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WO1996012011A1 - Reporter cell line - Google Patents

Reporter cell line Download PDF

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Publication number
WO1996012011A1
WO1996012011A1 PCT/EP1995/004032 EP9504032W WO9612011A1 WO 1996012011 A1 WO1996012011 A1 WO 1996012011A1 EP 9504032 W EP9504032 W EP 9504032W WO 9612011 A1 WO9612011 A1 WO 9612011A1
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WO
WIPO (PCT)
Prior art keywords
expression
gene
reporter gene
die
cells
Prior art date
Application number
PCT/EP1995/004032
Other languages
French (fr)
Inventor
Stefan Nilsson
Original Assignee
Karo Bio Ab
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Karo Bio Ab filed Critical Karo Bio Ab
Priority to JP8512938A priority Critical patent/JPH10509584A/en
Priority to AU38420/95A priority patent/AU3842095A/en
Publication of WO1996012011A1 publication Critical patent/WO1996012011A1/en

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    • 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

Definitions

  • This invention relates to a reporter cell line and is particularly, though not exclusively,
  • Glucocorticoids exert profound effects on the inflammatory and immune responses. They
  • Agonistic glucocorticoids inhibit transcription of these genes by interfering with the API transcription factor, the
  • GR hormone activated glucocorticoid receptor
  • Glucocorticoid induces a combination of high levels of parathyroid hormone, PTH, and
  • a cell line including a
  • first reporter gene arranged to express an assayable first gene product, expression of the
  • first reporter gene being mediated by a transcription factor
  • second reporter gene
  • the invention provides a cell line including a first reporter gene arranged to express an assayable first gene product, expression of d e first reporter gene being AP-1 mediated, and a second reporter gene arranged to express a second assayable gene
  • the present invention provides a convenient one cell line-based assay system which
  • the cell line allows the testing of two activities in a single cell whereas previously two cell lines would be required.
  • the cell line is derived from human, more preferably HeLa cells.
  • One of the reporter genes may be arranged to express an alkaline phosphatase such as
  • human placental alkaline phosphatase and the other reporter gene may be arranged to
  • a method of testing a compound for glucocorticoid-like activity comprising providing cells in accordance with
  • API activity may be stimulated by TPA or EGF.
  • Fig. 1A shows the nucleotide sequence of d e five API responsive elements inserted in tandem upstream of the mouse mammary tumour virus long terminal repeat (MMTN
  • ALP phosphatase
  • Fig IB shows the ALP reporter vector 5AP ⁇ T-ALP containing the nucleotide sequence
  • Fig 2 shows the glucocorticoid receptor controlled reporter vector MMTV-hGH2
  • hGH human growth hormone polypeptide
  • Fig.3 illustrates the effect of dexamethasone on API -dependent ALP expression and GR dependent hGH reporter gene transactivation, respectively and the effects of Epidermal
  • EGF Growth Factor
  • Fig. 4 illustrates the effect of different corticosteroids on glucocorticoid receptor dependent transactivation of hGH expression and repression of AP-1 -dependent ALP
  • HeLa tk " cells were initially stably transformed using conventional techniques with a
  • reporter vector 5APNT-ALP comprising five API response elements arranged in tandem
  • Figs. 1A and B show that secretion of the ALP protein into the medium is indicative of AP-1 mediated transcription.
  • Fig. 1A shows that secretion of the ALP protein into the medium is indicative of AP-1 mediated transcription.
  • MMTV terminal repeat
  • alkaline phosphatase ALP
  • 5APNT-ALP alkaline phosphatase
  • ALP is underlined.
  • the resulting cells were termed GRAP cells.
  • the level of ALP reporter protein expressed and secreted into d e medium can be determined indirectly by a chemiluminescence assay.
  • the facility to use a chemiluminescence assay The facility to use a chemiluminescence assay.
  • me ALP based reporter assay particularly convenient to use compared to intracellular reporters such as chloramphenicol acetyltransferase (CAT) and luciferase
  • CAT chloramphenicol acetyltransferase
  • the reporter vector MMTV-hGH2 comprises a GR-regulated promoter (MMTV) fused to a reporter
  • hGH human growth hormone
  • the level of glucocorticoid- induced hGH expression is determined immunologically by a Delfia assay (Wallac OY,
  • the GRAPF cells contain two exogenous transcription units whose reporter genes
  • the ALP transcription unit is controlled by, and responds to,
  • d e hGH transcription unit responds to ligand activated GR resulting in an
  • TPA or EGF induced API activity indirectly determined by increased ALP
  • glucocorticoids as well as dieir potency as inhibitors of API -dependent transcription, indirectly reflecting their potency as anti-inflammatory drugs.
  • the expression of the ALP reporter protein in these cells is controlled by the API transcription factor, induced by phorbol esters such as TPA or die growth factor EGF.
  • inflammatories was demonstrated using die synthetic glucocorticoid dexamethasone.
  • GRAPF cells were seeded in 96-well microtiter plates in
  • the relative levels of ALP expressed were determined by a chemiluminescent assay as follows: a lO ⁇ l aliquot of die cell culture medium was mixed wid 200 il of assay buffer (lOmM die ianolamine pH 10.; ImM MgCl 2 and 0.5mM AMPPD) in accordance with
  • Luminoskan Labsy stems, Finland The setting of the Luminoskan luminometer was
  • the alkaline phosphatase activity is expressed in light units (LU).
  • hGH expression was monitored immunologically with the Delfia assay mentioned above.
  • the cells were treated wid lOOng/ml EGF to induce the /as and jun genes (and
  • reporter expression is induced in a hormone-dependent manner irrespective of the
  • the reporter cell line and process of die invention may be used to test a wide variety of

