USE OF THE JAK-STAT SYSTEM IN CULTURED CELLS TO TRACE EFFECTS OF TESTED COMPOUNDS

Abstract: The invention relates to the use of a JAK-STAT system in cultured cells to trace effects of biologically active entities on living organisms by monitoring any prolongation of the JAK-STAT signal as well as a method to trace said effects.

Description: NEW USE OF THE JAK-STAT SYSTEM BACKGROUND OF THE INVENTION Cellular stress is believed to be an important part of a pathological cellular response. Such cellular stress can be inflicted by several different factors/situations such as oxygen depletion, metabolic dysfunction or chemical compounds. A cellular stress causes the cell to alter its cellular function by an interference with signalling molecules. A prolonged cellular stress can cause disease manifestations in e.g. cardiovascular and metabolic disorders. One of the key signaling pathways in many cells consists of the JAK-STAT pathway. Janus Kinases (JAKs) are intra cellular tyrosine kinases and Signal Transducers and Activators of Transcription (STATs) are transcription factors activated by tyrosine phosphorylation and again different molecular forms. The JAK-STAT pathway is preferentially activated by the, so-called, cytokine system. Cytokines include e.g. growth hormone (GH), prolactin (Prl), leptin, erytropoetin (Epo), colony stimulation factors, different interleukins and interferons. All of these activate the JAK-STAT pathway through specific membrane bound receptors and it its relevant to note that different forms of JAK molecules exist such as JAK1, JAK2, JAK3 and TYK1. Also different STAT molecules have been isolated, e.g. STAT1, STAT2, STATS, STAT4, STAT5, STAT5a, STAT5b and STAT6. Individual JAK-STAT combinations can be used in a specific signal transduction chain (for reviews, see references 1 and 2). The JAK- STAT pathway also includes other signaling molecules e.g. MAP kinases and IRS. According to this invention the JAK-STAT pathway is defined as cellular effects induced by JAK. An important aspect of cytokine signaling by receptors that use the JAK-STAT pathway is the mode of shutting the receptor signal off. Recently a new class of molecules, Suppressors of Cytokine Signaling (SOCS), has been described. SOCS proteins are induced as a consequence of a JAK-STAT activation and function to turn individual cytokine receptors off. As with other molecules in the JAK-STAT pathway several different SOCS have been described e.g. SOCS1, SOCS2, SOCS3 and CIS (references 3 and 4). THE PRESENT INVENTION As one outcome of our long investigations of cytokine signalling, we have surprisingly, become aware of that the JAK-STAT pathway can be modulated by a number of different chemical compounds. This fact became evident after a detailed investigation of shut off mechanisms of GH signals. By said research work we found that the JAK-STAT-SOCS pathway is extremely sensitive to chemical compounds that cause a cellular stress. A probable reason that this pathway displays such a sensitivity resides in the fact that the shut off mechanism i.e. the SOCS function is executed by a very rapid turnover of the SOCS proteins. The present invention relates to an assay system, based on the JAK-STAT-SOCS pathway, which can be used to monitor compounds that cause a cellular stress. Cellular stress can be monitored in several other ways e.g. by measuring heat shock response proteins or apoptosis. However, the analysis of the JAK-STAT-pathway in relation to actions of chemical compounds is advantageous as the JAK-STAT-pathway is an important intra cellular pathway and pharmacological compounds can influence the JAK-STAT pathway without causing overt heat shock responses and apoptosis. The technology described in the present invention can be used to screen chemical, biological, and physical entities, and particularily established drugs or potential drug candidates, for their effects on the JAK-STAT-SOCS pathway. If a particular compound scores positive in this assay it may cause cellular stress, which might lead to side effects if intact organisms are treated with said compound. In the prior art the existence and biological importance of the JAK-STAT-SOCS pathway is well documented. We have, however, not been able to find any documentation in the prior art showing that this pathway can be used as a read out for side effects of biologically active entities such as drugs. It is becoming increasingly important, at an early stage, to identify whether biologically active entities or other entities give side