WO1996017927A2

WO1996017927A2 - Reporter cell line

Info

Publication number: WO1996017927A2
Application number: PCT/EP1995/004819
Authority: WO
Inventors: Stefan Nilsson
Original assignee: Karo Bio Ab
Priority date: 1994-12-05
Filing date: 1995-12-05
Publication date: 1996-06-13
Also published as: GB9424497D0; AU4713496A; WO1996017927A3

Abstract

The present 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 NFλB. 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 potential anti-inflammatory activity. According to a further aspect of the invention there is provided a method of testing a compound for both AP1 and NFλB inhibitory activity, the method comprising providing cells in accordance with the first aspect of the invention, stimulating AP1 and NFλB activity in the cells and contacting the cells with the compound to be tested and monitoring expression of the first and second gene products wherein inhibition of AP1 expression of the first gene product, and inhibition of NFλB mediated expression of the second gene product is indicative that the compound has both AP1 and NFλB-inhibitory activity.

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.

Intracellular receptors and their ligands play key roles in various hormone dependent

diseases such as cancers, osteoporosis and cardiovascular and inflammatory

malfunctions. A number of pharmaceutical drugs that can modulate the function and

activity of intracellular receptors have been developed and are today used for the

treatment of major clinical indications: for example glucocorticoids as anti-inflammatory

drugs, estrogen agonists, vitamin D and thyroid hormone for hormone replacement

therapy, estrogen antagonists in the treatment of breast cancer and androgen antagonists

in prostate cancer therapy.

However, for many indications there are either no hormonal drugs available or current

drugs have undesired side effects due to poor receptor selectivity or tissue specificity.

For example, Tamoxifen, a potent anti-estrogen used in adjuvant therapy in breast cancer, causes an increased frequency of primary endometrial cancer in long term

treatment; or anti-inflammatory glucocorticoids for systemic treatment which frequently

induce osteopenia. Thus there is a need for safer drugs with an improved therapeutic profile, that is to say

drugs having a more defined specificity and tissue selective agonist/antagonist activity.

Glucocorticoids exert profound effects on the inflammatory and immune responses. The

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 throug

modulation of the transcription of specific sets of genes such as metalloproteases and

inflammatory mediators such as the tumour necrosis factor TNF-o, and the interleukin

IL-1,2,3, 5,6 and 8. 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, (Angel et al (1987), Cell 49 729-739; Boumpas (1991) et al Clinical

and Experimental Rheumatology 9, 413-423; Yang Yen et al, (1990) Cell 62,1205-1215

and references cited therein) and the NFKB transcription factor, the heterodimeric

complex composed of two subunits, p50 and p65, involved in the expression of a number of genes including immunoglobulin K, IL-2 receptor, and class 1

histocompatibility genes (Baeurle,(1991) Biochim. Biophys.Acta 1072 63-80; Stolpe et

al (1994) J. Biol. Chem. 269:6185-6192; Ray and Prefontaine (1994) Proc. Natl Acad.

Sci 91 752-756; Stein et al (1994) EMBO J. 12: 3879-3891; Caldenhoven et al. (1994)

Mol. Endocrinol. (in press); and references cited therein).

Glucocorticoids also block the migration of leucocytes to the site of inflammation by having a direct inhibitory effect on the expression of adhesion molecules like ICAM an

E-selectin (Caldenhoven et al. (1994) Mol. Endocrinol. (in press); and references cited therein).

Experimental data suggests that the mechanism of API and NFKB dependent

transcription is suppressed by the hormone activated glucocorticoid receptor (GR)

through the formation of a GR: API and/or a GR: NFKB protein:protein complex,

respectively. (Jonat et al (1990) supra; Yang Yen et al (1990) supra; Schϋle et al

(1990) Cell 62, 1217-1226; Ray and Prefontaine (1994) supra; Caldenhoven et al.

(1994)supra; 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 (7990);Boumpas et al

(1991) supra; Yang- Yen et al (1990); and Schϋle et al (1990) supra; unpublished

results).

However, one of the most severe side effects of long term glucocorticoid treatment is

steroid induced osteoporosis (in Osteoporosis 1990 edited by Christiansen C. and Overgaard K 1529-1538). A glucocorticoid induced negative calcium balance resulting

in secondary hyperparathyroidism and inhibition of osteoblast precursor maturation is

believed to be the major mechanisms of glucocorticoid induced bone loss (Hahn, T.J.

