AGAROSE FORMAT FOR MAMMALIAN CELL BASED REPORTER ASSAYS

Abstract: The present invention provides a novel method of using mammalian cells under a layer of agarose or embedded in agarose for transcription activation assays, where a functional response can be detected from the cells under a layer of agarose or embedded in agarose. The invention for the first time provides an agarose format for mammalian cell-based reporter assays useful in high throughput screening.

Description: AGAROSE FORMAT FOR MAMMALIAN CELL BASED REPORTER ASSAYS This application is a nonprovisional application that claims priority to provisional application Serial No. 60/096,431 filed August 13, 1998. Field of the Invention The present invention relates to the use of a semi-solid agarose matrix for transcription activation assays, where a functional response can be detected from mammalian cells embedded in agarose or under two layers of agarose. Background of the Invention Several types of assay systems are known and commonly used to measure gene expression. In reporter assays, the function of a promoter is measured by joining the promoter to a second gene (reporter) which codes for a readily assayable protein, commonly as enzymatic function. Berger et al., Gene 66:1 (1988) describe the use of secreted placental alkaline phosphatase ("SEAP") as a reporter gene. In this formulation, cells are plated in tissue culture treated vessels and treated with the appropriate agents; an aliquot of the medium is then removed and mixed with a substrate, before measuring the enzyme activity (e.g., absorbance, fluorescence, chemiluminescence, etc.). All of the assay systems available in the art, however, suffer from several drawbacks such as contamination of cells and loss of cells during washing. Moreover, the prior art procedures are laborious and require extensive manipulation, leading to low reproducibility and low throughput. Because of these limitations, prior art assays are not useful for the high throughput screening processes used in the modern research environment. Applicants have developed a new technology that permits one to significantly increase the throughput of the assay, as well as its reproducibility, by using a semi-solid agarose matrix. Pluznik et al. (Experimental Cell Research 43:553 (1966)), describe a system wherein single hematopoietic mouse cells are grown and cloned in soft agar. A cell suspension is added to medium containing agar, and this agar/cell suspension is then poured on a lower agar layer (0.5% agar in medium). This technique has been widely used to assess clonogenic capacity of hematopoietic cells, and of a variety of cancer cells, but not to detect cellular responses. Dulbecco et al. (Proc. Natl. Acad. Sci. (Wash.) 38:747 (1952)) describe a system consisting of a plaque technique for titrating animal viruses. A monolayer of mammalian cells (usually embryo fibroblasts) is infected with virus and, after a suitable incubation period, is overlaid with a nutrient agar. The agar prevents the products of virus multiplication from spreading throughout the medium as it would in a fluid culture. The viral products infect and damage adjacent fibroblasts, until the focus of infected cells (plaque) is large enough to be distinguished as an unstained area in a background of healthy cells which take up a vital dye. Chakrabarti et al. (Molecular and Cellular Biology 5(12):3403-3409 (1985)) teach a modification of Dulbecco's plaque assay. A β-galactosidase ("β-Gal") gene is inserted into Vaccinia Virus and used as a selection marker in the plaque assay. To detect viral recombinants producing β-Gal, infected cells are overlaid with agarose containing the substrate X-Gal. The plaques expressing the β-galactosidase gene turn blue. This assay also does not detect cellular responses, and does not involve the use of embedded cells. Jayawickreme et al. (The Journal of Biological Chemistry 269(25) :29846 (1994)) describe an assay system wherein frog melanophores are overlaid with 0.9% agarose. Compounds can then be applied to the surface of the agarose and a cellular response, in the form of pigment aggregation or dispersion, is detected by formation of dark or light zones in the plate. Drillien et al., in U.S. Patent 5,180,675, describe a system similar to the Chakrabarti publication cited above, wherein viral recombinants containing the β- galactosidase gene are detected in the plaque assay by adding X-Gal in the agar layer. As described above, prior art assays use a monolayer of virus-infected mammalian cells that have been overlaid by agarose, before using a vital dye to detect zones of inhibition or plaques. (Dulbecco, supra) Or, in a modification of the plaque assay, recombinant virus particles expressing different reporter genes have been used to infect monolayers of mammalian cells. The presence of recombinant virus particles is detected by adding specific substrates to the agar layer. (Chakrabarti, supra) Others ~ have used frog melanophores, under one layer of agarose, to detect the response to agonists or antagonists of the α-Melanocyte-Stimulating Hormone receptor, applied