effects or not. It might be any compound, biological or chemical or physical agent to which a living organism can be exposed. Biologically active entities are defined as any molecule that have or in the future may have a therapeutic, nutritional or environmental utility or on the contrary be detrimental to the organisms health. Said entities include drugs used in the clinic, candidate drug compounds with a potential clinical use, natural or synthetic nutritional or environmental chemicals that causes biological effects, radiation and similar means that might influence a living organism. As exemplified below an analysis of the JAK-STAT-SOCS pathway can be used to reveal potential side effects of test compounds. The bearing principle is to first activate a JAK- STAT-SOCS pathway in a cell and then add a test compound. As stated above, many factors can activate the JAK-STAT pathway in cultured cell and the actual nature of these, as well as doses required, are known to any person skilled in the art. A non-limiting list of factors that activate the JAK-STAT pathway include growth hormone, prolactin, erytropoetin, leptin, erythropoetin, a variety of interleukins, colony stimulation factors and interferons. A person skilled in the art also knows that there are several different methods available to detect components of the JAK-STAT pathway, these include protein analysis of activated, i.e. phospho- rylated, JAKs or STATs as well as the measurement of levels of SOCSs with antibody techniques or related protein-binding techniques. Alternative assays are gel shift analysis or similar DNA binding techniques that can be used to detect STAT DNA binding and different types of hybridisation techniques that can be used to analyse SOCS mRNA levels. In addition genetic tagging techniques where components in the JAK-STAT-SOCS pathway are modified with a tag to simplify detection in the cell are applicable. The present invention can thus be carried out using a variety of methods. The terms JAK-STAT pathway or JAK-STAT system is herein defined as any cellular event that depends on the activation of JAK. There are also other cellular parameters than JAKs or STATs that can be used to measure a cytokine signal such as activation of MAP kinases and IRS. The principle of the invention relies in the use of isolated cells in which the JAK-STAT pathway is activated. In case a cell is exposed to a test compound the interference of such a test compound with the JAK-STAT pathway will be detected by a temporal analysis of JAK phosphorylation or by STAT-5 DNA binding (or alternative methods) or by a temporal analysis of SOCS protein/mRNA levels. If a compound interacts in the JAK-STAT pathway it will score positive if the time for JAK-STAT activation is prolonged or if levels of SOCS are reduced. The term "temporal analysis" means a comparison of the duration of the JAK-STAT signal at the activation of the JAK-STAT pathway in the presence of a biologically active entity with the duration of a JAK-STAT signal caused by a cytokine in the absence of a biologically active entity. A significant change, normally a prolongation of the duration of the JAK-STAT pathway is assayed for. The use of the different end point read outs, here exemplified as STAT activation and SOCS levels can be converted into high through-put assays by a person skilled in the art. In practice, cells can be grown, preferably in a 96 well format, then exposed to a cytokine such as growth hormone and either the STAT activation or levels of SOCS measured. Initial experiments are needed to establish the time kinetics for cytokine activation of the JAK-STAT pathway with regard to the particular cytokine used and the particular cell line used. It is to be expected that the activation is transient which means that one time point where the JAK-STAT pathway is maximally active can be identified and another, later, time point, where the signal has been significantly reduced. If GH is used as the stimulatory cytokine in liver cells the activation of the JAK-STAT pathway is maximal within a time period of one hour (lh) and the signal is significantly reduced after four hours (4h). This information may vary between different cells and cytokines. After the initial parameters have been established, test compounds can be added in conjunction with the activation of the JAK-STAT pathway. A suitable time period to analyse components of the JAK-STAT pathway in the GH system can be 4h. At this time period one can assay for a prolongation of the JAK-STAT pathway consisting