesterol.
- beneficial effects such as preventing osteoporosis and reducing serum cholesterol.
- agonist activity may also be harmful.
- tamoxifen an estrogen in certain contexts, sometimes increases endometrial tumor incidence (lino et al. (1991) Cancer Treat. & Res. 53: 228-237) or switches from inhibition to stimulation of estrogen dependent growth in breast tumor progression (Parker, M.G.
- Estrogen action is opposed by progestins and glucocorticoids in several physiologic and pathophysiologic processes. For example, estrogen stimulates uterine growth and DNA synthesis, while glucocorticoids block these uterotrophic effects (Bibsby (1993) J. Steroid
- estrogen treatment is associated with increased levels of circulating corticosterone (Burgess et al. (1992) Endocrinol., 131 : 1261-1269), whereas glucocorticoids down-regulate hypothalamic pituitary-adrenal axis activation to reduce circulating glucocorticoid levels.
- Estrogen treatment is also associated with lesion-induced neuronal sprouting in vivo (Morse et al. (1992) Exptl. Neurol, 118: 47-
- glucocorticoids in excess are associated with dendritic atrophy and cell death in pyramidal neurons of the hippocampus (Sapolsky et al. (1990) J. Neurosci., 10: 2897-2902).
- estrogen blocks osteoclast development and activity (Oursler et al. (1994) Proc. Natl. Acad. Set, USA, 91: 5227-5231).
- the glucocorticoid agonist dexamethasone (Dex) induces osteoclast formation (Shuto et al. (1994) Endocrinol, 134: 1121-1126).
- estrogen promotes growth, while glucocorticoids inhibit it (Zhou et al. (1989) Mol Cell. Endocrinol, 66: 189-197).
- Ligands for nuclear transcription factors are used in the treatment of a wide variety of pathological conditions.
- antiestrogens e.g., tamoxifen
- estrogens are used in the treatment of osteoporosis
- progestins and estrogens are used in the regulation of fertility
- glucocorticoids are used in the treatment of certain anemias.
- nuclear transcription factor ligands vary in their ability to interact with estrogen pathway, and with their activity, depending on physiological state of the organism, tissue or cell location, and other poorly understood factors. It is thus desirable to identify or clarify the modes of activity of known nuclear transcription factor ligands, and agonistic or antagonistic analogues of such ligands. It is also desirable to identify new transcription factors ligands whose activity is better understood, and/or shows less interaction with other transcription factors, and/or is more predictable in mode of action.
- the prior art fails to provide methods for quickly and easily testing nuclear transcription factor ligands or ligand analogues for the specificity of their activity and/or their interaction with estrogen activation of a target gene. This invention addresses this and other problems in the art.
- the present invention provides methods for screening nuclear transcription factor ligands (e.g, progestins, glucocorticoids, retinoic acid, vitamin D, prostaglandins, mineralcorticoids, and their analogues) for the ability to modulate estrogen activation at an AP-1 site.
- the method includes the steps of: a) providing a first cell containing an estrogen receptor, a receptor for the nuclear transcription factor ligand, and a promoter comprising an API site which regulates expression of a first reporter gene; b) contacting the first cell with the transcription factor ligand and with a compound having API mediated estrogenic activity; and c) detecting expression of the first reporter gene.
- the method can entail detection of the transcription factor ligand's ability to activate a gene under control of an estrogen response element (ERE).
- the method may further include the steps of: d) providing a second cell containing an estrogen receptor, a receptor for the nuclear transcription factor ligand (e.g., its cognate receptor), and a promoter comprising an estrogen response element (ERE) that regulates expression of a second reporter gene; e) contacting the second cell with the transcription factor and with the compound having AP-1 mediated estrogenic activity; and f) detecting expression of the second reporter gene.
- steps d) through e) can be performed as a completely independent assay.
- steps d) through e) may still be performed using a different cell.
- the assay would entail the use of two different cell types; one cell containing the first reporter gene construct and a second cell containing the second reporter gene construct.
- both constructs can be contained within a single cell.
- the methods may also entail detection of the transcription factor ligand's ability to activate a gene under control of its own response element (e.g., in the presence of a compound having estrogenic activity).
- the method may further include the steps of: d) providing a second cell containing a cognate receptor of the transcription factor ligand, and a promoter comprising an response element for the cognate receptor that regulates expression of a second reporter gene; e) contacting the second cell with the transcription factor ligand and with the compound having AP-1 mediated estrogenic activity; and f) detecting expression of the second reporter gene.
- steps d) through e) can be performed as a completely independent assay.
- steps d) through e) may still be performed using a different cell.
- the assay would entail the use of two different cell types; one cell containing the first reporter gene construct and a second cell containing the second reporter gene construct.
- both constructs can be contained within a single cell.
- Preferred cells include one or more AP-1 proteins (e.g., jun or fos).
- the proteins can be endogenous to the cell, the cell can be recombinantly engineered to express the protein(s) (e.g., transfected with an expression cassette capable of expressing an AP-1 protein), or exogenous AP-1 proteins can be supplied to the cell.
- the ER receptor can be endogenous to the cell, and/or the cell can be recombinantly engineered to express an ER receptor (e.g., transfected with an expression cassette that expresses an ER receptor).
- the ER receptor can be a native ER receptor or a modified ER receptor as described herein.
- the transcription factor ligand can be virtually any nuclear transcription factor ligand as long as the cell contains a cognate receptor for that ligand. It will be appreciated that the transcription factor is a factor ligand other than the compound having AP-1 mediated estrogenic activity so that the cell is in effect contacted with two different transcription factor ligands, the transcription factor ligand and the compound having AP-1 mediated estrogenic activity. As with the ER receptor, the transcription factor (e.g., the cognate receptor of the transcription factor ligand) can be one that is endogenously expressed by the cell and/or the cell can be recombinantly engineered to express the receptor.
- the transcription factor e.g., the cognate receptor of the transcription factor ligand
- Preferred transcription factor ligands and their cognate receptors include, but are not limited to a glucocorticoid and a glucocorticoid receptor (GR), a protestin (eg., progesterone) and a progestin receptor, retinoic acid and a retinoic acid receptor, vitamin D, and a vitamin D receptor, an androgen and an androgen receptor, a mineralcorticoid and a mineralcorticoid receptor, and so forth.
- GR glucocorticoid and a glucocorticoid receptor
- protestin eg., progesterone
- progestin receptor eg., prog., progesterone
- retinoic acid and a retinoic acid receptor retinoic acid receptor
- vitamin D and a vitamin D receptor
- an androgen and an androgen receptor a mineralcorticoid and a mineralcorticoid receptor
- the reporter gene is a luciferase, a green fluorescent protein (GFP) or a ⁇ -galactosidase.
- Preferred promoter/reporter gene combinations include, but are not limited to, any of the promoter/reporter gene combinations described herein. It will be appreciated that where the first and second reporter gene are located in different cells, both reporter genes can be the same type of reporter gene. Conversely, where both reporter genes are located in the same cell, the reporter genes are preferably different reporter genes (e.g., providing different distinguishable signals).
- Suitable cells include, but are not limited to HeLa cells, MCF-7 cells
- MDA453 cells ZR-75-1, ERC1 cells, ERC2 cells, or ERC3.
