WO1998019158A1

WO1998019158A1 - Assay and cell line for the identification of growth hormone mimetics

Info

Publication number: WO1998019158A1
Application number: PCT/US1997/019350
Authority: WO
Inventors: Charles I. Rosenblum; Aurawan Vongskierttikachor
Original assignee: Merck & Co., Inc.
Priority date: 1996-10-31
Filing date: 1997-10-27
Publication date: 1998-05-07
Also published as: EP0935753A4; CA2270179A1; EP0935753A1

Abstract

A stably transformed cell line is provided for the identification of growth hormone mimetics wherein the cell line is transformed to express a reporter gene construct comprised of a promoter and a growth hormone response element operatively linked to a reporter gene.

Description

TITLE OF THE INVENTION

ASSAY AND CELL LINE FOR THE IDENTIFICATION OF GROWTH

HORMONE MIMETICS

CROSS-REFERENCE TO RELATED APPLICATIONS Not Applicable

STATEMENT REGARDING FEDERALLY-SPONSORED R&D Not Applicable

REFERENCE TO MICROFICHE APPENDIX Not Applicable

FIELD OF THE INVENTION This invention relates to a novel recombinant cell line and to its use in an assay for the identification of growth hormone-like compounds, to the assay methodology, and to compounds identified using this assay.

BACKGROUND OF THE INVENTION

Growth hormone (GH) has been implicated in a broad range of physiological activities influencing both an organism's growth and its intermediary metabolism. Precisely how these activities are set into motion, though, has evaded observation and, thus, has become a subject of great interest. Research efforts, such as those conducted by Yoon et al, 1990 J. Biol. Chem. 265(32):19947-19954, Sotiropoulos et al., 1994 Endocrinology 135(4):1292-1298, and Goujon et al, 1994 Proc. Nat'l Acad. Sci. USA 91:957-961, have advanced knowledge to some degree focusing primarily on the Spi 2.1 gene and the receptor. Yoon et al. scanned regions of the serine protease inhibitor 2.1 (Spi 2.1) gene to isolate those sequences responsible for GH induction of transcription. Sotiropoulos et al. and Goujon et al. concentrated their work on identifying cytoplasmic domains of the growth hormone receptor responsible for Spi 2.1 activation. Contributing somewhat to our limited knowledge on the mechanism behind growth hormone receptor activation is the availability of an efficient, reliable assay. Methods currently in use are either crude and inaccurate in their finding or impracticably inconvenient and time-consuming.

Radio ImmunoAssay (RIA), one method available for the type of studies contemplated here, is incapable of distinguishing between bioactive and bioinactive forms. Some active forms are not even recognized.

Treatment of hypophysectomized rats with known amounts of GH or a putative GH mimetic requires a given number of days or weeks for the body weight or bone (usually the tibia) to be measured and a conclusion rendered. The problem with this is that, while the results give one an accurate picture of a molecule's activity, these tests are overburdenly time consuming and consume large amounts of hormone.

An in vitro bioassay described by Foster et al., 1993, Endocrinology, 132:2073-2082 employs cultures of 3T3-F442A preadipocytes which are converted to adipocytes. GH-induced suppression of glucose conversion to intracellular lipid is determined over a 24 hour incubation in the presence of radiolabelled glucose. This assay requires long term culture sufficient to allow for the conversion of preadipocytes to adipocytes; an inconvenient process involving time consuming extraction steps to complete the lipid isolation procedure.

Rincon-Limas et al., 1993 Biochim Biophys Acta 1172:49-54 describe an in vitro bioassay which uses 35 mm^ or 60 mm^ culture dishes of 3T3-F442A preadipocyte cells. Here, treatment with GH or a compound exhibiting GH-activity results in conversion of preadipocytes to adipocytes within a 12-15 day period. Clearly, this assay is not suitable for fast results. A more efficient assay is still necessary.

Enberg e£ al., 1994 J. Mol. Endocrinology 12:39-46, has proposed a method whereby various cell lines transfected with growth hormone receptor cDNA could be transformed to express a reporter construct containing the growth hormone response element identified in Yoon et al. The response element now present within the cell could then be triggered by addition of growth hormone into the cell preparation; growth hormone bioactivity being detected by expression of the reporter gene. No indications were made within the reference that this method could potentially be used as a screen for ligands capable of eliciting the same reaction as Growth Hormone. Instead, the focus of the article was on providing a useful system for future studies intended on elucidating the mechanism by which Growth Hormone regulates the SPI 2.1 gene. In any case, this method involves the introduction of growth hormone receptor-encoding nucleic acids; a step deemed unnecessary in the present invention, where the goal is to provide a screen capable of deriving accurate, precise results with greater speed and efficiency.

