Cell model and method for screening c-Fms kinase inhibitors

Abstract

The present invention provides a cell model and a method for screening c-Fms tyrosine kinase inhibitors. Specifically, the present invention provides a cell that expresses macrophage colony stimulating factor receptor and STAT1 protein simultaneously. The present invention further provides a method for screening c-Fms tyrosine kinase inhibitors, a method for evaluating the inhibiting activity of a compound or a composition against c-Fms tyrosine kinase, and use of the cell in screening c-Fms tyrosine kinase inhibitors. The cell model established in the present invention is sensitive, highly effective and reliable, and can be used in high-throughput screening and/or high-content screening of c-Fms tyrosine kinase inhibitors.

Claims

1. A cell strain, which was deposited in China General Microbiological Culture Collection Center, with an accession number of CGMCC No. 4688, and a deposit date of Mar. 22, 2011.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1: GFP-STAT1 nuclear translocation induced by hrM-CSF in cells expressing human c-Fms (FIG. 1A-B).

(2) FIG. 2: The dose-effect relationship of GFP-STAT1 nuclear translocation induced by hrM-CSF. n=6, EC.sub.50 is 43.503.68 ng/mL (FIG. 2A-B).

(3) FIG. 3: Reliability analysis of the drug screening system based on GFP-STAT1 nuclear translocation induced by hrM-CSF. n=6, the mean Z value is 0.734.

(4) FIG. 4: The effect of the time for treating cells with hrM-CSF on GFP-STAT1 nuclear translocation.

(5) FIG. 5: The effect of the seeding density of U2OS-GFP-STAT1/CSF-1R cells on GFP-STAT1 nuclear translocation.

(6) FIG. 6: A drawing shows the inhibitory effect of c-Fms inhibitor GW2580 on nuclear translocation in the cell model wherein GFP-STAT1 nuclear translocation is induced by hrM-CSF. (n=3; IC.sub.50 of GW2580 is 30.742.9 nM).

(7) FIG. 7: A drawing shows the inhibitory effect of tyrosine kinase inhibitor Sutent on nuclear translocation in the cell model wherein GFP-STAT1 nuclear translocation is induced by hrM-CSF. (n=3; IC.sub.50 of Sutent is 14.850.55 nM).

(8) FIG. 8: A drawing shows the inhibitory effect of tyrosine kinase inhibitor Imatinib on nuclear translocation in the cell model wherein GFP-STAT1 nuclear translocation is induced by hrM-CSF. (n=3; IC.sub.50 of Imatinib is 196.121.7 nM).

ABOUT THE REFERENCE OF DEPOSIT OF THE BIOLOGICAL MATERIAL

(9) The invention relates to the following biological material:

(10) Human osteosarcoma cell strain (U20S-GFP-STAT1/CSF-1R), which was deposited in China General Microbiological Culture Collection Center (CGMCC) on Mar. 22, 2011, with an accession number of CGMCC No. 4688. The deposit address is NO. 3 of yihaoyuan, Beichen West Road, Chaoyang District, Beijing 100101, China.

SPECIFIC MODE FOR CARRYING OUT THE INVENTION

(11) (1) The embodiments of the invention are illustrated in detail via the following examples. However, a person skilled in the art would understand that the following examples are only intended to illustrate the invention, rather than being considered to limit the scope of the invention. Under the circumstance wherein the specific techniques or conditions are not indicated in the Examples, the inventions are carried out according to the techniques or conditions described in the literature of the art (for example, Sambrook. J. et al., Molecular Cloning: A laboratory Manual, translated by Huang Peitang et al., 3rd edition, Science Press) or according to the specifications of the products. The reagents or apparatuses, of which the manufacturers are not indicated, are conventional products that are commercially available.

(12) Main drugs and reagents: Plasmid MiniPrep Kit, purchased from Omega Bio-tek Co.; restriction endonucleases, T4 ligase, purchased from TaKaRa Co.; liposome 2000, hoechst33342 dye, purchased from Invitrogen Co.; DMEM (High glucose), purchased from Gibco Co.; fetal bovine serum purchased from Tianjin Chuanye Biochemicals Co., Ltd.; neomycin (G418), puromycin, purchased from Merck Co.; hrM-CSF factor, purchased from Peprotech Co.; BSA, HEPES, purchased from Amresco Co.; all the kinase inhibitors used in the invention were synthesized by the inventors; the rest agents are analytical reagents produced in China.