- this invention provides methods of screening an agent (e.g., a test compound that is to be screened for agonistic or antagonistic transcription factor activity (e.g., a steroid or steroid analogue, or steroid inhibitor)) for the ability to alter modulation of estrogen activation at an AP-1 site by a nuclear transcription factor.
- agents e.g., a test compound that is to be screened for agonistic or antagonistic transcription factor activity (e.g., a steroid or steroid analogue, or steroid inhibitor)) for the ability to alter modulation of estrogen activation at an AP-1 site by a nuclear transcription factor.
- kits for the practice of the methods described herein include a first cell containing an estrogen receptor, a receptor for a nuclear transcription factor, and a promoter comprising an AP-1 site that regulates expression of the first reporter gene.
- the kits also preferably include instructional materials detailing protocols for the practice of the assay methods described herein.
- an antiestrogen is a compound that substantially inhibits estrogen activity as measured in a standard assay for estrogenic activity, for example, cellular assays as described in Webb et al. Mol. Endocrinol, 6:157-167 (1993). More generally, a "transcription factor antagonist” is a compound that substantially inhibits transcription factor activity as measured in a standard assay for that transcription factor activity.
- a "nuclear transcription factor” as used herein refers to members of the nuclear transcription factor superfamily. This is a family of receptors that are capable of entering the nucleus of a cell and once there, effecting the up-regulation or down-regulation of one or more genes.
- nuclear transcription factor ligand is a compound that binds to a nuclear transcription factor.
- Preferred nuclear transcription factors are typically steroids, however, the group is not so limited.
- Nuclear transcription factor ligands include, but are not limited to estrogen, progestins, androgens, mineralcorticoids, glucocorticoids, retinoic acid, vitamin D, and prostaglandins.
- Transcription factor ligands also include analogues of naturally occurring factors and blocking agents (antagonists) of such factors.
- Transcription factors also include, as they are identified, the ligands that bind orphan receptors (those nuclear transcription factors which have been identified by sequence homology, but whose ligand is yet unidentified).
- the nuclear transcription factor ligand when used in the context of a modulator of estrogen activity, is typically one other than estrogen (or other than the estrogen or estrogen agonist whose activity is being modulated). Nuclear transcription factors typically mediate their activity through binding of a cognate receptor in the cell nucleus.
- the term cognate receptor refers to a receptor of the type that is typically bound by the transcription ligand in question.
- the cognate receptor for an estrogen is an estrogen receptor
- the cognate receptor for a glucocorticoid is a glucocorticoid receptor
- the receptor for a progestin is a progestin receptor
- the cognate receptor includes the native (naturally occurring) form as well as modified receptors.
- modulate estrogen activation or “modulation of estrogen activation” refer to alteration of the estrogen induced expression of a particular gene. Where the phrase additionally recites “at an AP-1 site or at an ERE” the phrase refers to alteration of the level of estrogen induced expression of one or more genes under control of the AP-1 site or ERE site respectively.
- detecting expression when used with reference to a reporter gene refers to detection of presence or absence of expression of the reporter gene or to quantification of expression level of the reporter gene. The quantification can be either an absolute measurement or a relative measurement (e.g., in comparison to another expressed gene).
- operably linked refers to functional linkage between a nucleic acid expression control sequence (such as a promoter, signal sequence, or transcription factor binding site) and a second nucleic acid sequence, wherein the expression control sequence affects transcription and/or translation of the nucleic acid corresponding to the second sequence.
- a nucleic acid expression control sequence such as a promoter, signal sequence, or transcription factor binding site
- a second nucleic acid sequence wherein the expression control sequence affects transcription and/or translation of the nucleic acid corresponding to the second sequence.
- recombinant when used with reference to a cell indicates that the cell replicates a heterologous nucleic acid, or expresses a peptide or protein encoded by a heterologous nucleic acid. Recombinant cells can express genes that are not found within the native (non-recombinant) form of the cell.
- Recombinant cells can also express genes found in the native form of the cell wherein the genes are modified and re-introduced into the cell by artificial means.
- Recombinant expression refers to the expression of the heterologous nucleic acid by such a recombinant cell.
- heterologous sequence or a “heterologous nucleic acid”, as used herein, is one that originates from a foreign source (or species) or, if from the same source, is modified from its original form.
- a heterologous nucleic acid operably linked to a promoter is from a source different from that from which the promoter was derived, or, if from the same source, is modified from its original form. Modification of the heterologous sequence may occur, e.g., by treating the DNA with a restriction enzyme to generate a DNA fragment that is capable of being operably linked to the promoter. Techniques such as site- directed mutagenesis are also useful for modifying a heterologous sequence.
- a “recombinant expression cassette” or simply an “expression cassette” is a nucleic acid construct, generated recombinantly or synthetically, with nucleic acid elements that are capable of effecting expression of a structural gene in hosts compatible with such sequences.
- Expression cassettes include at least promoters and optionally, transcription termination signals.
- the recombinant expression cassette includes a nucleic acid to be transcribed (e.g., a nucleic acid encoding a desired polypeptide), and a promoter. Additional factors necessary or helpful in effecting expression may also be used as described herein.
- an expression cassette can also include nucleotide sequences that encode a signal sequence that directs secretion of an expressed protein from the host cell.
- AP-1 mediated estrogenic/agonist activity refers to activation of a gene under the control of an AP-1 site (also referred to as an AP-1 response element) mediated by the interaction of a nuclear transcription factor with the AP-1 site.
- an AP-1 site also referred to as an AP-1 response element
- the pathway is referred to as the indirect estrogen response (in contrast to the classical estrogen response which is mediated through an ERE).
- a general description of the API site is found in Angel & Kann, Biochem. Biophys. Ada., 1072: 129-157 (1991) and Angel, et al, Cell, 49: 729-739 (1987).
- a "compound having AP-1 mediated estrogenic activity” refers to a compound that, when present in a cell containing a gene under control of an AP-1 site and
- AP-1 proteins activates transcription of the gene under control of the AP-1 site.
- Figs. 1A , IB, and 1C illustrate that estradiol and the glucocorticoid dexamethasone modulate each others transcriptional properties at the AP-1 response element.
- Fig. 1A shows the structure of collagenase reporter and steroid receptor vectors used.
- Fig. IB shows HeLa cells co-transfected with the Coll73-LUC reporter gene (5 ⁇ g) and the human ER expression vector, pHEO (5 ⁇ g) and treated were treated with vehicle, Dex, estradiol, or Dex+Estradiol (10" ⁇ M, each steroid) for approximately 40 hrs and then assayed for luciferase activity. The data are from three experiments.
- FIG. 1C shows that three point mutations in the AP-1 site of the collagenase promoter markedly attenuated steroid effects on transcriptional activation.
- HeLa cells were transfected with 5 ⁇ g of either the intact (Coll517-CAT) or mutated (Coll517mAP-l-CAT) collagenase reporter genes along with GR (l ⁇ g) and ER (3 ⁇ g) expression vectors. Data are from two experiments. Columns represent the average. (B&C) Error bars represent the standard deviation.