SUMMARY OF THE INVENTION Not Applicable

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to a novel assay for detecting GH-type bioactivity present in a sample, comprising: contacting a cell endogenously expressing growth hormone receptors with a compound suspected of having GH-type activity, said cell having been transformed to contain a reporter gene construct comprised of a promoter and a growth hormone response element operably linked to a reporter gene; and determining if transcription of the reporter gene occurs. One embodiment of the present invention is a method of quantifying GH-type bioactivity, comprising: contacting a cell endogenously expressing growth hormone receptors with a compound suspected of having GH-type activity, said cell having been transformed to contain a reporter gene construct comprised of a promoter and a growth hormone response element operably linked to a reporter gene; and measuring the amount of the characteristic gene product of the reporter gene.

A further embodiment of this invention are cell lines endogenously expressing growth hormone receptors which have been transformed with a reporter gene construct comprising a promoter and a growth hormone response element operably linked to a reporter gene.

Yet another embodiment of this invention are the molecules comprising GH-type activity identified through the methods described herein. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a diagram of the construction of the plasmid pSPI2.1-luc used to transform the stable cell line of the present invention. Figure 2 is a flow diagram illustrating the detailed steps involved in preparing a 96 well plate for the assay.

Figure 3 is a diagram illustrating the response of a stable clone of Example 3 to various class I cytokines. Cells were treated with murine interleukin-3, ovine prolactin, and human GH. Figure 4 is a diagram illustrating the time-course of luciferase expression in cells following treatment with 10 nM human GH for 24 hours.

Figures 5A and 5B are both diagrams indicating that luciferase is synthesized by the clone of Example 3 in the presence of rat or human GH in a dose-responsive manner. Cells were treated with hormones for 24 hours.

As used throughout the specification and claims, the following definition shall apply: "GH-type activity": activity of the same type as human growth hormone, although the response may be weaker or stronger than that evoked with human growth hormone.

"Characteristic gene product": The gene product or identifying characteristic, e.g., the luminescence of luciferase. "GH": Growth hormone.

"GHR": Growth hormone receptor. "Growth hormone mimetic" or "GH-like compound": a compound other than Growth Hormone which binds to a GH receptor and triggers a cascade of intracellular reactions which ultimately result in transcription of the DNA under the control of the promoter.

"Growth hormone response elements": DNA sequences located within or near a promoter region which are responsive to a GH receptor binding event, and in the presence of such an event, allow for transcription of the DNA under the control of the promoter. "Promoter region": DNA located upstream of a protein or peptide encoding sequence, and includes at least one response element. "Promoter": This term includes full length promoters, minimal promoters and promoters which are less than full length, but include more nucleic acids than minimal promoters.

"SPI" or "Spi": Serine protease inhibitor.

"Luc": Luciferase.

"CAT": Chloramphenicol Acetyltransferase.

"Sufficiently similar response": A response capable of triggering the same intracellular cascade which occurs on the interaction of growth hormone with its receptor.

The novel assay of the present invention employs a transformed cell line in the identification of compounds eliciting an intracellular response sufficiently similar to that observed upon the interaction of growth hormone with its receptor. The cells of interest are transformed to express a reporter gene which is under the control of a promoter such that expression of the gene can be readily determined. The promoter governing expression of the reporter gene, in turn, responds to the binding of growth hormone or a growth hormone mimetic to the growth hormone receptor located in the cell membrane. This response triggers transcription of the DNA and, ultimately, effects the cell's growth or metabolism.

In accordance with this invention, it has been found that the binding of growth hormone with growth hormone receptor initiates a cascade of events which induces transcription of the serine protease inhibitor 2.1 (Spi 2.1) gene. Thus, one preferred promoter of this invention is the Spi 2.1 promoter, although others may be used if combined with the response element of the Spi 2.1 promoter. Because a fragment of the Spi 2.1 gene was shown to contain the elements conferring maximal responsiveness to growth hormone (Le Cam et al. , 1994 J Biol Chem 34:21532-21539), a suitable growth hormone response element could be a region comprised of basepairs -175 to +59.

Sequences encoding for the growth hormone response element are preferably placed proximal to the promoter region. Intervening sequences may be present between the growth hormone response element and the promoter, provided they do not interfere with the functioning of the growth hormone response element. In a preferred embodiment, the promoter region comprises a plurality of sequences representing the growth hormone response element of Spi 2.1 promoter; more preferably, at least three such sequences. When a plurality of growth hormone response elements are present, they are preferably joined in tandem. Again, intervening sequences may be present as long as they do not interfere with the functioning of the growth hormone response elements.