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention provides a cell line including a first reporter gene arranged to express an assayable first gene product, expression of the first reporter gene being AP-1 mediated, and a second reporter gene arranged to express a second assayable gene product, expression of the second reporter gene being mediated by the glucocorticoid receptor (GR). The invention also provides a method of testing a compound for glucocorticoid-like activity comprising providing cells in accordance with the first aspect of the invention, stimulating AP1 activity in the cells and contacting the cells with the compound and monitoring expression of the first and second gene products.

Description

REPORTER CELL LINE
This invention relates to a reporter cell line and is particularly, though not exclusively,
concerned with a cell line useful for testing compounds as potential anti-inflammatory
drugs.
The role of steroid/thyroid hormones and vitamins in oncogenesis and other types of
disorders is well known. Synthetic hormonal drugs acting as agonists or antagonists are
today used in the treatment of several different diseases. However, for many indications there are no hormonal drugs available or already existing drugs have severe side effects
due to poor receptor specificity or tissue selectivity.
Glucocorticoids exert profound effects on the inflammatory and immune responses. They
affect growth, differentiation and function of a broad range of cells involved in these
processes. The principal mechanism whereby they exert their powerful effects is through
modulation of the transcription of specific sets of genes such as metalloproteases and inflammatory mediators such as TNF-o, IL-1, 2, 3, and 6. Agonistic glucocorticoids inhibit transcription of these genes by interfering with the API transcription factor, the
heterodimeric complex formed by the c-fos and c-jun oncoproteins, the major enhancer
of the metalloprotease and lymphokine promoters (Angel et al , (1987) Cell 49 : 729-
739; Boumpas et al (1991) Clin. Experim. Rheum. 9 : 413-423; Yang Yen et al, (1990)
Cell 62 : 1205-1215, and references cited therein). Experimental data suggests that the mechanism of API dependant transcription is
suppressed by the hormone activated glucocorticoid receptor (GR) through the formation
of a GR: API complex which mutually knocks out the ability of both the GR and the
API factors to act as transcriptional regulators through their DNA response elements,
respectively (Jonat et al (1990) Cell 62 : 1189-1204; Yang- Yen et al, (1990) Cell 62 :
1205-1215; Schϋle et al, (1990) Cell 62 : 1217-1226; and references cited therein).
It has been indicated from experimental data that the relative potency of synthetic and
naturally occurring glucocorticoids as anti-inflammatory agents corresponds with their potency in inhibiting API -dependent transcription (Jonat et al, (1990) Cell 62 : 1189-
1204; Yang- Yen et al (1990) Cell 62 : 1205-1215; Schϋle et al (1990) Cell 62 : 1217-
1226; Boumpas et al (1991) Clin. Experim. Rheum. 9 : 413-423; unpublished results).
Glucocorticoids which are presently clinically used have side effects. One of the most
severe side effects of long term steroid use is steroid induced osteopenia (in
Osteoporosis 1990 editors Christiansen C. and Overgaard K. p. 1529-1538; Luckert
B.P. and Raisz L.G. , (1990) Annals of Internal Medicine 112 : 352-364). Glucocorticoid induces a combination of high levels of parathyroid hormone, PTH, and
the inhibition of matrix synthesis in bone which leads to a rapid rate of bone loss. The
severe and negative side effects of existing, clinically used glucocorticoids points to the
need for novel anti-inflammatory drugs with reduced toxicity. Many of the large pharmaceutical companies have accumulated libraries exceeding
100,000 compounds which are of no market value until proven valuable for a clinical
application. The traditional testing of potential anti-inflammatories on animals is time-
consuming and expensive and is nowadays considered undesirable and in certain cases
may give inaccurate results.
According to one aspect of the present invention there is provided a cell line including a
first reporter gene arranged to express an assayable first gene product, expression of the
first reporter gene being mediated by a transcription factor, and a second reporter gene
arranged to express a second assayable gene product, expression of the second reporter
gene being mediated by a receptor whereby the effect of a compound on the expression
of the two genes can be determined.