- Figs. 2A, 2B, 2C, and 2D illustrate ER and GR competition at the AP-1 site.
- HeLa cells were transfected with the Coll73-LUC reporter gene (5 ⁇ g) and the expression vectors illustrated in Fig. 1A as follows:
- Fig. 2A shows GR (l ⁇ g) and increasing amounts of ER as indicated. Columns represent an average of three treatment points from one experiment.
- Fig.2B shows the average of three experiments expressed as fold induction.
- Fig. 2C shows the results of cells transfected with ER (10 ⁇ g) and increasing amounts of GR. Columns represent the average of three treatment points.
- Fig. 2D shows the average of two experiments not including the experiment shown in Fig. 2C.
- Figures 2A through 2D the error bars represent standard deviation. (RLU) Relative light units.
- Figs. 3A and 3B show that Dex inhibits both Tamoxifen stimulation and the constitutive activity of the ER deleted of the ligand binding domain (HE15).
- HeLa cells were transfected with the Coll73-LUC reporter gene as in Fig. 1 A and IB.
- Cells were transfected with GR (l ⁇ g) and increasing amounts of HE0. They were treated with vehicle, Dex, tamoxifen (5xlO" ⁇ M), or Dex+tamoxifen.
- Fig. 3 A the columns represent the average of three treatment points.
- Fig. 3B shows the results of cells transfected with increasing amounts of HE15 and treated with vehicle or Dex.
- Fig. 4 shows that Dex inhibits the ER deleted of its DNA binding domain.
- HeLa cells were transfected with the Coll73-LUC reporter gene as in Fig. 1 and expression vectors as follows: Empty expression vector (pSG5; 5 ⁇ g), ER (HEO; 5 ⁇ g), and the ER deleted of its DNA binding domain (HE11 ; 3 and 5 ⁇ g). Cells were treated with steroids as in Fig. 1. Columns represent an average of three treatment points, error bars the standard deviation. The data are representative of similar experiments performed 3 times or more.
- Figs 5A and 5B show that co-transfected c-Jun potentiates steroid effects; and co-transfected c-Fos further potentiates c-Jun effects on estradiol stimulation.
- HeLa cells were transfected with the Coll73-LUC reporter gene and treated with steroids as in Fig. 1.
- Cells were co-transfected with ER and GR expression vectors (1 ⁇ g each) and increasing amounts of c-Jun (Fig. 5 A) or c-Fos (Fig. 5B). All columns and error bars represent the average of three treatment points except in (B) in which the c-Jun and c-Jun+c-Fos data represent one transfection with one treatment point each. Error bars represent standard deviation.
- Figs. 6A and 6B show that PMA and TNF- ⁇ differentially alter steroid responses at Coll73.HeLa cells were transfected with the Coll73-LUC reporter gene as in Fig. 1. They were co-transfected with ER (5 ⁇ g) and GR (l ⁇ g) and treated with steroids as in Fig. 1 in the presence or absence of PMA (Fig. 6 A) or TNF- ( Fig. 6B) at the doses indicated.
- Fig. 6A Note: the scale for PMA treated cells is lOx that of cells not treated with PMA ("No PMA"). Columns represent an average of three treatment points, error bars represent standard deviation. The data are representative of similar experiments performed 3 times or less.
- B Columns represent an average of three experiments. (A&B) error bars represent standard deviation.
- Fig. 7 illustrates that Dex inhibits estradiol stimulation of transcription through the AP-1 response element in a hypothalamic cell line.
- GTl-1 cells were transfected with ColALuc (5 ⁇ g), HEO (5 ⁇ g), GR (l ⁇ g), and c-Jun (3 ⁇ g). Cells were treated with steroids 4 hrs. after transfection and harvested 36 hrs. later. The data are expressed as per cent estradiol stimulation. Columns represent the average of three experiments. Error bars represent the standard deviation.
- Fig. 8A and 8B show that estradiol and the progestin RU5020 modulate each others transcriptional properties at the AP-1 response element.
- FIG. 8 A HeLa cells were transfected with ColALuc (5 ⁇ g), ER (l ⁇ g), PR-A or PR-B (l ⁇ g), and c-Jun (3 ⁇ g). Data are from four separate transfections from three experiments for PR-A and from two transfections from two experiments for PR-B. Columns represent the average fold induction.
- FIG. 8B CV-1 cells were transfected with ColALuc (5 ⁇ g), ER (HEO, 0.5 ⁇ g), PR-A (l ⁇ g) and c-Jun (3 ⁇ g). Data is from one experiment. Columns represent the average of two treatment points. Similar experiments have been repeated 3 times or less.
- FIGs. 8A and 8B Cells were treated with steroids immediately after transfection and harvested 40 hrs later. Error bars represent standard deviation.
- Nuclear transcription factor ligands are used in the treatment of a wide variety of pathological conditions.
- estrogens are used in the treatment of osteoporosis and other aspects (e.g., vasomotor instability) of menopause, in the treatment of hypoestrogenism, and in the regulation of fertility.
- Glucocorticoids are used in the treatment of pure red cell anemia, acute renal failure due to acute glomerulonephritis or vasculitis, lyrnphocytic leukemias, lymphomas, and other conditions.
- Progestins or progestational agents such as medroxyprogesterone or megestrol acetate are used in the treatment of endometrial carcinoma and breast carcinoma, and are used in the regulation of fertility.
- transcription factor ligands are complex and vary with physiological context.
- agonistic activity of the transcription factor ligand, ligand analogue agonist, or putative blocking agent (antagonist) may be mediated through a variety of pathways.
- antiestrogens can exhibit agonist estrogenic activity mediated through an AP-1 response element (see, e.g., Webb, et al (1995) Mol. Endo., 9: 443-456).
- the transcription factor ligands are known to interact.
- progestins and glucocorticoids are known to oppose estrogen activity in various physiologic and pathophysiologic processes.
- nuclear transcription factor ligand when contemplating use of a nuclear transcription factor ligand to evaluate the activity of that ligand on estrogen activated transcription.
- This permits the identification of transcription factor agonists or antagonists that either act on or act independently of the estrogen response.
- This facilitates the design and administration of therapeutics whose activity is well characterized in the physiological context in which they are to be administered.
- liganded nuclear transcription factors can interact with estrogen activation at the level of transcription and this activity is detectable through AP-1 mediated transcription.
- This invention therefore provides assays (methods of screening) nuclear transcription factor ligands, and putative or known transcription factor ligand agonists or antagonists for the ability to modulate estrogen activation at an AP-1 site.
- the methods involve providing a cell containing an estrogen receptor, a receptor for the nuclear transcription factor (e.g., glucocorticoid (GR) receptor), and a promoter comprising an AP-1 site operably linked to a reporter gene.
- the cell is then contacted with a compound having estrogenic activity (e.g., ⁇ -estradiol) and with the nuclear transcription factor ligand, transcription factor ligand agonist or antagonist, (test compound that is to be screened) and the effect on estrogen mediated activation of transcription through the AP-1 site is evaluated.
- the level of AP-1 mediated transcription is determined by detecting presence, absence, or level of expression of the reporter gene.
- the level of expression of the reporter gene from the assay is compared to the level of expression from a control that either lacks the transcription factor ligand (test compound) and/or to a control that contains the test compound in a known different concentration.