(13) Main apparatus: In Cell Analyzer 1000 or In Cell Analyzer 2000 GE healthcare life science.

(14) Cell line: U2OS-GFP-STAT1 human osteosarcoma cell Thermo (Bio Image).

Example 1: Establishment of a Cell Line Stably Expressing Human c-Fms and STAT1

(15) (1) A eukaryotic expression vector pCORON/puro-cfms comprising full-length cDNA of human c-Fms (donated by Dr. Charles Sherr and Dr. Martine Roussel from St. Jude Children's Research Hospital, USA) was constructed, and a human osteosarcoma cell line wherein GFP and STAT1 were fused and expressed, was transfected with the vector by lipofectin.

(16) A new cell line stably expressing human c-Fms was obtained by means of resistance screening and limiting dilution. DMEM medium that contains 4500 mg/L glucose, 10% fetal bovine serum, 4 mM L-glutamine, 500 m/mL G418 and 2 g/mL puromycin was used, and the cell line was cultured in an incubator at 37 C., 5% CO.sub.2, 80% humidity.

(17) Compared with the cells prior to transfection, the established cell had no significant change in cellular morphology, and had a stable growth state.

(18) The cell line prepared in this example (i.e. the human osteosarcoma cell line of the present invention which was deposited with an accession number of CGMCC No. 4688) can be used as a cell model for screening tyrosine kinase inhibitors or evaluating the inhibiting activity of a compound against tyrosine kinase.

Example 2: Characterization of the Newly Established Cell Line

(19) The cell line prepared in the Example 1 was treated with recombinantly expressed human M-CSF(hrM-CSF), so as to activate c-Fms and induce GFP-STAT1 nuclear translocation. In the established cell line, human macrophage colony stimulating factor receptors were dimerized upon binding to the specific ligand M-CSF, and meanwhile the steric conformation was changed and the kinase domain was activated. The downstream signal pathway of c-Fms was activated by transphosphorylation. The phosphorylated GFP-STAT1 were also dimerized and entered nucleus, namely, nuclear translocation occurred.

(20) In order to quantitatively detect the extent of nuclear translocation of GFP-STAT1 induced by hrM-CSF, in this Example, in Cell Analyzer 1000 or In Cell Analyzer 2000 was used to obtain the cell image of GFP-STAT1 nuclear translocation. The cell image obtained was analyzed by using Nuclear Trafficking Analysis Module, wherein the translocation index represents the extent of nuclear translocation of GFP-STAT1. Translocation index equals to the ratio of the mean fluorescence intensity of nucleus to the mean fluorescence intensity of cytoplasm of the cells obtained per well. The steps were as follows: 1) seeding the cells to a 96-well culture plate (which was black and transparent at the bottom), culturing in an incubator at 37 C., 5% CO.sub.2, 80% humidity for 24 hours; 2) washing the cell twice with serum-free DMEM medium that contains 4500 mg/L glucose, 4 mM L-glutamine, 0.2% BSA and 10 mM HEPES (hereafter referred to as cell analytic liquid), wherein the cell analytic liquid was added at 100 L/well for each time; after that, adding 50 L cell analytic liquid to each well; 3) adding the cell analytic liquid that contains hrM-CSF at 50 L/well, to obtain a final hrM-CSF concentration of 100 ng/mL, and incubating the cells in an incubator at 37 C. for 30 minutes; 4) fixing the cells with 8% formaldehyde diluted with 1PBS solution and preheated at 37 C. at 100 L/well, and placing the plate in dark at room temperature for 20 minutes; 5) discarding the cell fixation solution, adding 1PBS solution that contains hoechst33342 at 200 L/well, placing the plate in dark at room temperature for 30 minutes; 6) obtaining the cell image by using In Cell Analyzer 1000, analyzing the extent of nuclear translocation of GFP-STAT1 by using Nuclear Trafficking Analysis Module.

(21) In the presence of hrM-CSF at a concentration of 100 ng/mL, significant GFP-STAT1 nuclear translocation occurred in the U2OS-GFP-STAT1/CSF-1R cells expressing human c-Fms, as compared to the U2OS-GFP-STAT1 cells expressing no human c-Fms (FIG. 1A-B).