A further aspect of this invention is a reporter gene operatively linked to the promoter region of above. The reporter gene may be any gene which encodes a peptide which is easily detected or otherwise allows for easy detection of transcription or translation. It generally encodes a protein which does not naturally occur in the host cell or only is produced in small amounts by the host cell. Examples of well known reporter genes include: chloramphenicol acetyl transferase (CAT), green fluorescent protein (GFP), luciferase (either bacterial or firefly), and other enzyme-based detection systems such as b- galactosidase, alkaline phosphatase, and the like. In a particularly preferred embodiment, luciferase is the reporter gene. Even more preferred is firefly luciferase. In alternative embodiments, the mRNA transcribed from the reporter gene DNA may be measured rather than the translation product.

The reporter gene construct comprising (a) the promoter region comprised of the promoter and at least one growth hormone response element, and (b) the reporter gene operatively linked to the promoter region forms yet another aspect of this invention. It is preferably placed in an appropriate vector and used to transfect a host cell. This vector comprises another aspect of this invention. The vector may be any known vector, including plasmids, cosmids and viral vectors which can function in a chosen host cell.

Comprising yet another aspect of the present invention is the transformed host cell. Any cell line which is amenable to cell culture and which endogenously expresses growth hormone receptors on its membrane may be transformed for this invention. Examples of such cell lines include IM-9 human lymphoblastoid cell lines and RIN5- AH rat pancreatic cells. One especially preferred cell line is the murine 3T3-F442A (sometimes referrered to in the literature as 3T3-442A) preadipocyte cell line. In accordance with this invention, compounds which are suspected of being growth hormone receptor ligands can be assayed. Cells naturally expressing growth hormone receptor are, first, transfected with the reporter gene construct. The putative growth hormone receptor ligand is placed in contact with the transfected cells, and the presence of the reporter gene transcription or translation product is detected. This may be compared to the amount of transcription or translation determined for a control assay, where an identically transfected cell is placed in contact with growth hormone. Also using the embodiments of this assay, growth hormone antagonists may be identified. A growth hormone antagonist is a compound which can bind to the growth hormone receptor, but produces a lesser response than that of native growth hormone. Such compounds would be useful in the treatment of diabetes. The assay described herein offers many attributes not present in the prior art. One such advantage is that the assay of the present invention is dose-responsive; i.e. as more ligand-growth hormone receptor binding occurs, transcription and/or translation of reporter gene increases, and therefore allows for a quantitative determination of growth hormone receptor binding activity.

Furthermore, the assay may be carried out in a 96 well format, using a 3 hour incubation procedure which permits rapid, high throughput screening of chemical entities. In addition, only very small amounts (i.e., nanograms or picograms) of compound are required for screening assays when using the 96 well format.

In sum, a stable cell line, in accordance with this invention, provides many advantages over the current methods utilized in the art. Certain characteristics such as stability, uniformity and ease of use can not be attained with the transient assays suggested in the art. With a stable cell line, there is no need for passage, no need for transfection every time, and no variability due to the introduction of foreign genes. Further, the novel assay of the present invention exhibits a low coefficient of variation; a plus when considering the robustness of an assay. The following non-limiting Examples are presented to better illustrate the invention.

EXAMPLE 1

Construction of GH-responsive expression vector

A fragment of the rat serine protease inhibitor 2.1 gene promoter, bps -175 to +59, was shown to contain the elements required for maximal responsiveness to growth hormone (Le Cam et al. 1994). This region was isolated using polymerase chain reaction (PCR). Two primers were designed. The first primer , δ'-AGATCTAATAAAAG- GTGTGCCGCAGG-3', contains a Bgl II site provided for subcloning purposes adjoining nucleotides encoding rat serine protease inhibitor 2.1 gene promoter from bp -175 to bp -157. The second primer, 5'-AAG- CTTTGCCAGCCTCTTGTGTTG-3', contains a HinD III site provided for subcloning purposes adjoining nucleotides encoding rat serine protease inhibitor 2.1 gene promoter from bp +59 to bp +42. A PCR reaction was performed using rat lung genomic DNA. The DNA template, Taq DNA polymerase and reaction buffers including primers were incubated at 94° C for 2 minutes. The fragment was amplified in a series of 35 cycles consisting of a 94° C incubation for 1 minute, a 50° C incubation for 1 minute and a 72° C incubation for 25 seconds. The resulting product, a single band, was subcloned into the plasmid vector, pGEM-T, using the manufacturer's protocol (Promega, Madison, WI). Authenticity of the amplified fragment was confirmed by determination of the DNA sequence using Sequenase 2.0 T7 DNA polymerase according to the manufacturer's protocol. The fragment was released from pGEM-T by digestion with HinD III and Bgl II, purified and subcloned into pGL2Basic which was previously digested with HinD III and Bgl II.