- the control can be performed using the same cell before or after the test assay. However, in a preferred embodiment, the control is performed using a different cell and is most preferably performed simultaneously with the test assay.
- this invention provides assays (methods of screening) nuclear transcription factor ligands, transcription factor ligand analogues, and potential transcription factor antagonists for the ability to modulate estrogen activation at an estrogen response element (ERE).
- the method is identical to the screen for activation at an AP-1 site, however in this embodiment, the cell contains a promoter comprising an estrogen response element (ERE) site operably linked to a reporter gene instead of the AP-1/reporter gene construct.
- the assay is conducted in exactly the same manner.
- Detection of the presence, absence, or quantification of the reporter gene product provides a measure of the estrogen-mediated activation of transcription through an ERE (classical pathway) and of the effect of the nuclear transcription factor ligand on such estrogen mediated activation. It will be appreciated that these assays allow one to determine the extent to which a given nuclear transcription factor ligand (e.g., a progestin, glucocorticoid, or other steroid) maintains its ability to block or agonize estrogen activation at an AP-1 site or at an ERE.
- a given nuclear transcription factor ligand e.g., a progestin, glucocorticoid, or other steroid
- estrogen agonists By varying the estrogen agonists in these assays, it is possible to identify those estrogens (or estrogen analogues) that can or cannot be inhibited or agonized by a nuclear transcription factor ligand at an AP-1 site, at an ERE, or at both sites.
- the two assays described above can be used in conjunction with each other to assay activation through both the "indirect pathway (at an AP-1 site) and the direct or classical pathway (at an ERE).
- a combined assay preferably utilizes two different cell types; one cell type containing the AP-1/reporter gene construct and another cell type containing the ERE/reporter gene construct.
- different reporter genes can also be used. In this case, both assay systems may be performed in one cell.
- a single cell type can be used containing both an API/reporter gene construct (e.g., Coll73-LUC or C0II6O-LUC) and an ERE/reporter gene construct (e.g., EREcoll ⁇ OCAT and EREcoll73CAT, see, Webb, et al (1995) Mol. Endo., 9: 443-456, and USSN 08/410,807).
- an API/reporter gene construct e.g., Coll73-LUC or C0II6O-LUC
- ERE/reporter gene construct e.g., EREcoll ⁇ OCAT and EREcoll73CAT, see, Webb, et al (1995) Mol. Endo., 9: 443-456, and USSN 08/410,807.
- this invention provides methods of screening for agents that inhibit the modulation of estrogen activation at an AP-1 or ERE site by a nuclear transcription factor ligand.
- the assay is performed as described above, with the addition of the agent that is to be screened.
- the effect of the agent on transcription factor ligand modulation of estrogen activation at the AP- 1 or ERE is then determined by detecting the presence, absence, or level of expression of the reporter regulated by the AP-1 site, the reporter gene regulated by the ERE, or both.
- Appropriate controls in this case may include the same assay with all components except the agent being screened and/or the same assay with all components including the agent being screened at a known concentration different from that in the test assay.
- this invention provides assays for screening an orphan receptor for the ability to modulate estrogen activation at an AP-1 site.
- This assay involves: providing a first cell containing an estrogen receptor, and orphan receptor, and a promoter comprising an AP-1 site that regulates expression of a first reporter gene; b) contacting the first cell with a compound having AP-1 mediated estrogenic activity; and detecting expression of the first reporter gene.
- the effect of the compound on orphan receptor mediated modulation at the AP-1 site is then determined by detecting the presence, absence, or level of expression of the reporter regulated by the AP-1 site.
- the orphan receptor it is possible to use a transcription activator or co-activator.
- a given cell would preferably contain receptors for both estrogen and the transcription factor (e.g., both ER and GR where the transcription factor in question is a glucocorticoid).
- Cells that naturally express both receptor types can be used in the assays of this invention.
- cells can be modified (e.g., through recombinant DNA techniques) to express the ER and transcription factor receptor of choice.
- Suitable cells for practicing the methods of this invention include, but are not limited to cells derived from a uterine cervical adenocarcinoma (HeLa) , a hypothalamic cell line (GTl-1 (Mellon et al. (1990) Neuron, 5: 1-10), MCF-7 cells (ATCC No. HTB 22), MDA453 cells (ATCC No. HTB 131), ZR-75-1 cells (ATCC No. CRL 1500) or ERC1 cells described in Kushner et al, Mol. Endocrinol, 4:1465-1473 (1990). ERC2 and ERC3 cells as described by Webb, et al. Mol Endocrinol, 6:157-167 (1993). It will be appreciated that the invention is not limited to practice in mammalian cells and may be practiced, for example in yeast and insect cells, transfected with the appropriate genes and recombinant constructs. Cells naturally expressing two or more receptor types.
- ER and glucocorticoid receptors co-exist in the endometrium (Prodi et al. (1979) Tumori. 65: 241-253).
- maps of ER and GR immunoreactivity and mRNA localization suggest co-localization in certain cerebral nuclei such as the paraventricular nucleus of the hypothalamus, the hypothalamic arcuate nucleus, and the central nucleus of the amygdala (Fuxe et al.
- Cells recombinantly modified to express two or more receptor types are provided.
- Cells normally lacking the ER or the other transcription factor cognate receptor, or both can be recombinantly modified to express both receptors and optionally, additional receptors. Typically this involves transfecting the cell with an expression cassette comprising a nucleic acid encoding the receptor and culturing the cell under conditions where the receptor is expressed (e.g., in the presence of an appropriate inducer if the promoter regulating expression of the receptor is inducible). Typically, the cassette is selected to provide constitutive expression of the receptor.
- a cell that naturally expresses one receptor need only be modified to express the second receptor. However, if the cell expresses neither receptor, it may be transfected with expression cassettes expressing both receptors. Even where a cell naturally expresses one or both receptors, it may be recombinantly modified to express those receptors at a higher level (e.g., by introducing expression cassettes encoding the receptor(s) whose expression level it is desired to increase).
- the cells need not contain "native" receptors, but may be modified to provide truncated or chimeric receptors to provide increased affinity and/or sensitivity of the assay.
- the cells of this invention preferably express one or more AP-1 proteins (the Jun or Fos proteins or other members of that protein family, see bohmaan, et al. (1987) Science, 238: 1386-1392).
- the cells can naturally express the AP-1 protein(s) or they can be modified
- AP-1 proteins are well known to those of skill in the art (see, e.g., Turner et al. (1989) Science 243:1689-1694 and Cohen et al. (1989) Genes & Dev., 3: 173-184, and Example 1). Cells that naturally express one or more AP-1 proteins may still be so modified to increase intracellular jun and/or fos levels.
- the assays of this invention utilize cells containing an estrogen receptor and a receptor for a nuclear transcription factor (typically a transcription factor other than estrogen).
- the factor can be one that is expressed endogenously by the cell or, alternatively, the cell can be modified (e.g., a recombinant cell) so that it expresses the receptor.