(1990), in Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism

First edition, published by the American Society for Bone and Mineral Research p.158-

162; Luckert B.P. and Raisz L.G., (1990) Annals, of Internal Medicine 112: 352-364; and references cited therein).

The severe and negative side effects of existing, clinically used glucocorticoids points t

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.

Most of the drugs in current clinical use have been developed using classical techniques

like in vitro binding to target receptors in tissue extracts or cells. Lead compound

optimization has been performed by traditional medicinal chemistry. In addition, the lac

of relevant in vitro assays for test of the biological activity of drugs have often

necessitated large and extensive animal studies to prove their effects for the indicated

clinical application. Initial screening of a large number of compounds on animals is time-consuming, expensive and is nowadays considered undesirable and. in certain

cases, may give misleading results.

Recent advances in molecular biology and molecular endocrinology have made it possible to develop tools by which novel drugs can be discovered and evaluated in a

rational way. It is today possible to do screening and evaluation of large compound

libraries using genetically engineered, cell based transcription assays (reporter assays).

This type of mechanistic in vitro assay not only discloses whether a compound interacts with a specific hormone receptor but also the biological consequences of that interaction

by indirectly deteπnining the rate of transcription of a selected target gene (reporter

gene).

According to a first 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 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 NFKB.

API and/or NFKB activity in the cells may be stimulated and the cells subsequently

contacted with a compound to be tested. Inhibition of either or both activities in the thus

stimulated cells is indicative that the compound has at least potential anti-inflammatory

activity.

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 potential anti-inflammatory

activity.

Preferably, the cell line is derived from human, more preferably HeLa cells. The invention is not, however, limited to mammalian cells. For example, the cell line may

be derived from yeast or insect 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 readil

assayed for, but other suitable reporter genes may be selected by the skilled worker.

According to a second aspect of the invention there is provided a method of testing a

compound for both API and NFKB inhibitory activity, the method comprising providi

cells in accordance with the first aspect of the invention, stimulating API and NFKB

activity in the cells and contacting the cells with the compound to be tested and

monitoring expression of the first and second gene products wherein inhibition of API

expression of the first gene product, and inhibition of NFKB mediated expression of th

second gene product is indicative that the compound has both API and NFKB -inhibito

activity.

API activity in the cells may be stimulated by the phorbol ester (TPA), epidermal

growth factor (EGF), pyrrolidine dithiocarbamate (PDTC) or N-acetyl-L-cysteine

(NAC).

NFKB activity may be stimulated by TPA, TNFα, IL-l_p, H₂O₂ or any combination

thereof.

According to a third aspect of the invention there is provided a method of testing a

compound for API inhibitory activity comprising providing cells in accordance with t first aspect of the invention, stimulating API activity in the cells, and contacting the

cells with the compound to be tested and monitoring expression of the first and second

gene products wherein inhibition of API-mediated expression of the first gene product

and no effect or substantially no effect on the expression of the second gene product is

indicative that the compound has API inhibitory action.

According to a fourth aspect of the invention there is provided a method of testing a

compound for NFKB inhibitory activity comprising providing cells in accordance with

the first aspect of the invention, stimulating NFKB activity in the cells, and contacting

the cells with the compound to be tested and monitoring the expression of the first and

second gene products wherein inhibition of NFκB-mediated expression of the second

gene product and no effect or substantially no effect on the expression of the first gene

product is indicative that the compound has NFKB inhibitory activity.

API activity or NFKB activity respectively may be stimulated in the methods according

to the latter two aspects of the invention as described above in relation to the method in

accordance with the second aspect of the invention.

The production of a cell line in accordance with the invention and its use to test

compounds in methods in accordance with the invention will now be described, by way

of example only, with reference to the accompanying Figures, Fig.s 1 to 11 in which:

Fig. 1A shows the nucleotide sequence of the five API responsive elements inserted in tandem upstream of the mouse mammary tumour virus long terminal repeat (MMTV

LTR) promoter and the gene encoding the secreted form of human placental alkaline

phosphatase (ALP);

Fig IB shows the ALP reporter vector 5APNT-ALP containing the nucleotide sequence

of Fig lA;

Fig 2A shows the nucleotide sequence of the two NFKB responsive elements inserted in

tandem upstream of the mouse mammary tumour virus long terminal repeat (MMTV

LTR) promoter;

Fig.2B shows the NFKB controlled reporter vector SNKB2-1.GH2 encoding the human

growth hormone polypeptide (hGH);

Fig. 3 A illustrates the effect of dexamethasone on cells from a cell line in accordance

with the invention in which AP-1 activity has been stimulated;