Example 3: Determination of the Median Effective Concentration of hrM-CSF for Inducing GFP-STAT1 Nuclear Translocation

(22) The steps were as follows: 1) using the cells prepared in the Example 1 to prepare a cell suspension of 110.sup.5 cells/mL, seeding the cells to a 96-well culture plate (which was black and transparent at the bottom) at 100 L/well; 2) culturing the cells in an incubator at 37 C., 5% CO.sub.2, 80% humidity for 24 hours; 3) washing the cells twice with the cell analytic liquid at 100 L/well for each time, discarding the solution, and adding the cell analytic liquid at 50 L/well; 4) adding hrM-CSF diluted with the cell analytic liquid, to obtain a final concentration of 1, 3, 10, 30, 100, 300, 600, 1000 ng/mL respectively; 5) after incubating the cells in an incubator at 37 C., 5% CO.sub.2, 80% humidity for 30 minutes, adding the cell fixation solution preheated at 37 C. at 100 L/well, slightly shaking the culture plate to mix the mixture uniformly, and placing the plate in dark at room temperature for 20 minutes; 6) discarding the solution, adding 1PBS solution that contains hoechst33342 at 200 L/well, and placing the plate in dark at room temperature for 30 minutes; and 7) obtaining the cell image by using In Cell Analyzer 2000, and analyzing the extent of nuclear translocation of GFP-STAT1 by using Nuclear Trafficking Analysis Module.

(23) As shown in FIG. 2A-B, when the concentration of hrM-CSF was 10 ng/mL, nuclear translocation of GFP-STAT1 fusion protein began; when the concentration of hrM-CSF was 600 ng/mL, the extent of nuclear translocation of GFP-STAT1 reach the maximum. The S-type curve was fitted by Origin 7.5 software sigmoidal Fit to obtain the median effective concentration (EC.sub.50) of hrM-CSF, at which the c-Fms kinase in the U2OS-GFP-STAT1/CSF-1R cells was activated to induce GFP-STAT1 nuclear translocation, and which was 43.503.68 ng/mL.

Example 4: Evaluation of the Reliability of the U2OS-GFP-STAT1/CSF-1R Cell Model

(24) In this example, the reliability of the new cell model established in the example 1 was evaluated by Z factor. Z factor, as an important parameter for evaluating reliability of an experimental system, is widely applicable when evaluating the stability and reliability of high-throughput screening, high content analysis experimental systems. Z factor is calculated according to the following formula:

(25) $Z^{}=1-\frac{\left(3{}_{C+}+3{}_{C-}\right)}{.Math.{}_{C+}-{}_{C-}.Math.}$
wherein represents standard deviation, represents mean signal, C+ represents positive control, C represents negative control.

(26) Z factor is within 01. When Z is 0, it indicates that the experimental system is not tenable. When 0.5>Z>0, it indicates that the experimental system has poor stability and is not reliable. When 1>Z0.5, it indicates that the experimental system has good stability and reliability. If Z factor is 1, then the standard deviation between positive control and negative control is 0, which indicates an ideal experimental system (Ji-Hu Zhang, Thomas D. Y. Chung and Kevin R. Oldenburg. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays [J]. J Biomol. Screen. 1999.4: 67).

(27) The steps for evaluating the reliability of the experimental system established in the present invention were as follows: 1) preparing a cell suspension of 110.sup.5 cells/mL, seeding the cells to a 96-well culture plate (which was black and transparent at the bottom) at 100 L/well, and culturing the cells in an incubator at 37 C., 5% CO.sub.2, 80% humidity for 24 hours; 2) washing the cells twice with the cell analytic liquid at 100 L/well for each time, discarding the solution, and adding the cell analytic liquid at 50 L/well; 3) adding hrM-CSF diluted with the cell analytic liquid, to obtain a final concentration of 200 ng/mL, setting the cell analytic liquid containing no hrM-CSF as the control, and incubating the cells in an incubator at 37 C., 5% CO.sub.2, 80% humidity for 30 minutes; 4) adding the cell fixation solution preheated at 37 C. at 100 L/well, slightly shaking the culture plate to mix the mixture uniformly, and placing the plate in dark at room temperature for 20 minutes; 5) discarding the solution, adding 1PBS solution that contains hoechst33342 at 200 L/well, placing the plate in dark at room temperature for 30 minutes; 6) obtaining the cell image by using In Cell Analyzer 2000, analyzing the extent of nuclear translocation of GFP-STAT1 by using Nuclear Trafficking Analysis Module, and calculating the Z factor.