- An estrogen receptor includes an estrogen receptor in its native (naturally occurring) form as well as modified estrogen receptors. Numerous modifications of estrogen receptors are known to those of skill in the art. These include, but are not limited to VP16-ER, V-ER, a chimeric receptor comprising the strong VP16 transcriptional activation domain linked to the amino terminus of the ER, V-ER in which the
- ER DNA binding domain (DBD) is deleted, HI 1 an ER lacking the DNA binding domain, and the like (see e.g., Kumar et al, Cell, 51 : 941-951 (1987) and Elliston et al. (1990) JBiol Chem 265:11517-21).
- Nuclear transcription factor ligand and cognate receptor Nuclear transcription factor ligand and cognate receptor
- the cells preferably contain a cognate receptor for the nuclear transcription factor ligand whose interaction with the estrogen activation pathways is to be assayed.
- cognate receptor refers to a receptor of the type that is typically bound by the transcription factor ligand in question.
- the cognate receptor for an estrogen is an estrogen receptor
- the cognate receptor for a glucocorticoid is a glucocorticoid receptor
- the receptor for a progestin is a progestin receptor
- the cognate receptor includes the native (naturally occurring ) form as well as modified receptors.
- Natural and modified cognate receptors for nuclear transcription factor ligands, particularly for steroid nuclear transcription factors, are well known to those of skill in the art. These include, but are not limited to the glucocorticoid receptors, the progestin receptors (e.g., PR-A, PR-B (see, e.g., Law et al. (1987) Proc.
- the cells of this invention are transfected with reporter genes in which a response element (either the AP-1 site or ERE) regulates expression of a reporter gene.
- a response element either the AP-1 site or ERE
- two different reporter genes are used. One gene reports transcription induced by the classical estrogen response system, while the other gene reports transcription induced by the indirect estrogen response.
- the two reporter genes and response elements are typically placed in separate cells, but the methods can also be used with both constructs in the same cell. Promoter Constructs.
- the methods of this invention involve providing a cell containing an estrogen receptor a receptor for a nuclear transcription factor, and a promoter comprising an AP-1 site that regulates expression of a reporter gene (also referred to herein as the reporter gene for the indirect estrogen response pathway (see, e.g., U.S.S.N. 08/410,807 and Webb, et al (1995) Mol. Endo., 9: 443-456).
- a reporter gene also referred to herein as the reporter gene for the indirect estrogen response pathway
- the reporter gene for the indirect estrogen response pathway contains an AP- 1 site preferably upstream of the target promoter and capable of regulating (i.e., operably linked to) that promoter.
- the AP-1 site are sites that are bound by AP-1 (the Jun and Fos proteins) or other members of that protein family.
- the consensus AP-1 site (or AP-1 response element) is TGA(C/G)TCA (SEQ ID NO: 1).
- API site used is not a critical aspect of the invention. Any sequence capable of being bound by API or members of that family and regulating a promoter is suitable. This would include promoters which encompass a naturally occurring API site. Typical promoters include, but are not restricted to metalloprotease genes such as stromelysin, gelatinase, matrilysin, and the human collagenase gene.
- promoters may be constructed which contain a non-naturally occurring AP-1, or related, binding site. This facilitates the creation of reporter gene systems that are not typically found under the control of AP-1.
- promoters may be constructed which contain multiple copies of the AP-1 site thereby increasing the sensitivity or possibly modulating the response the reporter gene system.
- the methods of this invention involve providing a cell containing a promoter comprising an estrogen response element that regulates expression of a reporter gene (also referred to herein as the reporter gene for the direct or classical estrogen response pathway (see, e.g., U.S.S.N. 08/410,807 and Webb, et al (1995) Mol. Endo., 9: 443-456).
- a reporter gene also referred to herein as the reporter gene for the direct or classical estrogen response pathway
- the reporter gene for the direct or classical estrogen response pathway see, e.g., U.S.S.N. 08/410,807 and Webb, et al (1995) Mol. Endo., 9: 443-456.
- the estrogen response element is upstream of the target promoter and capable of regulating that promoter.
- the ERE may be the consensus estrogen response element AGGTCACAGTGACCT (SEQ ID NO: 2) from the Xenopas vitellogenin A2 gene.
- the particular ERE used in the cell is not a critical aspect of the invention and the present invention is not limited to the use of any one particular ERE. Suitable EREs are well known to those of skill. For instance, other sources of naturally occurring EREs include the vitellogenin B2 gene, the chicken ovalbumin gene, and the PS2 gene. Alternatively, non-naturally occurring EREs may be inserted into particular promoters.
- the consensus ERE from the Xenopus vitellogenin A2 gene is widely used for this purpose, but other EREs may be used as well.
- the present invention is not limited to a particular reporter gene. Any gene that expresses an easily assayable product will provide a suitable indicator for the present assay.
- Suitable reporter genes are well known to those of skill in the art. Examples of reporter genes include, but are not limited to CAT (chloramphenicol acetyl transferase) (Alton and Vapnek (1979), Nature 282: 864-869), luciferase, and other enzyme detection systems, such as beta-galactosidase; firefly luciferase (deWet et al. (1987), Mol. Cell. Biol. 1:125-131); bacterial luciferase (Engebrecht et al (1984), Proc. Natl Acad.
- AP-1 site can be used in combination with any promoter and reporter gene compatible with the cell being used.
- the promoter will preferably be one susceptible to regulation by the AP-1 site.
- the promoter/reporter expression cassettes and, other expression cassettes described herein can be constructed according to ordinary methods well known to those of skill in the art. Construction of these cassettes is variously exemplified in Example 1, in USSN 08/410,807, in Webb et al (1995) Mol Endo. 9: 443-456, and in other references cited herein.
- the constructs can all be created using standard amplification and cloning methodologies well known to those of skill in the art. Examples of these techniques and instructions sufficient to direct persons of skill through many cloning exercises are found in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology 152 Academic Press, Inc., San Diego, CA (Berger); Sambrook et al.
- Detection of the reporter genes of this invention is by standard methods well known to those of skill in the art. Where the reporter gene is detected through its enzymatic activity this typically involves providing the enzyme with its appropriate substrate and detecting the reaction product (e.g., light produced by luciferase). The detection may involve simply detecting presence or absence of reporter gene produce, or alternatively, detection may involve quantification of the level of expression of reporter gene products. The quantification can be absolute quantification, or alternatively, can be comparitive e.g., with respect to the expression levels of one or more "housekeeping" genes. Methods of quantifying the expression levels of particular reporter genes are well known to those of skill in the art.
- high throughput screening systems are commercially available (see, e.g., Zymark Corp., Hopkinton, MA; Air Technical Industries, Mentor, OH; Beckman Instalments, Inc. Fullerton, CA; Precision Systems, Inc., Natick, MA, etc.). These systems typically automate entire procedures including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay. These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization.
- kits for the practice of the methods of this invention preferably include assay cells comprising an estrogen receptor, and a promoter comprising an API site which regulates expression of a first reporter gene.
- the cell may additionally include a cognate receptor for a nuclear transcription factor.
- the kits may additionally include a second cell comprising an estrogen receptor, and a promoter comprising an ERE site which regulates expression of a first reporter gene.
- the kits can include one cell type that contains both the API/reporter and the ERE/reporter constructs.