of e.g. maintained STAT DNA binding and/or JAK phosphorylation and/or levels of SOCS mRNA or SOCS protein. The use of different cell types i.e. derived from different cell linages, makes it possible to perform a risk assessment if a test compound has a potential to cause side effects through the JAK-STAT system. With different cell linages is meant that cells can be of different embryo- logical origin such as from endoderm, ektoderm, mesenchyme and so forth. Primary cell cultures as well as established cell lines can be used. The main features will be to use cells commonly used in the laboratory and a list of such cells can be obtained from the ATCC (American Tissue Culture Collection). In certain cases it might be useful to analyse cells, e.g. fibro- blasts or tumor cells, from individual patients to characterise the response to chemical compounds. In the examples below, the model has been JAK2 activation of STAT-5 in liver cells; a prolongation of STAT-5 DNA binding has been the read out for test compounds or a reduction of SOCS 2, SOCS 3 and CIS. The time kinetics as well as the model used in the examples are only meant as illustrations of the invention and are not intended to limit the use of this invention. Variations in experimental designs such as the used cell, the time kinetics applicable and/or the component of the JAK/STAT/SOCS pathway used as an end point measurement, fall within the scope of the present invention as long as the screening system is cell based, a cell in which the JAK-STAT-SOCS system is activated used and a temporal analysis of the effects of the test compounds on this pathway conducted. All of the features disclosed in the claims are herewith included by reference. Figure 1 relates to the transient nature of STAT5 activation by GH measured using Gel mobility shift assay. BRL4 cells were grown for 8 hours under serum free conditions. Recombinant hGH (50nM) was then added, the cells were harvested at the indicated points of time and nuclear extracts were prepared. STAT5 and STAT1 binding activity were determined using electrophoresis mobility shift assay with a 32 P-labeled SPIGLE1 probe. Relative DNA binding levels were measured by autoradiography. This experiment shows that GH activates STAT5 DNA binding in a transient fashion. After GH stimulation a STAT DNA binding activity is rapidly increased followed by a rapid decrease to background level after 2 hours. Table 1. Effect of various drugs on GH induced STAT-5 activation Cells were grown to 90% confluence, starved for 8h and incubated for 30 min with different drugs: D609 (50 mg/ml) (B) H7 (200uM), BAPTA-AM (25nM), Verapamile (100 uM), A23187 (5nM), Actinomycin D(lug/ml) Calphostin C (ImM) and Bisindolylmaleimide I (2mM). Recombinant hGH (50 nM) was then added, the cells where harvested at the indicated times (30 min or 4h) and nuclear extracts were prepared. STAT5 binding activity determined using electrophoresis mobility shift assay with 32 P-labeled SPIGLE1 probe. Activated STAT5 was visualized and quantitated by autoradiography. The table summarized the results obtained with different drugs. Significant detection of STAT5 DNA binding is denoted as +. None of the drugs caused STAT activation when added alone. When added in combination with GH the drugs didn't have major effects on the binding activity when measured after 15 min but some of said drugs could prolong the GH-induced STAT5 activation at the 4h period. Table 2. Effect of various drugs on GH-induced activation of SOCS Cells were grown to 90% confluence, starved for 8h and incubated for 30 min with different drugs as described in Table 1. Recombinant hGH (50 nM) was then added and cells were harvested at different times and used to prepare total RNA. SOCS-2, SOCS-3 and CIS mRNA was measured using solution hybridization assay in a time course study. SOCS proteins were analysed using Western blots. The table summarized the results obtained with different drugs. EXAMPLES 1. An assay to monitor drug induced effects on STAT activation BRL-4 cells (a rat liver cell) that respond to GH were used (5). GH was added to these cells at a concentration of 100 mg/ml. As shown in Fig 1 this cause a transient activation of STAT-5 measured in a gel shift analysis (6); a maximum STAT-5 binding was observed after 30 minutes and after 4h the response was lost. A large variety of chemical compounds were tested for their ability to prolong STAT-5 activation and a surprising finding was that so may compounds could prolong a STAT-5 activation in the presence