to the surface of the agarose. Cellular responses, in the form of pigment aggregation or dispersion, are visualized as dark or light zones in the plate. (Jayawickreme, supra) Mammalian cells embedded in agarose have been used in clonogenic assays, to assess clonogenic capacity of hemopoietic cells, and of a variety of cancer cells. Most mammalian cell reporter based assays, however, are performed in a liquid medium. The present disclosure, for the first time, teaches the use of mammalian cells grown under a layer of agarose or embedded in agarose for reporter assays. The present invention solves many of the problems confronted by prior art assays by providing for the first time an innovative screening technology for reporter- based mammalian cell assays utilizing a solid agarose format. Applicants' invention teaches the use of mammalian cells embedded in agarose for transcription activation assays, where a functional response can be detected from mammalian cells embedded in agarose or under a layer of agarose, and the application of such technology to high throughput screening. The present invention solves many of the problems inherent to prior mammalian cell based assays. First, cells are immobilized under a first layer of agarose or embedded in an agarose matrix, thus avoiding the loss of loosely attached cells upon media removal and washes. In the embodiment of the present invention wherein cells are immobilized under a layer of agarose, a plurality of wells are formed on the surface of the first layer of agarose to accept one or more test compounds. Second, because the agarose layer acts as a barrier between the cells and potential sources of environmental contamination, this adaptation enables these screens to be run in automated fashion using robotic systems in a non-sterile environment. Third, due to the diffusion of the test compound in the agarose layer, a concentration gradient is formed in proximity of the cells, thereby minimizing false negatives due to low test concentration or to cytotoxicity. Fourth, the assay of the present invention can tolerate 20% DMSO versus the customary 1-10% used in mammalian cell-based assays, allowing a better compound solubilization. Finally, the procedure eliminates most of the washing and incubation steps which are usually required for liquid-format reporter assays thus greatly increasing the throughput, or number of plates, that can be processed per day. Summary of the Invention Accordingly, the present invention provides a novel reporter-based mammalian cell assay. The assay is useful in detecting functional responses from cells cultured as a monolayer under two layers of agarose or embedded in agarose. Applicants' invention provides the ability to screen a plurality of compounds using a reporter-based mammalian cell assay on a single tray or dish. Applicants' assay diminishes loss of cells from washing and minimizes any chance of contamination. Additionally, the assay system of the present invention is useful and effective in high throughput screening technology. All references cited herein, whether supra or infra, are hereby incorporated herein in their entirety. Brief Description of the Figures Figure 1 shows the results from Example 1 using the assay of the present invention to test HEK293/GLP1 cells for their response to the Glucagon-like Peptide 1 (7-36). This is the physiological agonist of the GLP-1 receptor. The substrate added is BCIP NBT, for a colorimetric measurement of secreted alkaline phosphatase (SEAP). The agonist added on top of the agar diffuses through the agar creating a concentration gradient. Therefore, the potency of the added compound is represented by the diameter of the blue zone of activity around the compound application point. The diameter of the "activity zone" increases with increasing concentration of the agonist. The figure shows the average and standard deviation of three replicates from a representative experiment. Figure 2 shows the representative results from Example 4, using an assay of the present invention wherein cells are embedded in agarose. HEK293/GLP1 cells were tested for production of secreted alkaline phosphatase (SEAP) in response to stimulation with different concentrations of GLP-1 (7-36) amide. The amount of SEAP was measured using the MUP fluorescent substrate. The fluorescence intensity is proportional to the concentration of agonist added into the well. Detailed Description of the Invention The assay of the present invention is carried out using a cell sample, preferably mammalian cells. The cells, or agarose containing the cells, are grown and tested in a cellular container (e.g., a dish or tray). Any type of container known in the art may be used, preferably a tray or dish (e.g., a 96-well or 24-well titer plate), used to screen for cellular activity. One skilled in the art will be able to use the present invention in any type of cellular container adaptable for a specific use. The present invention provides an assay useful to test cells, preferably mammalian cells, for activity in response to a given stimulus. The present invention is