- the cells may additionally express high levels of fos and/or jun.
- the kits may optionally contain any of the buffers, reagents, culture media, culture plates, reporter gene detection reagents, and so forth that are useful for the practice of the methods of this invention.
- kits may include instructional materials containing directions (i.e., protocols) for the practice of the assay methods of this invention.
- instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention.
- Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like.
- Such media may include addresses to internet sites that provide such instructional materials.
- Coll73-LUC and Coll60-CAT have been previously described (Webb et al (1995) Mol. Endo., 9: 443-456; Lopez et al. (1993) Mol. Cell Biol, 13: 3042-3049).
- Coll73-LUC consists of -73 to +63 base pairs of the collagenase promoter upstream of the Luciferase reporter gene.
- Coll517-CAT and Coll517mAP-l-CAT each contain -517 to +63 of the collagenase promoter (Webb et al. (1995) Mol. Endo., 9: 443-456).
- Coll517mAP-l-CAT contains three point mutations in the consensus AP-1 response element (TGAGTCA mutated to GTACTCA).
- ColALuc contains the collagenase AP-1 response element upstream of the minimal drosophila alcohol dehydrogenase promoter (Starr et al, (1996) Genes & Dev., 10: 1271-1283).
- the ER expression vectors have been previously described: PHE0 (Green et al. (1986) Nature, 320: 134-139), PHEG0 (Tora et al. (1989) EMBOJ.
- PHEG0 is the wild type ER. PHE0 has reduced affinity for estrogens which allows for studies in cell culture without inadvertent activation.
- the protein coding regions of the ER plasmids were cloned into the multiple cloning site of the pSG5 expression vector.
- pRSVhGR (McEwan et al. (1993) Mol. Cell Biol, 13: 399-407) consists of a cDNA encoding the human GR coding region inserted into an expression vector driven by the Rous sarcoma virus promoter.
- the PR-A (PhPR-60) and PR-B (hPR65) plasmids were derived from T47D cDNA and genomic DNA (Law et al (1987) Proc. Natl Acad. Sci., USA, 84: 2877-2881 ; Wei et al (1988) Mol Endo., 2: 62-72) and cloned into an expression vector derived from pLEN (Kushner et al. (1990) Mol Endocrinol 4: 1465-1473).
- the beta-actin-hCG construct has been previously described (Lopez et al. (1993) Mol Cell Biol, 13: 3042-3049).
- the pJ3- LacZ plasmid is pBR322-based and contains an SV40 promoter which activates LacZ.
- Transfection Cells were transfected by electroporation as previously described (Webb et al.
- Cells were treated either immediately or up to 6 hours after transfection. They were then harvested at approximately 40 hrs. after plating. Dexamethasone, estradiol, and R5020 were all used at 10 "7 M. Tamoxifen was used at 5 x 10 "6 M. PMA (Sigma) was suspended in DMSO and cells were treated at 10 "7 M; TNF- ⁇ (R&D Systems, Minneapolis, MN) was resuspended in 0.1% BSA and cells were treated at 10 ng/ml.
- CAT, luciferase, and hCG assays were performed as described (Webb et al. (1995) Mol. Endo., 9: 443-456; Lopez et al (1993) Mol. Cell. Biol, 13: 3042-3049).
- a commercial luminescent assay (Tropix; Bedford, MA) was used for ⁇ -galactosidase measurements.
- GR inhibits ER transcriptional activation through the AP-1 response element.
- HeLa cells were transfected with ER (HEO) and the truncated collagenase promoter (Coll73-LUC) (Fig. 1A), then treated with Dex, estradiol, or Dex+ estradiol. As previously reported, Dex inhibited and estradiol stimulated transcription through this promoter. When both steroids were added, GR blocked estradiol-stimulated transcription (Fig. IB). A similar ER-GR interaction occurs with both HEO and HEG0, which encodes the wild type receptor.
- the steroid responses were evaluated at a longer form of the collagenase promoter in the presence of an intact or mutated AP-1 response element (Coll517 or Coll517mAP-l, respectively). As was the case with Coll73, Dex blocked estradiol activity through an intact AP-I response element. The steroid responses were abrogated when the promoter bearing the mutated AP-1 response element was used (Fig. IC; (Webb et al. (1995) Mol. Endo., 9: 443-456; Ponta et al. (1992) Biochem. Biophys. Ada, 1129:255-261, and references therein)).
- Fig. IC The minor steroid effects seen in Fig. IC are not reproducible. Steroid effects were also attenuated when HeLa cells were transfected with Coll73-CAT deleted of the AP-1 response element (C0II6O-LUC; (Id.)). Dex, then, is able to block estradiol stimulation of transcriptional activation mediated by the AP-1 response element.
- ER:GR a given proportion of ER:GR might resulting in the cancellation of any estrogen or glucocorticoid effects at all.
- Dexamethasone inhibits estradiol- and tamoxifen- mediated ER activation through the AP-1 response element.
- ER stimulation of transcription through the AP-1 response element occurs through more than one pathway (Webb et al. (1995) Mol. Endo., 9: 443-456).
- the alpha pathway is characterized by tamoxifen-induced transcriptional activation and a requirement for the ER DNA binding domain.
- Dex inhibited tamoxifen activation (Fig. 3 A).
- the degree of Dex inhibition diminished in the presence of high levels of co-transfected ER (Fig. 3 A).
- a C-terminally deleted ER (HE 15) serves as a model of tamoxifen activation. It lacks the activation function in the C-terminus and activates transcription through the activation function in the N-terminal domain.
- Dexamethasone inhibits the beta pathway characterized by estradiol activation and the lack of a requirement for the ER DNA binding domain.
- HE11 which lacks the DNA binding domain
- cells were treated with estradiol, Dex, or Dex+estradiol, as above.
- Dex inhibited estradiol activation through HE11 (Fig. 4). Since Dex inhibited tamoxifen stimulation, the constitutive activity of the ER deleted of its C- terminal domain, and estradiol-activated ER deleted of its DNA binding domain (Figs. 3 and 4), it is inferred that glucocorticoids can inhibit both alpha and beta pathways of ER stimulation.
- c-Jun and c-Fos differentially alter estradiol and Dex effects It has been demonstrated that individual members of the AP-1 family differentially change the pattern of steroid receptor activation at a hormone response element. For example, increasing amounts of c-Jun and c-Fos progressively attenuate ER activation at an estrogen response element (ERE) in MCF-7 cells whereas transfected JunD does not (Doucas et al (1991) EMBO J., 10: 2237-2245). In addition, the ratio of Jun:Fos in a given cell will change the steroid response to Dex at the AP-1 site (Teurich et al, (1995) Chem. Senses, 20: 251-255) and the proliferin composite (GRE/AP-1) response element (Diamond et al. (1990) Science, 249: 1266-1272).
- GRE/AP-1 proliferin composite
- c-Jun increased estradiol transcriptional activation at CoII73 (Fig. 5 A). At levels of co-transfected c-Jun which resulted in slightly increased AP-1 activated transcription, estradiol stimulation was potentiated. At levels of co-transfected c-Jun which resulted in marked stimulation of AP-1 activated transcription, further estradiol stimulation of AP-1 activation was no longer present. Dex treatment alone restricted transcriptional activity to low levels at all amounts of transfected c-Jun.