Fig 3B illustrates the effect of dexamethasone on cells from a cell line in accordance

with the invention in which NFKB activity has been stimulated;

Fig 4 illustrates the effect of dexamethasone on cells from a cell line in accordance with

the invention in which AP-1 and NFKB activity has been stimulated; Fig 5 illustrates the effect of betamethasone on cells from a cell line in accordance with

the invention in which AP-1 and NFKB activity has been stimulated;

Fig 6 illustrates the effect of beclomethasone on cells from a cell line in accordance with

the invention in which AP-1 and NFKB activity has been stimulated;

Fig 7 illustrates the effect of aldosterone on cells from a cell line in accordance with the

invention in which AP-1 and NFKB activity has been stimulated;

Fig 8 illustrates the effect of corticosterone on cells from a cell line in accordance with

the invention in which AP-1 and NFKB activity has been stimulated;

Fig 9 illustrates the effect of cortisone on cells from a cell line in accordance with the

invention in which AP-1 and NFKB activity has been stimulated;

Fig 10 illustrates the effect of cortexolone on cells from a cell line in accordance with

the invention in which AP-1 and NFKB activity has been stimulated; and

Fig 11 illustrates the effect of RU486 on cells from a cell line in accordance with the

invention in which AP-1 and Nfi B activity has been stimulated.

1 Generation of combined AP-1 /NFKB 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 tande

and fused 5' to the core promoter sequences of the mouse mammary tumour virus long

terminal repeat (MMTV LTR) and the gene encoding the secreted form of human

placental alkaline phosphatase (ALP) (as shown in Figs 1A and B) whereby secretion o

the ALP protein into the medium is indicative of AP-1 mediated transcription.

Fig. 1A shows the nucleotide sequence of the five API responsive elements (over lined

inserted in tandem upstream of the TATA-box in the mouse mammary tumour virus lo

terminal repeat (MMTV LTR) promoter and the beginning of the gene encoding the

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 the 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 the ALP based reporter assay particularly convenient to use compared

intracellular reporters such as chloramphenicol acetyltransferase(CAT) and luciferase

(Alam J. and Cook J.L.(1990) Analytical Biochemistry 188, 245-254). Additionally, th

high sensitivity of the chemiluminescent ALP assay enables growth of cells and testing

of compounds in 96- well microtiter plates.

Then a second, NFKB regulated reporter vector SNKB2-hGH2 shown in Fig 2B was

introduced into the GRAP cells using conventional techniques for the stable transfectio of mammalian cells to produce a new cell line - termed NAP cells. The reporter vector

SNKB2-hGH2 comprises a NFκB-regulated promoter (MMTV) fused to a reporter gene

encoding human growth hormone (hGH). The hGH reporter protein is secreted into the

cell culture medium like the ALP reporter protein. The level of NFκB-induced hGH

expression is determined immunologically by a Delfia assay (Wallac OY, Finland).

Thus the NAP cells contain two exogenous transcription units whose reporter genes are

transcriptionally induced by the two distinct signal-activated transcription factors API

and NFKB, respectively. The ALP transcription unit is controlled by, and responds to,

the presence of elevated levels of the API transcription factor induced by TPA, EGF,

NAC or PDTC by increased ALP expression. On the other hand, the hGH transcription

unit responds to signal activated NFKB resulting in an increase in hGH expression.

2 Determining the effects of dexamethasone on the API-dependent ALP

expression and NFKB dependent hGH reporter gene transactivation. in NAP cells

The use of the NAP cells in determining the use of various compounds as anti-

inflammatories can be demonstrated using the synthetic glucocorticoid dexamethasone.

Cells are cultured in MEM supplemented by 10%FCS, ImM pyruvate and 1 % non

essential amino acids.

Before exposure to compounds, NAP cells are seeded in 96-well microtiter plates in Ham's F12 ( without phenol red) supplemented with 0.25% bovine serum albumin and

50μg/ml gentamicin. The AP-1 -dependent ALP reporter gene expression and the NFK

dependent hGH reporter gene expression, respectively, was induced by 2 μM phorbol

ester TPA in Ham's F12 supplemented with 0.5% FCS and increasing concentrations o

the synthetic glucocorticoid, dexamethasone.