Preferably, the invention provides a cell line including a first reporter gene arranged to express an assayable first gene product, expression of d e first reporter gene being AP-1 mediated, and a second reporter gene arranged to express a second assayable gene
product, expression of the second reporter gene being mediated by the glucocorticoid
receptor.
Thus the present invention provides a convenient one cell line-based assay system which
can be used to rapidly test a large number of compounds for anti-inflammatory activity.
Furthermore, the cell line allows the testing of two activities in a single cell whereas previously two cell lines would be required. Preferably, the cell line is derived from human, more preferably HeLa cells.
One of the reporter genes may be arranged to express an alkaline phosphatase such as
human placental alkaline phosphatase and the other reporter gene may be arranged to
express human growth hormone. These gene products are preferred as they are readily
assayed for.
According to another aspect of the invention there is provided a method of testing a compound for glucocorticoid-like activity comprising providing cells in accordance with
the first aspect of the invention, stimulating API activity in the cells and contacting the
cells with the compound and monitoring expression of the first and second gene products. Inhibition of API -mediated expression of the first gene product and
stimulated expression of the second gene product being indicative of glucocorticoid-like
behaviour of the compounds.
API activity may be stimulated by TPA or EGF.
The production and use of a cell line to test compounds in accordance with the invention will now be described, by way of example only, with reference to the accompanying Figures, Fig.s 1A to 4 in which:
Fig. 1A shows the nucleotide sequence of d e five API responsive elements inserted in tandem upstream of the mouse mammary tumour virus long terminal repeat (MMTN
LTR) promoter and the gene encoding the secreted form of human placental alkaline
phosphatase (ALP);
Fig IB shows the ALP reporter vector 5APΝT-ALP containing the nucleotide sequence
of Fig 1 A;
Fig 2 shows the glucocorticoid receptor controlled reporter vector MMTV-hGH2
encoding the human growth hormone polypeptide (hGH);
Fig.3 illustrates the effect of dexamethasone on API -dependent ALP expression and GR dependent hGH reporter gene transactivation, respectively and the effects of Epidermal
Growth Factor (EGF) on hGH expression in cells in accordance with the invention; and
Fig. 4 illustrates the effect of different corticosteroids on glucocorticoid receptor dependent transactivation of hGH expression and repression of AP-1 -dependent ALP
reporter gene transcription in GRAPF cells.
1 Generation of combined AP-l/GR reporter cell line
HeLa tk" cells were initially stably transformed using conventional techniques with a
reporter vector 5APNT-ALP comprising five API response elements arranged in tandem
and fused 5' to the core promoter sequences of the mouse mammary tumour virus long terminal repeat (MMTN LTR) and die gene encoding d e secreted form of human placental alkaline phosphatase (ALP) (as shown in Figs. 1A and B) whereby secretion of the ALP protein into the medium is indicative of AP-1 mediated transcription. Fig. 1A
shows the nucleotide sequence of die five API responsive elements (over lined) inserted
in tandem upstream of the TATA-box in the mouse mammary tumour virus long
terminal repeat (MMTV) promoter and the beginning of the gene encoding die secreted
form of alkaline phosphatase (ALP) in plasmid 5APNT-ALP. The ATG (start codon) for
ALP is underlined. The resulting cells were termed GRAP cells.
The level of ALP reporter protein expressed and secreted into d e medium can be determined indirectly by a chemiluminescence assay. The facility to use a
chemiluminescence based assay and the fact that the ALP is secreted into the culture
medium, makes me ALP based reporter assay particularly convenient to use compared to intracellular reporters such as chloramphenicol acetyltransferase (CAT) and luciferase
(Alam J. and Cook J.L., (1990) Analyt. Biochem. 188 : 245-254). Additionally, the
high sensitivity of the chemiluminescent ALP assay enables growth of cells and testing
of compounds in 96- well microtiter plates.
Then a second, GR regulated reporter vector MMTV-hGH2 shown in Fig 2 was
introduced into the GRAP cells using conventional techniques for the stable transfection of mammalian cells to produce a new cell line - termed GRAPF cells. The reporter vector MMTV-hGH2 comprises a GR-regulated promoter (MMTV) fused to a reporter
