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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Biomol Screen. Author manuscript; available in PMC 2012 April 1.
Published in final edited form as:
PMCID: PMC3237679

Development of a high-throughput cell-based reporter assay for screening JAK3 inhibitors


JAK3 has become an ideal target for the therapeutic treatment of immune-related diseases, as well as for the prevention of organ allograft rejection. A number of JAK3 inhibitors have been identified by in vitro biochemical enzymatic assays, but the majority display significant off-target effects on JAK2. Therefore, there is an urgent need to develop new experimental approaches to identify compounds that specifically inhibit JAK3. Here, we showed that in 32D/IL-2Rβ cells, STAT5 becomes phosphorylated by IL-3/JAK2- or IL-2/JAK3-dependent pathway. Importantly, the selective JAK3 inhibitor CP-690,550 blocked the phosphorylation as well as the nuclear translocation of STAT5 following treatment of cells with IL-2, but not with IL-3. In an attempt to use the cells for large-scale chemical screens to identify JAK3 inhibitors, we established a cell line 32D/IL-2Rβ/6×STAT5 stably expressing a well-characterized STAT5 reporter gene. Treatment of this cell line with IL-2 or IL-3 dramatically increased the reporter activity in a high-throughput format. As expected, JAK3 inhibitors, CP-690,550 and JAK3 inhibitor VI, selectively inhibited the activity of the 6×STAT5 reporter following treatment with IL-2. By contrast, the pan-JAK inhibitor Curcumin non-selectively inhibited the activity of this reporter following treatment with either IL-2 or IL-3. Thus, this study indicates that our STAT5 reporter cell line can be used as an efficacious cellular model for chemical screens to identify low-molecular-weight inhibitors specific for JAK3.

Keywords: Assay development, Cytokine, JAK3 inhibitors, STAT5 reporter, high-throughput chemical screening


The Janus kinases (JAKs) play key roles in numerous cytokine- and growth factor-mediated signaling pathways.1, 2 In mammals, the JAK family has four members; JAK1, JAK2, JAK3, and TYK2. In particular, JAK3 is preferentially expressed in lymphoid cells and mediates signals through a common gamma (γc) chain shared by receptors for IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, implicating JAK3 function in the lymphoid development and the homeostasis of the immune system.3 Consistent with these observations, disruption of JAK3 or γc causes severe combined immunodeficiency in humans and mice,4, 5 and persistent JAK3 activation is correlated with human autoimmune disorders.6 Furthermore, JAK3 may also contribute to the pathogenesis of hematopoietic neoplasms. Somatic mutations in JAK3 were reported in minority of acute megakaryoblastic leukemia patients,7 in a childhood acute lymphoblastic leukemia (ALL) case,8 and in cutaneous T-cell lymphoma patients.9 Furthermore, functional analyses of a subset of these alleles showed that each of the mutations can cause lethal hematopoietic malignancies in animal models, suggesting that these activating alleles of JAK3 can contribute to the pathogenesis of various hematopoietic neoplasms.

Several JAK3 inhibitors have recently been developed and shown to function as a new class of immunosuppressive agents. In fact, two in particular- PNU15804 and CP-690,550-significantly prolonged survival in animal models for organ transplantations.10, 11 In addition, another inhibitor WHI-P131 effectively prevented mast cell-mediated allergic reactions as well as asthmatic responses in animal models.12 These studies raise the important issue that inhibition of JAK3 function may ameliorate the debilitating symptoms of patients with these diseases. However, these compounds display varying degrees of inhibition on JAK2, due at least in part to the significant structural homology between JAK2 and JAK3.13, 14 JAK2 knockout mice die during embryonic development due to the absence of definitive erythropoiesis and JAK2-/- cells fail to respond to erythropoietin, thrombopoietin, IL-3 or granulocyte/macrophage colony-stimulating factor.15 Consistent with an important role of JAK2 in normal hematopoiesis, high doses of JAK2 inhibitors in a clinical setting are associated with myelosuppression as an adverse side effect.16 Therefore, identifying highly selective JAK3 inhibitors with reduced JAK2 off-target effects remains an important challenge for the treatment of JAK3-dependent disorders. Here, we describe the generation of a myeloid cell line 32D/IL-2Rβ/6×STAT5 stably expressing a STAT5 reporter gene that can be used as an excellent cellular model for the discovery of selective JAK3 antagonists in a high-throughput format. Our cell-based experimental approach affords a simple, sensitive, and cost-effective mean for the identification of small molecule inhibitors with high specificity for JAK3 over JAK2.

Materials and Methods

Cell culture

Murine IL-3-dependent myeloid progenitor 32D cells stably expressing IL-2Rβ (32D/IL-2Rβ) were maintained in RPMI 1640 medium containing 10% FBS, 2mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin, and 5% WEHI-3 cell-conditioned medium as a source of IL-3. 32D/IL-2Rβ/6×STAT5 cells (this study) that stably express a STAT5 reporter gene were grown in the same medium but supplemented with 300 μg/mL hygromycin.