The relative levels of ALP expressed were determined by a chemiluminescent assay as

follows: a 10/il aliquot of the cell culture medium was mixed with 200μl of assay buffe

(lOmM diethanolamine pH 10; lmM MgCl₂ and 0.5mM AMPPD) in accordance with

the procedures of Tizard et al (1990) Proc. Natl Acad Sci.87 4514-4518) and Alksnis

al (1991) J. Biol. Chem. 266 10078-10085), in white microtiter plates and incubated

37 °C for 20 minutes before being transferred to a microplate format luminometer

(Luminoskan Labsystems, 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 abov

The cells were treated with 2μM TPA to induce the heterodimeric fos/jun transcription

factor (and therefore API activity) and NFKB activity which was indirectly determined

by an increased expression of ALP (Fig3A) and hGH (Fig3B), respectively. In the

presence of increasing concentrations of dexamethasone, the AP-1 dependent ALP expression and the NFKB dependent hGH expression, respectively, were inhibited in a dose dependent manner.

3 Determining the effects of various mineralicorticoids and glucocorticoids on

the API-dependent ALP expression and NFKB dependent hGH reporter gene

transactivation in NAP cells

Fig.s 4 to 10 illustrate the effect of various mineralicorticoids and glucocorticoids on

NAP cells in which AP-1 and NFKB activity was previously stimulated as described

above. It will be seen that each compound inhibited both AP-1 and NFKB dependent

reporter gene expression to a greater or lesser effect. For example, the lower degree of

inhibition shown in Figs 7 and 8 suggests that the mineralicorticoids tested, aldosterone

and cortisone, are less likely to begood anti-inflammatories.

The cell line and the method of the present invention can be used to test a wide variety

of compounds for API and/or NFKB inhibitory activity and can be used in a compact

convenient assay format.

Claims

1 A cell line including a first reporter gene arranged to express an assayable first

gene product, expression of the first repoπer 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 NFKB.

2 A cell line according to claim 1 in which API and/or NFKB activity in the cells

can be stimulated and the cells subsequently contacted with a compound to be tested.

3 A cell line according to claim 2 in which inhibition of either API or NFKB

activity in the thus stimulated cells is indicative that the compound has anti-

inflammatory activity.

4 A cell line according claim 1 or 2 in which inhibition of both API and NFKB activity in the thus stimulated cells is indicative that the compound has anti-

inflammatory activity.

5 A cell line according to any preceding claim which is derived from human cells.

6 A cell line according to claim 5 which is derived from HeLa cells.

7 A cell line according to any preceding claim in which one of the reporter genes

arranged to express an alkaline phosphatase. 8 A cell line according to claim 7 in which the alkaline phosphatase is human

placental alkaline phosphatase.

9 A cell line according to any preceding claim in which one or the other of the

reporter genes is arranged to express human growth hormone.

10 A method of testing a compound for both API and NFKB inhibitory activity, the

method comprising providing cells from a cell line in accordance with any preceding

claim, stimulating API and NFKB activity in the cells and contacting the cells with the

compound to be tested and monitoring expression of the first and second gene products,

wherein inhibition of API -mediated expression of the first gene product, and inhibition

of NFκB-mediated expression of the second gene product is indicative that the compound

has both API and NFKB -inhibitory activity.

11 A method according to claim 10 in which an indication that the compound has

both API and NFKB -inhibitory activity is indicative that the compound has an anti-

inflammatory activity.

12 A method according to claim 10 or 11 in which API activity in the cells is

stimulated by the phorbol ester (TPA), epidermal growth factor (EGF), pyrrolidine

dithiocarbamate (PDTC) or N-acetyl-L-cysteine (NAC). 13 A method according to claim 10, 11 or 12 in which NFKB activity is stimulated

by TPA, H₂O₂, INFα, IL-1_B or a combination of TPA and H₂O₂.

14 A method of testing a compound for API inhibitory activity, the method

comprising providing cells from a cell line in accordance with any one of claims 1 to 9,

stimulating API activity in the cells, and contacting the cells with the compound to be

tested and monitoring expression of the first and second gene products wherein

inhibition of API -mediated expression of the first gene product and no effect or

substantially no effect on the expression of the second gene product is indicative that the

compound has API inhibitory action.

15 A method of testing a compound for NFKB inhibitory activity, the method comprising providing cells from a cell line in accordance with any one of claims 1 to 9,

stimulating NFKB activity in the cells, and contacting the cells with the compound to be

tested and monitoring the expression of the first and second gene products wherein

inhibition of NFκB-mediated expression of the second gene product and no effect or substantially no effect on the expression of me first gene product is indicative that the

compound has NFKB inhibitory activity.