gene encoding human growth hormone (hGH). The hGH reporter protein is also secreted
into d e cell culture medium like the ALP reporter protein. The level of glucocorticoid- induced hGH expression is determined immunologically by a Delfia assay (Wallac OY,
Finland).
Thus the GRAPF cells contain two exogenous transcription units whose reporter genes
are transcriptionally induced by the two distinct signal-activated transcription factors
API and GR, respectively. The ALP transcription unit is controlled by, and responds to,
the presence of elevated levels of die API transcription factor induced by d e phorbol ester TPA or the epidermal growdi factor EGF by increased ALP expression. On the
other hand, d e hGH transcription unit responds to ligand activated GR resulting in an
increase in hGH expression.
In the presence of both elevated levels of API and ligand activated GR bodi transcription
units are silenced due to die formation of AP1:GR complexes that mutually inhibit
interaction of GR and API with their corresponding response elements (see references
supra).
However, TPA or EGF induced API activity, indirectly determined by increased ALP
expression, does not interfere with glucocorticoid-dependent hGH expression in the GRAPF cells although hormone-activated GR interferes wi i API -dependent
transcription. Thus the GRAPF cells make it possible in one and d e same experiment to
evaluate the agonist/antagonist profile and potency of compounds to act as
glucocorticoids as well as dieir potency as inhibitors of API -dependent transcription, indirectly reflecting their potency as anti-inflammatory drugs. The expression of the ALP reporter protein in these cells is controlled by the API transcription factor, induced by phorbol esters such as TPA or die growth factor EGF.
Exposure of the GRAP cells to TPA or EGF results in the distinct induction of ALP
expression that can be inhibited by a factor of 60% in die presence of lOOnM
dexamethasone.
2 Determining the effects of dexamethasone on the API-dependent ALP expression
and GR-dependent hGH reporter gene transactivation. in GRAPF cells
The use of the GRAPF cells in determining d e use of various compounds as anti-
inflammatories was demonstrated using die synthetic glucocorticoid dexamethasone.
Cells were cultured in MEM supplemented by 10%FCS, ImM pyruvate and 1 % non
essential amino acids.
Before exposure to compounds, GRAPF cells were seeded in 96-well microtiter plates in
Ham's F12 ( without phenol red) supplemented with 10% FCS (stripped widi dextran-
coated charcoal). On the second day the cells were rinsed and refed wid 100 μl Coons
F12 (without phenol red) supplemented with 5 % serum substitute containing additives
as detailed below.
The relative levels of ALP expressed were determined by a chemiluminescent assay as follows: a lOμl aliquot of die cell culture medium was mixed wid 200 il of assay buffer (lOmM die ianolamine pH 10.; ImM MgCl2 and 0.5mM AMPPD) in accordance with
the procedures of Tizard et al (1990) Proc. NatlAcad Sci. 87 : 4514-4518) and Alksnis
et al (1991) J. Biol. Chem. 266 : 10078-10085), in white microtiter plates and incubated
at 37 °C for 20 minutes before being transferred to a microplate format luminometer
(Luminoskan Labsy stems, Finland). The setting of the Luminoskan luminometer was
integral measurement with 1 second reading of each well. The alkaline phosphatase activity is expressed in light units (LU).
hGH expression was monitored immunologically with the Delfia assay mentioned above.
The cells were treated wid lOOng/ml EGF to induce the /as and jun genes (and
dierefore API activity) which was indirectly determined by an increased expression of
ALP (open circles Fig.3). In the presence of increasing concentrations of dexamethasone
("dex"), die API -dependent ALP expression is inhibited whilst die GR-controlled hGH
reporter expression is induced in a hormone-dependent manner irrespective of the
presence or absence of EGF.
The effect of different corticosteroids on glucocorticoid reporter dependent
transactivation of hGH expression and repression of AP-1 -dependent ALP reporter
protein expression in GRAPF cells is shown in Fig. 4. It can be seen that diere is no suppressive effect on the efficacy of the glucocorticoids on glucocorticoid receptor- mediated transcription (hGH), but substantial inhibitory activity of the API function
(ALP). The reporter cell line and process of die invention may be used to test a wide variety of
compounds for anti-inflammatory activity and can be used in a compact convement assay
format.