(28) In the presence of 200 ng/mL hrM-CSF, the screening system established in the present invention had a mean Z factor of 0.734 (FIG. 3).

(29) The result shows that the cell of the present invention is very reliable and feasible as a cell model for screening tyrosine kinase inhibitors or evaluating the inhibiting activity of a compound against tyrosine kinase.

Example 5: The Effect of the Time for Treating Cells with hrM-CSF on GFP-STAT1 Nuclear Translocation

(30) The steps were as follows: 1) using the cells prepared in the example 1 to prepare a cell suspension of 110.sup.5 cells/mL, seeding the cells to a 96-well culture plate (which was black and transparent at the bottom) at 100 L/well; and culturing the cells in an incubator at 37 C., 5% CO.sub.2, 80% humidity for 24 hours; 2) washing the cells twice with the cell analytic liquid at 100 L/well for each time, discarding the solution, and adding the cell analytic liquid at 50 L/well; 3) adding hrM-CSF diluted with the cell analytic liquid every 5 minutes, to obtain a final concentration of 200 ng/mL, setting the cell analytic liquid containing no hrM-CSF as the control, incubating the cells in an incubator at 37 C., and treating the cells with the cytokine for 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 minutes, respectively; 4) adding the cell fixation solution preheated at 37 C. at 100 L/well, slightly shaking the culture plate to mix the mixture uniformly, and placing the plate in dark at room temperature for 20 minutes; 5) discarding the solution, adding 1PBS solution that contains hoechst33342 at 200 L/well, and placing the plate in dark at room temperature for 30 minutes; and 6) obtaining the cell image by using In Cell Analyzer 2000, and analyzing the extent of nuclear translocation of GFP-STAT1 by using Nuclear Trafficking Analysis Module.

(31) Once hrM-CSF factor was added, the GFP-STAT1 nuclear translocation quickly occurred. During the period while the cells were treated with the factor from 5 to 30 minutes, the extent of GFP-STAT1 nuclear translocation increased continuously. At the point of 30 minutes, the extent of GFP-STAT1 nuclear translocation reached the maximum. After 30 minutes, as the time for treating the cells with the factor increased, the extent of GFP-STAT1 nuclear translocation reduced gradually instead (FIG. 4).

Example 6: The Effect of the Cell Seeding Density on GFP-STAT1 Nuclear Translocation

(32) The steps were as follows: 1) seeding the cells prepared in the example 1 to a 96-well culture plate (which was black and transparent at the bottom), at 1000, 2000, 4000, 6000, 8000, 10000, 12000, 14000 cells/mL respectively, and culturing the cells in an incubator at 37 C., 5% CO.sub.2, 80% humidity for 24 hours; 2) washing the cells twice with the cell analytic liquid at 100 L/well for each time, discarding the solution, and adding the cell analytic liquid at 50 L/well; 3) adding hrM-CSF diluted with the cell analytic liquid at 50 L/well, to obtain a final concentration of 200 ng/mL, setting the cell analytic liquid containing no hrM-CSF as the control, and incubating the cells in an incubator at 37 C. for 30 minutes; 4) adding the cell fixation solution preheated at 37 C. at 100 L/well, slightly shaking the culture plate to mix the mixture uniformly, and placing the plate in dark at room temperature for 20 minutes; 5) discarding the solution, adding 1PBS solution that contains hoechst33342 at 200 L/well, and placing the plate in dark at room temperature for 30 minutes; and 6) obtaining the cell image by using In Cell Analyzer 2000, and analyzing the extent of nuclear translocation of GFP-STAT1 by using Nuclear Trafficking Analysis Module.

(33) As shown in FIG. 5, when the cell seeding density increased from 2000 cells/well to 12000 cells/well and the cells were treated with 200 ng/mL hrM-CSF for 30 minutes, the induced extent of the GFP-STAT1 nuclear translocation reduced gradually. However, the reduction was not significant. The ratio of the mean fluorescence intensity of nucleus to the mean fluorescence intensity of cytoplasm in the blank control cells reduced as the cell seeding density increased.