useful in screening test compounds that may modulate cellular activity, for example agonists or antagonists. The compounds screened may be known compounds or unknown compounds, and may encompass chemical compounds, a library of compounds, proteins, peptides, and/or a peptide library. A peptide library may be, for example, generated on beads, e.g., resin beads. The primary, but not sole, use would be the high throughput screening for receptor agonists. Low melting point ("LMP") agarose is in the liquid state at 37°C, the optimal temperature for mammalian cell culture, and polymerizes at 24-28°C. In one embodiment of the present invention, a layer of LMP agarose in medium without serum and without phenol red is added to cells growing attached at the bottom of a tissue-culture treated plate (microtiter tray) to cover the cells with a first layer of agarose. As discussed above, any type of tissue culture treated tray or dish known to those skilled in the art may be used in practicing the present invention. While the agarose is gelling, wells are formed on the surface of the agarose. In one embodiment of the invention, a pin-lid (available from Polyfiltronics, Inc.) is added to the plate, to form wells, for example 96-wells, on the surface of the agarose. Test compounds are then added into the wells (6μl/well) and the plates are incubated at 37°C for 8-24 hours. A plurality of test compounds may be used, adding one or more compounds to each well in the surface of the first layer of agarose. Test compounds may be used, for example, to determine their capability to modulate the - interaction between the cells and a substrate added later. By "modulate" is meant that the test compound may inhibit or lower cellular activity, or may activate or increase a cellular activity. After the incubation, a second layer of LMP agarose containing the substrate(s) for an alkaline phosphatase reaction is added on top of the first layer and allowed to gel at room temperature for 15 minutes. Alternatively, the substrate(s) may be added to the tray in buffer, without agarose (e.g., a liquid medium). The substrate layer is in carbonate buffer, preferably about pH 9.5, in order to detect the activity due to the alkaline phosphatase secreted by responsive cells. Several substrates known and available in the art that give a good dose response may be used in practicing the present invention. Two such substrates, for example, that have been shown to give a good dose-response are: (1) BCIP (5-Bromo- 4-Chloro-3-indolyl Phosphate)/NBT (NitroBlue Tetrazolium) mixture, or (2) MUP (Methyl Umbelliferyl Phosphate). The plates are incubated at 37°C for 4 hours (MUP) or 24 hours (BCIP/NBT). The read-out is a blue (BCIP/NBT) or fluorescent (MUP) zone in and around the well containing the active compound. The fluorescent signal (MUP) peaks at 4 hours and is relatively stable up to 24 hours; the colorimetric signal appears after 24 hours and is stable at 37°C for at least 3 days. Plates that have developed the blue activity zone can be stored at 4°C for at least 6 months. For most assays, an activity zone can be detected by the eye, although an image analyzer may be used in some instances. In an alternate embodiment of the present invention, the cells are suspended in medium plus 0.4% agarose and plated in well plates, for example 24- or 96-well plates. The agarose is allowed to gel and the reporter assay is run in the wells. Test compounds and substrate can be added to the wells directly. A second layer of agarose is not necessary in this embodiment. Additionally, the method of the present invention can be used to find enzyme inhibitors or activators. For example, the present invention encompasses a method of assaying activity of an enzyme comprising: (a) providing said enzyme to at least one container; (b) adding an agarose solution to said container to form a first agarose layer; (c) forming at least one well on the surface of said first agarose layer; (d) adding at least one test compound into said at least one well on the surface of said firs ' t agarose layer; (e) adding a second layer of agarose solution comprising at least one substrate on top of said first agarose layer to form a second agarose layer; and (f) detecting any activity of said enzyme in response to said at least one substrate in said second agarose layer. Alternatively, the present invention encompasses a method of assaying activity of an enzyme comprising: (a) incorporating said enzyme into an agarose solution to form an enzyme/agarose mixture; (b) depositing an amount of said enzyme/agarose mixture into at least one well of a multiple well plate; (c) allowing the enzyme/agarose mixture to gel; (d) adding at least one test compound into said at least one well; (e) adding a solution comprising at least one substrate into said at least one well; and (f) detecting any activity of said enzyme in response to said at least one substrate. The following examples are for illustrative purposes only and do not limit the scope of the present invention in any way. Example 1 HEK293 cells are a Human Embryonic Kidney cell line which