- Activators of c-Jun differentially alter estradiol and Dex patterns of response at the AP-1 response element.
- HeLa cells were treated with estradiol and/or Dex in the presence or absence of either PMA (10 "7 M) or TNF- ⁇ (10 ng/ml). These doses resulted in maximal AP- 1 activation for each agent (data not shown). PMA treatment in the absence of steroids resulted in a ten fold stimulation of transcriptional activity (Fig 6 A, note difference in the scale of the No PMA and PMA axes). The pattern of steroid effects was maintained in the presence of PMA (Fig. 6A).
- estradiol stimulation was no longer apparent in the presence of TNF- ⁇ although Dex inhibition was maintained (Fig. 6B).
- the loss of estradiol stimulation was not a result of altering the functional activity of ER.
- Cells simultaneously transfected with both Coll73-LUC and ERE-C0II6O-CAT failed to show diminished activity of ER at an ERE (data not shown). Therefore, while both of these agents activate c-Jun, they each have different effects on estradiol responses at the AP-I response element.
- the GR inhibits ER stimulation in a hypothalamic cell line.
- GTl-1 cells were derived from a transgenic mouse whose GNRH neurons were targeted for transformation by the SV40 T antigen (Mellon et al. (1990) Neuron 5: 1-10). They express neuronal but not glial markers (Id.), GNRH (Id.), and the glucocorticoid receptor (Chandran et al, (1994) Endocrinol, 134: 1467-1474). GTl-1 cells were transfected with the reporter plasmid ColALuc (Starr et al.
- the progesterone receptor (PR interacts with ER at the AP-1 site.
- progestins oppose estrogen actions. Since it has been demonstrated that the PR inhibits PMA activated transcription through the AP-1 response element (Bamberger et al. (1996) Proc. Natl. Acad. Sci., USA, 93: 6169-6174), it was determined whether or not the PR could also interact with the ER at the AP-1 response element.
- the progesterone agonist R5020 inhibited the basal activity of an AP-1 site through both PR-A and PR-B (Fig. 8 A). As before, estradiol treatment stimulated transcription. Treatment with both steroids resulted in a loss of RU5020 inhibition. PR-A behavior was then evaluated in a different cell line.
- a cell may be capable of mounting an estrogen or glucocorticoid response at the AP-1 response element, whether or not the response will actually occur will depend on the relative levels of each receptor.
- Estrogen stimulation of AP-1 regulated genes may be blunted in the presence of glucocorticoids.
- glucocorticoid inhibition could be overcome by estrogen activation.
- the steroid response will be modulated by the levels and composition of the AP-1 protein complex in the cell. Transfected c-Jun and c-Fos differentially altered the estrogen and glucocorticoid pattern of transcription.
- the steroid responses will also be modified by the activation state of the cell.
- Certain activators of AP-1 may modulate a steroid response, e.g. TNF- ⁇ modulation of estrogen stimulation, while others may not.
- TNF- ⁇ modulation of estrogen stimulation e.g. TNF- ⁇ modulation of estrogen stimulation
- estradiol treatment increases the level of IGF- 1 mRNA and the increase is attenuated by prior administration of Dex (Sahlin (1995) J. Steroid Biochem. Molec. Biol, 55: 9-15).
- GTl cells contain endogenous GR, which apparently functions to down-regulate GnRH transcription in GTl cell lines in response to Dex (Chandran et al. (1994) Endocrinol, 134: 1467-1474).
- GnRH which contains an AP-1 response element in its promoter (Bruder et al. (1992) Endocrinol, 131: 2552-2558), could be regulated by estrogens and glucocorticoids in this manner.
- the AP-1 response element will integrate the effect of the ER with other members of the family, as well as to integrate the effects of other superfamily members with each other.
- Such integration might occur at Jun/Jun, Jun/Fos AP-1 complexes or through shared co-activators.
- CBP CREB-Binding Protein
- CBP has been shown to interact with several members of the steroid receptor superfamily as well as with members of the steroid receptor co-activator (SRC) family (Kamei et al, (1996) Cell, 85: ;403-414). Therefore, the functional interaction of the steroid receptors described at the AP-1 site could be mediated not only through AP-1 protein complexes but also through a number of co-activator proteins involved in transducing steroid receptor signals to the basal transcriptional machinery.
- SRC steroid receptor co-activator
- SEQ ID NO: 1 consensus AP-1 site.
- SEQ ID NO: 2 consensus estrogen response element AGGTCACAGTGACCT
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Tropical Medicine & Parasitology (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Endocrinology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU82622/98A AU757559B2 (en) | 1997-06-30 | 1998-06-24 | Methods for screening nuclear transcription factors for the ability to modulate an estrogen response |
EP98932825A EP1005533A4 (en) | 1997-06-30 | 1998-06-24 | Methods for screening nuclear transcription factors for the ability to modulate an estrogen response |
JP50566699A JP2002510210A (en) | 1997-06-30 | 1998-06-24 | Methods for screening nuclear transcription factors for their ability to modulate estrogen response |
CA002296015A CA2296015A1 (en) | 1997-06-30 | 1998-06-24 | Methods for screening nuclear transcription factors for the ability to modulate an estrogen response |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US5130997P | 1997-06-30 | 1997-06-30 | |
US60/051,309 | 1997-06-30 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1999000488A1 true WO1999000488A1 (en) | 1999-01-07 |
Family
ID=21970492
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1998/013089 WO1999000488A1 (en) | 1997-06-30 | 1998-06-24 | Methods for screening nuclear transcription factors for the ability to modulate an estrogen response |
Country Status (7)
Country | Link |
---|---|
US (1) | US20020098477A1 (en) |
EP (1) | EP1005533A4 (en) |
JP (1) | JP2002510210A (en) |
KR (1) | KR20010014368A (en) |
AU (1) | AU757559B2 (en) |
CA (1) | CA2296015A1 (en) |
WO (1) | WO1999000488A1 (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001053479A2 (en) * | 2000-01-24 | 2001-07-26 | Sangamo Biosciences, Inc. | Molecular switches ii |
WO2001055726A1 (en) * | 2000-01-25 | 2001-08-02 | Glaxo Group Limited | Assay for glucocorticoid receptor signalling pathway |
US6599741B1 (en) | 1999-09-14 | 2003-07-29 | Avontec Gmbh | Modulating transcription of genes in vascular cells |
WO2014142984A1 (en) * | 2013-03-15 | 2014-09-18 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Coincidence reporter gene system |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6994967B1 (en) * | 2000-07-05 | 2006-02-07 | California Institute Of Technology | Transcription factor regulators and methods for screening for same |
KR20030071162A (en) * | 2002-02-28 | 2003-09-03 | (주)지노첵 | A Target-Selective Transcriptional Coactivator |