Example 7: The Inhibitory Effect of GW2580 on GFP-STAT1 Nuclear Translocation in the Cell Model

(34) GW2580 is a selective macrophage colony stimulating factor receptor kinase inhibitor, which can effectively inhibit the phosphorylation of the receptor kinase. The c-Fms inhibitor screening model established in the Example 1 was used to evaluate the inhibitory activity of GW2580. The steps were as follows: 1) seeding the U2OS-GFP-STAT1/CSF-1R cells to a 96-well culture plate (which was black and transparent at the bottom), at 1.010.sup.4 cells/mL, and culturing the cells in an incubator at 37 C., 5% CO.sub.2, 80% humidity for 24 hours; 2) discarding the medium, and washing the cells twice with the cell analytic liquid at 100 L/well for each time, 3) adding the cell analytic liquid at 50 L/well; adding GW2580 dissolved in DMSO which was diluted with the cell analytic liquid at 50 L/well, and incubating the cells in an incubator at 37 C., 5% CO.sub.2, 80% humidity for 1 hour; 4) adding the cell analytic liquid that contains hrM-CSF at 25 L/well, to obtain a final hrM-CSF concentration of 200 ng/mL, and incubating the cells in an incubator at 37 C., 5% CO.sub.2, 80% humidity for 30 minutes; 5) adding the preheated formaldehyde (8%) diluted with 1PBS solution at 125 L/well, and placing the plate in dark at room temperature for 20 minutes; 6) discarding the solution, adding 1PBS solution that contains hoechst33342 at 200 L/well, and placing the plate in dark at room temperature for 30 minutes; obtaining the cell image by using In Cell Analyzer 2000, analyzing the extent of nuclear translocation of GFP-STAT1 by using Nuclear Trafficking Analysis Module; and fitting the S-type curve by Origin7.5 software sigmoidal Fit and calculating the IC.sub.50 value.

(35) The result was shown in FIG. 6. In the cell model for screening c-Fms inhibitors wherein hrM-CSF activated human c-Fms and induced GFP-STAT1 nuclear translocation, GW2580 could significantly inhibit GFP-STAT1 nuclear translocation (n=3; IC.sub.50=30.742.9 nM).

Example 8: The Inhibitory Effect of Sutent on GFP-STAT1 Nuclear Translocation in the Cell Model

(36) Sunitinib (SU11248, Sutent) is a multiple-targeted tyrosine receptor kinase inhibitor, and its main targets are vascular endothelial growth factor receptor (VEGFR), PDGFR, c-Kit. In clinical practice, sunitinib is mainly used to treat cancers such as malignant renal cell carcinoma, and gastrointestinal stromal tumor resistant to Imatinib. It is reported in literature that sunitinib can also effectively inhibit phosphorylation of c-Fms (Guo J, Marcotte Pa., McCall J O, et al. Inhibition of phosphorylation of the colony-stimulating factor-1 receptor (c-Fms) tyrosine kinase in transfected cells by ABT-869 and other tyrosine kinase inhibitors[J]. Mol. Cancer Ther. 2006, 5(4):1007-1013). Therefore, the inhibitory activity of sutent was evaluated by using the cell model for screening c-Fms inhibitors as established in the example 1. The activity of sutent was evaluated by using a method similar to that in example 7. The result showed that sutent could also effectively inhibit GFP-STAT1 nuclear translocation (n=3; IC.sub.50=14.850.55 nM, as shown in FIG. 7).

Example 9: the Inhibitory Effect of Imatinib on GFP-STAT1 Nuclear Translocation in the Cell Model

(37) Imatinib is a tyrosine kinase inhibitor developed by Novartis International AG, and is mainly used to treat myelogenous leukemia abnormally expressing Bcr-ab1 and gastrointestinal stromal tumor with abnormal c-Kit activity in clinical practice. The activity of imatinib was evaluated by using a method similar to that in example 7. The result showed that imatinib could also effectively inhibit GFP-STAT1 nuclear translocation (n=3; IC.sub.50=196.121.7 nM, as shown in FIG. 8).

(38) Although the specific modes for carrying out the Invention are described in detail, the skilled in the art would understand that various amendments and replacements may be made to the details on the basis of all the disclosed teachings, and the changes are within the scope of the present invention. The scope of the invention is defined by the attached claims and any equivalent thereof.