Claims

A cell line including a first reporter gene arranged to express an assayable first gene product, expression of the first reporter gene being mediated by a
transcription factor, and a second reporter gene arranged to express a second
assayable gene product, expression of die second reporter gene being mediated
by a receptor whereby die effect of a compound on die expression of d e two
genes can be determined.
A cell line according to claim 1 including a first reporter gene arranged to
express an assayable first gene product, expression of die first reporter gene being AP-1 mediated, and a second reporter gene arranged to express a second
assayable gene product, expression of the second reporter gene being mediated
by die glucocorticoid receptor.
A cell line according to claim 1 or 2 which is derived from human cells.
A cell line according to claim 3 which is derived from HeLa cells.
A cell line according to any preceding claim in which one of the reporter genes is
arranged to express an alkaline phosphatase.
A cell line according to claim 5 in which the alkaline phosphatase is human
placental alkaline phosphatase.
7 A cell line according to claim 5 or 6 in which the other reporter gene is arranged
to express human growth hormone.
8 A method of testing a compound for its effect on transcription factor-mediated
gene expression comprising:
providing cells including a first reporter gene arranged to express an assayable
first gene product, expression of die first reporter gene being mediated by a
transcription factor, and a second reporter gene arranged to express a second
assayable gene product, expression of die second reporter gene being mediated by a receptor;
stimulating transription factor activity in the cells and contacting the cells wid
the compound and monitoring expression of the first and second gene products.
9. A metiiod of testing a compound according to claim 8 in which the compound is
tested for anti-inflammatory activity comprising providing cells in accordance
widi any one of claims 1 to 8, stimulating API activity in the cells and contacting the cells with the compound and monitoring expression of die first and second
gene products.
10 A med od according to claim 8 in which inhibition of API-mediated expression of die first gene product and stimulated expression of die second gene product is indicative of anti-inflammatory behaviour of the compounds.
A mediod according to claim 10 in which the anti-inflammatory behaviour is
glucocorticoid-like activity.
The GRAPF cell line.
PCT/EP1995/004032 1994-10-14 1995-10-12 Reporter cell line WO1996012011A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP8512938A JPH10509584A (en) 1994-10-14 1995-10-12 Reporter cell line
AU38420/95A AU3842095A (en) 1994-10-14 1995-10-12 Reporter cell line

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9420735.4 1994-10-14
GB9420735A GB9420735D0 (en) 1994-10-14 1994-10-14 Reporter cell line

Publications (1)

Publication Number Publication Date
WO1996012011A1 true WO1996012011A1 (en) 1996-04-25