of GH. In the absence of GH these compounds did not exert any effect. As listed in Table 1 such compounds included CHX, D609, Bapta and H7. These compounds have very different mechanisms of action and such actions include inhibition of protein translation, tyrosine phosphorylation, calcium and lipases. The finding that all of these prolong STAT-5 activation indicates a relation between the action of the compounds as well as a prolongation of STAT5 and cellular stress. By the use of this cellular system a compound can be categorized for its ability to prolong STAT5 or not. Such information is important in the drug development process. One embodiment of the present invention is a cell based drug screen by which a drug might be categorized for a possible interaction with the JAK-STAT-SOCS pathway. A cytokine receptor would here be activated in a cell by one cytokine or a combination of different cytokines. Such cytokines could consist of proteins from varying sources and of varying purity, protein variants of cytokines or possibly of analogues of cytokines. The drug of interest is added in conjunction with (before, together or after) the cytokine stimulation. Subsequently the effect of said drug could be detected by analysing the STAT proteins in a period of time. Interference in the pathway will result in a prolongation of a cytokine response on STAT DNA binding activity or a prolongation of the presence of STAT tyrosine phosphorylation. As is well known to a person skilled in the art, such assays can be conducted in an automated or semi-automated fashion suitable for screening purposes. 2. An assay to monitor drug induced effects on SOCS expression The effect of different drugs to cause a prolongation of STAT-5 activation in example 1 is most easily explained by the possibility that SOCS proteins are reduced by drugs, a reduced level of SOCS would subsequently lead to a diminished "cytokines receptor shut off. This concept was tested by analysing different SOCS mR As using the technique of RNase protection/solution hybridisation (7,8). As shown in Table 2 several different drugs could reduce SOCS mRNA. In one case (BAPTA) the effect was, however, the opposite, namely an increase of SOCS mRNA. For this reason the effect of BAPTA on protein synthesis was measured and was found to be reduced. Consequently, all treatments described in table 2 will ultimately result in a reduction of SOCS proteins. According to one embodiment of the present invention the SOCS protein levels is subsequently measured. This can be achieved with different types of e.g. protein binding assays. In a screening assay, test compounds will score positive if they cause a reduction of SOCS proteins including SOCS1, SOCS2, SOCS3 and CIS. List of references. l.Wood TJ, Haldosen L-A, Sliva D, Sundstrδm M and Norstedt G Stimulation of kinase cascades by growth hormone; a paradigm for cytokine signalling. In; Progress in Nucleic Acid Research and Molecular Biology, 57: 73-94 (1997) 2.Heim MH. The Jak-STAT pathway: cytokine signalling from the receptor to the nucleus. J Recept Signal Transduct Res 1999 Jan-Jul;19(l-4):75-120 3. Starr R, Willson T A, Viney E M, Murray L J L, Rayner J R, Jenkins B J, Gonda T J, Alexander W S, Metcalf D, Nicola N A and Hilton D J A family of cytokine-inducible inhibitors of signaling Nature, 387: 917-921 (1997) 4. Endo T A, Masuhara M, Yokouchi M, Suzuki R, Sakamoto H, Mitsui K, Matsumoto A, Tanimura S, Ohtsubo M, Misawa H, Miyazaki T, Leonor N, Taniguchi T, Fujita T, Kanakura Y, Komiya S and Yoshimura A A new protein containing SH2 domain that inhibits JAK kinases Nature, 387: 921-924 (1997) 5. Norstedt G., Enberg B., Francis S.M., Hansson A.,Hulthen A, Lobie P.E., Sliva D., Wood T.J.J. Billestrup N. Cell transfection as a tool to study growth hormone action. Proc. Soc. Exp. Biol. Med. 206(3) 181 , 1994 6. Sliva D., Wood T.J., Schindler C, Lobie P.E., Norstedt G. Growth hormone specifically regulates serine protease inhibitor gene transcription via gamma-activated sequence. like DNA elements. J. Biol. Chem. 269:26208,1994. 7. Moller C, Arner P, Sonnenfeld T, Norstedt G: Quantitative comparision of insulin-like growth factor I (IGF-I) and IGF-II messenger RNA levels in human and rat tissues analysed by a solution hybridization assay. J. Molecular Endocrinology, 7:213, 1991. 8. Tollet-Egnell P, Flores-Morales A, Stavreus-Evers A, Sahlin L, Norstedt G. Growth hormone regulation of SOCS-2,SOCS-3 and CIS messenger ribonucleic acid expression in the rat. Endocrinolgy 140 3693- 3704 (1999).