WO2020081979A1 (en) * | 2018-10-19 | 2020-04-23 | Memorial Sloan-Kettering Cancer Center | Low dose radiation conditioning for immunotherapy |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5071773A (en) * | 1986-10-24 | 1991-12-10 | The Salk Institute For Biological Studies | Hormone receptor-related bioassays |
JPH06502532A (en) * | 1990-10-10 | 1994-03-24 | ラ ジョラ キャンサー リサーチ ファウンデーション | Method of transcription suppression using nuclear receptors |
US5723291A (en) * | 1993-09-01 | 1998-03-03 | The Regents Of The University Of California | Methods for screening compounds for estrogenic activity |
-
1998
- 1998-06-23 US US09/103,355 patent/US20020098477A1/en not_active Abandoned
- 1998-06-24 EP EP98932825A patent/EP1005533A4/en not_active Withdrawn
- 1998-06-24 AU AU82622/98A patent/AU757559B2/en not_active Ceased
- 1998-06-24 CA CA002296015A patent/CA2296015A1/en not_active Abandoned
- 1998-06-24 KR KR1019997012525A patent/KR20010014368A/en not_active Application Discontinuation
- 1998-06-24 WO PCT/US1998/013089 patent/WO1999000488A1/en not_active Application Discontinuation
- 1998-06-24 JP JP50566699A patent/JP2002510210A/en active Pending
Non-Patent Citations (3)
Title |
---|
GAUB M.-P., ET AL.: "ACTIVATION OF THE OVALBUMIN GENE BY THE ESTROGEN RECEPTOR INVOLVES THE FOS-JUN COMPLEX.", CELL, CELL PRESS, US, vol. 63., 21 December 1990 (1990-12-21), US, pages 1267 - 1276., XP002911618, ISSN: 0092-8674, DOI: 10.1016/0092-8674(90)90422-B * |
PONTA H., CATO A. C. B., HERRLICH P.: "INTERFERENCE OF PATHWAY SPECIFIC TRANSCRIPTION FACTORS.", BIOCHIMICA ET BIOPHYSICA ACTA., ELSEVIER, NL, vol. 1129., 1 January 1992 (1992-01-01), NL, pages 255 - 261., XP002911620, ISSN: 0006-3002 * |
WEBB P, ET AL.: "TAMOXIFEN ACTIVATION OF THE ESTROGEN RECEPTOR/AP-1 PATHWAY: POTENTIAL ORIGIN FOR THE CELL- SPECIFIC ESTROGEN-LIKE EFFECTS OF ANTIESTROGENS", MOLECULAR ENDOCRINOLOGY, THE ENDOCRINE SOCIETY, US, vol. 09, no. 04, 1 January 1995 (1995-01-01), US, pages 443 - 456, XP002911619, ISSN: 0888-8809, DOI: 10.1210/me.9.4.443 * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6599741B1 (en) | 1999-09-14 | 2003-07-29 | Avontec Gmbh | Modulating transcription of genes in vascular cells |
US7186556B2 (en) | 1999-09-14 | 2007-03-06 | Avontec Gmbh | Modulating transcription of genes in vascular cells |
WO2001053479A2 (en) * | 2000-01-24 | 2001-07-26 | Sangamo Biosciences, Inc. | Molecular switches ii |
WO2001053479A3 (en) * | 2000-01-24 | 2002-01-31 | Sangamo Biosciences Inc | Molecular switches ii |
WO2001055726A1 (en) * | 2000-01-25 | 2001-08-02 | Glaxo Group Limited | Assay for glucocorticoid receptor signalling pathway |
WO2014142984A1 (en) * | 2013-03-15 | 2014-09-18 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Coincidence reporter gene system |
Also Published As
Publication number | Publication date |
---|---|
US20020098477A1 (en) | 2002-07-25 |
CA2296015A1 (en) | 1999-01-07 |
KR20010014368A (en) | 2001-02-26 |
EP1005533A1 (en) | 2000-06-07 |
EP1005533A4 (en) | 2004-08-18 |
JP2002510210A (en) | 2002-04-02 |
AU757559B2 (en) | 2003-02-27 |
AU8262298A (en) | 1999-01-19 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Weigel et al. | Ligand-independent activation of steroid hormone receptors | |
Wen et al. | The A and B isoforms of the human progesterone receptor operate through distinct signaling pathways within target cells | |
US6599698B1 (en) | Mutated steroid hormone receptors, methods for their use and molecular switch for gene therapy | |
Aronica et al. | Stimulation of estrogen receptor-mediated transcription and alteration in the phosphorylation state of the rat uterine estrogen receptor by estrogen, cyclic adenosine monophosphate, and insulin-like growth factor-I. | |
Giguère et al. | Estrogen receptor β: re-evaluation of estrogen and antiestrogen signaling | |
US5639592A (en) | Functional antagonism between proto-oncoprotein c-Jun and hormone receptors | |
US5506102A (en) | Methods of using the A form of the progesterone receptor to screen for antagonists of steroid intracellar receptor-mediated transcription | |
Miesfeld | The Structure and Function of Steroid Receptor Protein | |
Maroder et al. | Cell-specific bifunctional role of Jun oncogene family members on glucocorticoid receptor-dependent transcription. | |
JPH09500023A (en) | Identification of ligands by selective amplification of cells transfected with the receptor | |
Somers et al. | Effects of okadaic acid, a protein phosphatase inhibitor, on glucocorticoid receptor-mediated enhancement. | |
AU757348B2 (en) | Differential ligand activation of estrogen receptors ERalpha and ERbeta at AP1 sites | |
Liu et al. | The dominant negative effect of thyroid hormone receptor splicing variant alpha 2 does not require binding to a thyroid response element. | |
AU757559B2 (en) | Methods for screening nuclear transcription factors for the ability to modulate an estrogen response | |
Maudsley et al. | Proline-rich tyrosine kinase 2 mediates gonadotropin-releasing hormone signaling to a specific extracellularly regulated kinase-sensitive transcriptional locus in the luteinizing hormone β-subunit gene | |
US20030124508A1 (en) | Method for testing the hormonal effect of substances | |
AU682398B2 (en) | Methods for screening antiestrogen compounds | |
US7074566B2 (en) | Method for testing hormonal effects of substances | |
US20030091978A1 (en) | Assay for glucocorticoid receptor signalling pathway | |
Xing et al. | An N-terminal deletion mutant of estrogen receptor exhibits increased synergism with upstream activators and enhanced binding to the estrogen response element | |
EP1944378B1 (en) | Diagnostic and screening method | |
AU724598B2 (en) | A molecular switch for a transgenic animal or a plant, and methods for their use. | |
Ince | Dominant negative mutants of the human estrogen receptor | |
Manning | The views, opinions and/or findings contained in this report are | |
Scott | Developmental regulation of proopiomelanocortin gene expression in the rat pituitary |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AU CA JP KR |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE |
|
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
ENP | Entry into the national phase |
Ref document number: 2296015 Country of ref document: CA Ref country code: CA Ref document number: 2296015 Kind code of ref document: A Format of ref document f/p: F |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1019997012525 Country of ref document: KR |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1998932825 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 82622/98 Country of ref document: AU |
|
WWP | Wipo information: published in national office |
Ref document number: 1998932825 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 1019997012525 Country of ref document: KR |
|
WWG | Wipo information: grant in national office |
Ref document number: 82622/98 Country of ref document: AU |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 1019997012525 Country of ref document: KR |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 1998932825 Country of ref document: EP |