grow as a monolayer attached to tissue-culture treated plastic. This cell line was stably transfected with the receptor for glucagon-like peptide 1 ("GLP-1"), as well as with the cDNA for Secreted Alkaline Phosphatase ("SEAP") as a reporter gene, under the control of the Cyclic AMP Responsive Element ("CRE"). Agonist binding activates the GLP-1 receptor, and leads to accumulation of cyclic AMP in a G-protein coupled pathway; cAMP in turn activates the transcription of the SEAP gene and secretion of alkaline phosphatase. This cell line, received from Cadus Pharmaceuticals, will be referred to as HEK/GLP1R cells. Cells were propagated in DMEM GlutaMAX I medium (Gibco BRL) containing 10% Newborn Calf Serum (Gibco BRL), 0.2 mg/ml Zeocin (Invitrogen) and 0.4 mg/ml Hygromycin B (Calbiochem). This medium is referred to hereinafter as "complete medium". Cells were harvested using Cell Dissociation Solution (Gibco BRL) and resuspended in complete medium at 0.5-lxl0 6 cells/ml. 20 ml were added per plate on Polyfiltronics plasma-coated plates (microtiter trays). Cells were allowed to grow ' to confluence for 2-4 days at 37° C, in 5% CO 2 atmosphere. When the cells reached confluence, they were subjected to serum starvation overnight to deplete the intracellular pools of cAMP and therefore reduce the background. The complete medium was aspirated from the plate and the cells were gently rinsed with 10 ml PBS. The complete medium was replaced with a DMEM medium without serum and without phenol red ("starvation medium") containing 1% Low Melting Point Agarose (27ml). A 3% Low Melting Point (LMP) agarose stock was prepared in distilled sterile H 2 O using a microwave oven and allowed to cool to 48°C. This was added to the 37°C starvation medium to a final concentration of 1%. 50μM homo-arginine was added from a 50 rtiM stock in H 2 0, to inhibit the activity of non specific alkaline phosphatases. While the agarose was gelling, a pin-lid (available from Polyfiltronics Inc.) was applied to the plates to form sample wells on the surface of the agarose. The agarose was allowed to gel with the pin-lid at room temperature for 15 min., after which the plates were incubated at 37°C in humidified 5% CO2 atmosphere. Cell viability was tested in selected plates by adding a solution of 0.04%o Trypan blue on top of the agarose layer. More than 99% of the cells are viable after 2 days under an agarose layer, based on microscopic observation using Trypan blue. After an overnight serum-starvation, test compound was added (6μl/well) and the plate was incubated at 37°C for 24 hours. At the end of the incubation, 15 ml of LMP agarose containing the substrates for the detection reaction was added, forming a second agarose layer on top of the first layer. The second layer of LMP agarose is used to avoid detachment of cells. The substrate is preferably added in the second layer of agarose, although substrate may be added in liquid form. (The inventors found that if the substrate in a liquid solution is used the cells are lifted from the bottom of the plate to the surface of the agarose - probably reflecting the poor adhesion capacity of this cell line especially after serum starvation). The substrate solution contained 1% LMP agarose, 160 μg/ml BCIP (5-Bromo-4-Chloro-3-indolyl Phosphate), 2 μg/ml NBT (NitroBlue Tetrazolium), and 50 μM homo-arginine, in 0.1M carbonate buffer, at a pH 9.5. An alternative substrate, for fluorescence readout, contained 50 μg/ml MUP (Methyl Umbelliferyl Phosphate) instead of the 160 μg/ml BCIP (5-Bromo-4-Chloro-3-indolyl Phosphate) and 2 μg/ml NBT (NitroBlue Tetrazolium). After addition of the substrate(s), the plates were incubated at 37°C for 4 hours (MUP) or 1-3 days (BCIP/NBT). The readout was a blue (BCIP/NBT) or a fluorescent (MUP) zone in and around the wells containing the active compounds. (Figure 1) This method was developed using Glucagon-like Peptide 1 (7-36), amide as agonist. This is the physiologic agonist of the GLP-1 receptor. Using this method we detected cellular responses to the Glucagon-like Peptide (7-36), amide (GLP-1, Bachem) in a dose-related manner using concentrations from 1.5 nM to 1.5 μM of ligand. Example 2 In this example HEK293 cells transfected with the Glucagon receptor instead of the GLP-1 receptor were used. This cell line was received from Cadus Pharmaceuticals. The microtrays were prepared as described in Example 1. After overnight serum-starvation, dilutions of glucagon were applied into the agarose wells and incubated for 24 hours. The substrate BCIP/NBT was applied as in Example 1 and the active wells were scored after 24 hour incubation. A positive signal (blue zone) can be detected using Glucagon concentrations between 1 nM and 1 μM. Example 3 In this example a different cell line was used. Baby Hamster Kidney ("BHK") cells were transfected with the Glucagon receptor and the CRE-SEAP construct (see Example l)(Cadus Pharmaceuticals). These cells were also grown as adherent cell line, and the protocol used is the same as in Example 1. A positive signal (blue zone) can be detected using Glucagon concentrations between 1 nM and 1 μM. Example 4 As described above, an additional embodiment of the present invention comprises an assay wherein cells are embedded in agarose. In this example, HEK293- GLP1R cells were grown in T175 flasks. The medium was removed from the flasks, the cells were rinsed with PBS, and then 50 ml of starvation medium (see Example 1) was added to the flasks. After 16 hours the cells were harvested using Cell Dissociation Solution (Sigma). Approximately 8x10 6 cells were mixed with 1.3ml of 2.5%o LMP agarose, 400μl of 2% gelatin and 8μl of 50mM homo-arginine in 8 ml of starvation medium (final lxlO 6 cells/ml, 0.4% 0 LMP agarose, 0.1% gelatin, 50μM homo-arginine). All components were previously equilibrated to 37°C to avoid immediate gelling. 