This construct was designated pSPI 2.1-luc. Bacteria were propagated and preserved using standard methods (Maniatis, et al., 1982). Plasmids were propagated in the Escherichia coli strain DH-5a purchased in transformation-competent form and transformed according to manufacturer's protocol (Life Technologies, Bethesda, MD). EXAMPLE 2

Cell Culture

3T3-442A cells were maintained in DMEM containing 2mM glutamine, 10 % bovine calf serum, 50 μg/ml streptomycin, and 50 U/ml penicillin (growth medium) (Life Technologies, Bethesda, MD) at 37° C in an atmosphere of 95% air, 5% CO2. Cells were seeded at approximately 3.3 x lO^/cm^ and passaged before confluence.

EXAMPLE 3

Stable Transfection of 3T3-442A Cells

18 hours before transfection, cells were plated at 2.2 x 10^ cells per 60 mm tissue culture dish. The plasmid pSPI 2.1-luc was introduced into cells using liposome-mediated transfection. 2 μg pSPI 2.1-luc, 1 μg pSV2neo, and 45 μg Lipofectamine™ (Life Technologies,

Bethesda, MD) were mixed in 2 mis of Optimem™ according to the manufacturer's protocol (Life Technologies, Bethesda, MD). Cells were washed twice with phosphate buffered saline. The DNA-liposome complex was applied to cells. Cells were incubated for 6 hours at which time 4 mis of growth medium was added to cells. 48 hours after transfection, cells were treated with medium supplemented with 500 μg G418/ml. Individual colonies of G418-resistant clones were collected for further analysis and one clone designated SPI-32 was used in subsequent studies.

EXAMPLE 4

Luciferase Assay

Cells were grown in growth medium to near confluence. Cells were then cultured for 16 to 20 hours in serum-free DMEM, 2 mM glutamine. The cells were then seeded at 2 x 10^ cells per well in Dynatech Microlite 96 well tissue culture plates (Dynatech, Chantilly, VA) in 100 μl of an assay culture medium consisting of DMEM, 2 mM glutamine, 5 ng/ml bovine transferrin, 5 ng/ml bovine insulin, 2 nM T3, 1 μM biotin, 500 nM dexamethasone, 500 μg/ml BSA, 40 μM β- mercaptoethanol, 50 μg/ml streptomycin and 50 U/ml penicillin or in later experiments, Optimem™ supplemented with 500 nM dexamethasone, 50 μg/ml streptomycin, and 50 U/ml penicillin. Cultures were coincubated with various concentrations of hormones or cytokines for 24 hours. The cell culture medium was then removed and cells were promptly prepared for assay with 20 μl Promega cell culture lysis reagent. Cell lysates were then assayed for luciferase activity using a Dynatech ML 3000 luminometer (Dynatech, Chantilly, VA) equipped with an automatic injector. The reaction was initiated by injection of 100 μl of Promega luciferase assay reagent. Light output at 25° C was integrated over a 20 second period. The intraassay coefficient of variation (CV) was 11.8% (n=18) and interassay CV was 14.4% (n=8).

EXAMPLE 5

Time Course of Luciferase Induction Cells were treated with lOnM human GH for various periods of time and were assayed for luciferase activity.

Claims

WHAT IS CLAIMED IS:

1. A method for detecting growth hormone-type bioactivity in compounds suspected of having such activity comprising: contacting a cell endogenously expressing growth hormone receptors with a compound suspected of having GH-type activity, said cell having been transformed to contain a reporter gene construct comprised of a promoter and a growth hormone response element operatively linked to a reporter gene; and determining if transcription of the reporter gene occurs.

2. A method according to Claim 1 wherein the assay is used to screen for growth hormone antagonists useful in the treatment of diabetes.

3. A method according to Claim 1 wherein the growth hormone response element is contained within base pairs -175 to +59 of the serine protease inhibitor 2.1 gene.

4. A method according to Claim 1 wherein the assay is carried out in a 96 well format using an incubation period of three hours or less.

5. A method according to Claim 3 wherein the promoter is a serine protease inhibitor 2.1 promoter.

6. A method according to Claim 3 wherein the cell line is the murine 3T3-442A preadipocyte cell line.

7. A method according to Claim 3 wherein the reporter gene is luciferase.

8. A method according to Claim 7 wherein the reporter gene is firefly luciferase.

9. A method according to Claim 7 wherein transcription is detected by luminescence.

10. Compounds identified by the method of Claim 1.

11. A cell line found to endogenously express growth hormone receptors which, in accordance with this invention, is transformed with a reporter gene construct comprising a promoter and a growth hormone response element operably linked to a reporter gene..

12. A method for quantifying the amount of growth- hormone- type bioactivity present in a sample comprising: contacting a cell endogenously expressing growth hormone receptors with a compound suspected of having GH-type activity, said cell having been transformed to contain a reporter gene construct comprising a promoter and a growth hormone response element operatively linked to a reporter gene; and measuring the amount of the characteristic gene product of the reporter gene.

13. A method according to Claim 12 wherein the assay is carried out in a 96 well format using an incubation period of three hours or less.