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Country Status (4)

Country Link
JP (1) JPH10509584A (en)
AU (1) AU3842095A (en)
GB (1) GB9420735D0 (en)
WO (1) WO1996012011A1 (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE29916160U1 (en) 1999-09-14 2000-03-09 Cardiogene Gentherapeutische Systeme AG, 40699 Erkrath Modulation of gene transcription in vascular cells
WO2000023581A1 (en) * 1998-10-22 2000-04-27 Signal Pharmaceuticals, Inc. Dual reporter system and methods of use therefor
KR20030046896A (en) * 2001-12-07 2003-06-18 학교법인 포항공과대학교 Transformant having a recombinant reporter gene whose expression being regulated by glucocorticoid and method for screening analogue and inhibitor of glucocorticoid using same
US6599741B1 (en) 1999-09-14 2003-07-29 Avontec Gmbh Modulating transcription of genes in vascular cells

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992005447A1 (en) * 1990-09-21 1992-04-02 The Salk Institute For Biological Studies FUNCTIONAL ANTAGONISM BETWEEN PROTO-ONCOPROTEIN c-JUN AND HORMONE RECEPTORS
WO1994001584A1 (en) * 1992-07-06 1994-01-20 President And Fellows Of Harvard College Methods and diagnostic kits for determining toxicity utilizing bacterial stress promoters fused to reporter genes
WO1994017208A1 (en) * 1993-01-21 1994-08-04 President And Fellows Of Harvard College Methods and diagnostic kits utilizing mammalian stress promoters to determine toxicity of a compound
WO1994023041A2 (en) * 1993-04-02 1994-10-13 Ribogene, Inc. Method for selective inactivation of viral replication

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992005447A1 (en) * 1990-09-21 1992-04-02 The Salk Institute For Biological Studies FUNCTIONAL ANTAGONISM BETWEEN PROTO-ONCOPROTEIN c-JUN AND HORMONE RECEPTORS
WO1994001584A1 (en) * 1992-07-06 1994-01-20 President And Fellows Of Harvard College Methods and diagnostic kits for determining toxicity utilizing bacterial stress promoters fused to reporter genes
WO1994017208A1 (en) * 1993-01-21 1994-08-04 President And Fellows Of Harvard College Methods and diagnostic kits utilizing mammalian stress promoters to determine toxicity of a compound
WO1994023041A2 (en) * 1993-04-02 1994-10-13 Ribogene, Inc. Method for selective inactivation of viral replication

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ALKSNIS ET AL.: "High level expression of functional full length and truncated glucocorticoid receptor in chinese hamster ovary cells", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 266, no. 16, 1991, MD US, pages 10078 - 10085, XP002000023 *
JONAT ET AL.: "Antitumor promotion and atiinflamation: downmodulation of AP-1 (FOS/JUN) activity by glucocorticoid hormone", CELL, vol. 62, 1990, NA US, pages 1189 - 1204, XP002000021 *
SCHÜLE ET AL.: "Functional antagonism between oncoprotein c-Jun and the GCR", CELL, vol. 62, 1990, NA US, pages 1217 - 1226, XP002000022 *
YANG-YEN ET AL.: "Transcriptional interference between c-Jun and the glucocorticoid receptor: mutual inhibition of DNA binding due to direct protein-protein interaction", CELL, vol. 62, 1990, NA US, pages 1205 - 1215, XP002000020 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000023581A1 (en) * 1998-10-22 2000-04-27 Signal Pharmaceuticals, Inc. Dual reporter system and methods of use therefor
DE29916160U1 (en) 1999-09-14 2000-03-09 Cardiogene Gentherapeutische Systeme AG, 40699 Erkrath Modulation of gene transcription in vascular cells
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
KR20030046896A (en) * 2001-12-07 2003-06-18 학교법인 포항공과대학교 Transformant having a recombinant reporter gene whose expression being regulated by glucocorticoid and method for screening analogue and inhibitor of glucocorticoid using same

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AU3842095A (en) 1996-05-06
GB9420735D0 (en) 1994-11-30
JPH10509584A (en) 1998-09-22

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