500μl of the cell/agarose mixture were added per well of a 24-well plate (Corning). The plate was allowed to gel at room temperature for 30 min. lOμl of GLP-1 (7-36) amide was added to each well, and the 24-well plate was incubated at 37°C for 6 hours. 500μl of the substrate mix (160μg/ml BCIP, 2μg/ml NBT, 50μM homo- arginine), or 50μg/ml MUP in carbonate buffer, pH 9.5, was added to each well. Since the cells were incorporated into the agarose, there is no need to add the substrate in a second agarose layer. The plate was incubated at 37°C for 4-24 hours (MUP), or 1-4 days (BCIP/NBT). The readout was a blue (BCIP/NBT) or a fluorescent (MUP) in the wells containing the active compounds. (Figure 2). Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

Claims: We claim: 1. A method of assaying activity of a mammalian cell sample comprising (a) providing said cell sample to at least one cellular container; (b) adding an agarose solution to said cellular container to form a first agarose layer; (c) forming at least one well on the surface of said first agarose layer; (d) adding at least one test compound into said at least one well on the surface of said first agarose layer; (e) adding a second layer of agarose solution comprising at least one substrate on top of said first agarose layer to form a second agarose layer; and (f) detecting any activity of said cell sample in response to said at least one substrate in said second agarose layer. 2. A method of assaying activity of a cell sample comprising (a) incorporating said cell sample into an agarose solution to form a cell/agarose mixture; (b) depositing an amount of said cell/agarose mixture into at least one well of a multiple well plate; (c) allowing the cell/agarose mixture to gel; (d) adding at least one test compound into said at least one well; (e) adding a solution comprising at least one substrate into said at least one well; and (f) detecting any activity of said cell sample in response to said at least one substrate. 3. The method of claim 2 wherein said multiple well plate is selected from the group consisting of a 24-well plate and a 96-well plate. 4. The method of claim 1 wherein said at least one test compound is capable of modulating cellular activity. 5. The method of claim 1 wherein a different test compound is added to each of at least one well on the surface of said first agarose layer. 6. The method of claim 1 wherein said test compound is an agonist. 7. The method of claim 2 wherein said at least one test compound is capable of modulating cellular activity. 8. The method of claim 2 wherein a different test compound is added to each of at least one well on the surface of said first agarose layer. 9. The method of claim 2 wherein said test compound is an agonist. 10. The method of claim 1 wherein said at least one test compound comprises a peptide library. 11. The method of claim 2 wherein said at least one test compound comprises a peptide library. 12. A method of assaying activity of an enzyme comprising (a) providing said enzyme to at least one container; (b) adding an agarose solution to said container to form a first agarose layer; (c) forming at least one well on the surface of said first agarose layer; (d) adding at least one test compound into said at least one well on the surface of said first agarose layer; (e) adding a second layer of agarose solution comprising at least one substrate on top of said first agarose layer to form a second agarose layer; and (f) detecting any activity of said enzyme in response to said at least one substrate in said second agarose layer. 13. A method of assaying activity of an enzyme comprising (a) incorporating said enzyme into an agarose solution to form an enzyme/agarose mixture; (b) depositing an amount of said enzyme/agarose mixture into at least one well of a multiple well plate; (c) allowing the enzyme/agarose mixture to gel; (d) adding at least one test compound into said at least one well; (e) adding a solution comprising at least one substrate into said at least one well; and (f) detecting any activity of said enzyme in response to said at least one substrate.

Inventors: BROWN DANIEL M, CIVOLI FRANCESCA, BROWN, DANIEL, M., CIVOLI, FRANCESCA
Applicants: SQUIBB BRISTOL MYERS CO, BRISTOL-MYERS SQUIBB COMPANY
Application year: 1999
Main classification: G01N33
IPC codes: C12N5, C12Q1, C12R1