Claims: CLAIMS 1. Use of a JAK-STAT system in cultured cells to trace effects of biologically active entities by adding said biologically active entities onto cells where the JAK-STAT pathway has been activated, whereby the effects of the biologically active entities can be traced by monitoring a prolongation of the duration of a JAK-STAT signal and be compared to the duration of the JAK-STAT signal in the absence of a biologically active entity. 2. The use according to claim 1 wherein the biologically active entities include compounds of different chemical compositions with a know or potential therapeutical use, such as drugs or drug candidates and chemical compounds of natural or synthetic origin that such as nutritional and environmental chemical entities and radiation and similar physical agents that can influence living organisms. 3. The use of cells according to claim 1 which cells are characterised by an eukaryotic origin, selected from different types of cell linages, consisting of primary or established cell lines and wherein components of the JAK-STAT pathway is present either naturally or as a consequence of gene transfer. 4. The use of cells according to claim 1 wherein the JAK-STAT pathway has been activated by any compound that normally or artificially can activate said cellular pathway, activators are selected from the group of cytokines or mimics of cytokines. 5. The use according to claim 4 of activators selected from growth hormone (GH), prolactin (Prl), erytropoetin (Epo), leptin, colony stimulating factors, interleukins and interferons or analogues thereof. 6. The use according to claim 4 and 5 of activators in biologically active dose ranges either as single molecules or as mixtures of molecules. 7. The use of cells according to claim 1 wherein the activation of the JAK-STAT pathway is monitored by an analysis of a cytokine induced modification of said pathway, said modification asseyed for by detecting protein phosporylation, DNA binding, altered levels of proteins, analysis of protein transport and/or analysis of tagged proteins. 8. The use of cells according to claim 1-7 wherein the analysis of the JAK-STAT pathway is monitored by an analysis of proteins dependent on JAK activation. 9. The use of cells according to claim 1-8 wherein the analysis of the JAK-STAT pathway comprise different forms of JAKs such as JAK1, JAK2, JAK3 and TYK1 characterised by being receptor associated tyrosine kinases, different forms of STATs, such as STAT1 , STAT2, STAT3, STAT4, STAT5a, STAT5b and STAT6, characterised by binding to STAT DNA elements, different forms of SOCS such as SOCS1, SOCS2, SOCS3, SOCS4 and CIS, characterised by SOCS motifs and SH2 domains, endogenously expressed in cells or artificially expressed after gene transfer. 10. The use of cells according to claims 1-9 wherein the biologically active entities are screened for their capacity to prolong a JAK-STAT signal, said prolongation selected from time periods ranging from minutes to several hours and the assay to detect said prolongation is selected from any type of assay protocol of a low, medium or high through put nature. 11. A method to trace effects of biologic active entities on living organisms by activating the JAK-STAT pathway in cells of the living organism, adding said biological active entity onto the activated cells and monitoring any potential prolongation of the duration of the JAK- STAT signal compared to the JAK-STAT signal obtained in the absence of the biologic active entity. 12. The method according to claim 1 1 wherein the cells are characterised by being of an eukaryotic origin, selected from different types of cell linages, consisting of primary or established cell lines and wherein components of the JAK-STAT pathway is present either naturally or as a consequence of gene transfer. 13. The method according to claim 1 1 wherein the cells are activated by any compound that normally or artificially can activate said cellular pathway, and selected from the group of cy- tokins or mimics of cytokines. 14. The method according to claim 11 wherein the activation of the JAK-STAT pathway is monitored by an analysis of a cytokine induced modification of said pathway, said modification asseyed for by detecting protein phosporylation, DNA binding, altered levels of proteins, analysis of protein transport and/or analysis of tagged proteins. 15. The method according to claims 1 1 -14 wherein the biologically active entities are screened for their capacity to prolong a JAK-STAT signal, said prolongation selected from time periods ranging from minutes to several hours and the assay to detect said prolongation is selected from any type of assay protocol of a low, medium or high through put nature.

Inventors: FLORES MORALES AMILCAR, NORSTEDT GUNNAR, FLORES MORALES, AMILCAR, NORSTEDT, GUNNAR
Applicants: FLORES MORALES AMILCAR, NORSTEDT GUNNAR, SAHLTECH AB, FLORES MORALES, AMILCAR, NORSTEDT, GUNNAR, SAHLTECH I GOETEBORG AB
Application year: 2000
Main classification: C12Q1
IPC codes: C12Q1, G01N33