Development of a stable STAT5 reporter cell line

We obtained pGL3-3xSTAT5-Luc plasmid containing a triple repeat of the STAT5 consensus site corresponding to the β-casein gene promoter.17 We employed polymerase chain reaction (PCR) to amplify the promoter region containing a triple repeat of the STAT5 consensus site using pGL3-3xSTAT5-Luc plasmid as a template and this primer set: 5′-GGTACCGAGCTCAGATTTCTAGGA-3′ (KpnI); 5′-AGATCTCGAGGATTTGAATTCC-3′ (XhoI). The PCR-amplified fragments were subcloned into the KpnI/XhoI sites of pGL4.26 [luciferase/minP/Hygro] vector (Promega, WI) to generate pGL4.26-3xSTAT5-Luc. We performed PCR again to amplify the promoter region using pGL3-3xSTAT5 plasmid as a template and this primer set: 5′-GATATCGGTACCGAGCTCAGATTTCTAGGA-3′ (EcoRV); 5′-AAGCTTAGATCTCGAGGATTTGAATTCC-3′ (HindIII). The resulting fragments were subcloned into the pGL4.26-3xSTAT5-Luc plasmid using EcoRV and HindIII sites to generate pGL4.26-6×STAT5-Luc. 32D/IL-2Rβ cells were transfected with 2 μg of pGL4.26-6×STAT5-Luc by electroporation (Amaxa, Germany). One day after transfection, the cells were transferred to a new flask and continually grown in the presence of hygromycin (300 μg/mL). After 4 weeks, luciferase activity was measured using the hygromycin-resistant cells treated with IL-2 or IL-3 at various concentrations to verify the stable transfection and to assess whether the reporter can respond to JAK/STAT signaling.

The STAT5 reporter assays in a 96-well plate format

The 32D/IL-2Rβ/6×STAT5 reporter cells were deprived of WEHI-3 cell-conditioned medium for 6 hours. The cells were then re-suspended in the absence of WEHI-3 cell-conditioned medium (4×105 cells/mL) and were treated with IL-2 (20 ng/mL) or IL-3 (1 ng/mL) to activate JAK3 or JAK2, respectively. 54 μl reporter cells (~2.2×104 cells) were then dispensed into each well of the 96-well Costar white solid bottom plates where 6 μl JAK inhibitors dissolved in 10% DMSO had already been delivered to the wells. The cells were then incubated for an additional 16 hours in the absence of WEHI-3 cell-conditioned medium. A Firefly Luciferase Assay Kit (Promega, MI) was used to measure Luciferase Activity. Briefly, 60 μl luciferase assay buffer containing substrate was added to each well. After 10 min incubation at room temperature, the luminescence of the samples was measured using the Clarity™ Microplate Luminometer (BioTeK, Winooski, VT) in the photon counting mode with the measurement time set to 1 second per well. The readings were expressed in RLU/s (Relative Light Units per second). The RLU is proportional to the number of photons emitted by sample and captured by luminometer.


CP-690,550 was purchased from Axon Medchem BV (The Netherlands). Curcumin and WP-1066 were purchased from LKT Laboratories (St. Paul, MN) and Enzo Life Sciences (Plymouth Meeting, PA), respectively. AG490 and JAK3 inhibitor VI were purchased from EMD Chemicals (Gibbstown, NJ).

Western blot analysis

Cell pellets were lysed on ice in 50 mM Tris-HCl, pH 7.4, 350 mM NaCl, 1% Triton X-100, 0.5% Nonidet P-40, 10% glycerol, 0.1% SDS, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 1 mM phenylmethylsulphonyl fluoride and phosphatase inhibitor cocktails. Whole-cell extracts were resolved on SDS-PAGE, transferred to nitrocellulose membrane, and probed with appropriate antibodies. Antibodies specific for phospho-STAT5, phospho-JAK2, and JAK2 were purchased from Cell Signaling Technology (Cambridge, MA). Antibodies specific for phospho-JAK3, JAK3, and STAT5 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).


32D/IL-2Rβ cells were deprived of WEHI-3 cell-conditioned medium for 6 hours. The cells were then seeded into 48-well plates and incubated with or without CP-690,550 (100 nmol/L) for 16 hours. Subsequently, the cells were treated with 100 ng/mL IL-2 for 15 minutes or 5 ng/mL IL-3 for 30 minutes. The 5×104 cells were re-suspended in PBS and cytospinned onto a slide glass. The cells were then fixed with 100% methanol for 15 minutes and permeabilized with 0.1% Triton X-100 in PBS (pH 7.4) for 10 minutes. After blocking with 2% BSA in PBS, the cells were incubated with antibody specific for phospho-Tyr694-STAT5 (1:100 dilution) overnight at 4°C. The cells were washed with PBS and incubated with FITC-conjugated secondary antibody (Jackson ImmunoResearch, PA) for 1 hour at room temperature. The cells were then counterstained with 4′-6-Diamidino-2-phenylindole (DAPI) to visualize the nuclei and imaged by fluorescence microscopy (Carl Zeiss, Thornwood, NY).

Results and Discussion

The JAK3 inhibitor CP-690,550 blocks the phosphorylation of STAT5 by IL-2/JAK3 but not by IL-3/JAK2

In murine IL-3-dependent myeloid progenitor 32D cells stably expressing the human IL-2 receptor β chain (32D/IL-2Rβ), JAK2 and JAK3 undergo transient phosphorylation in response to IL-3 and IL-2, respectively (Fig. 1). Subsequently STAT5 becomes activated by JAK2 or JAK3, suggesting that measuring STAT5 activity induced by IL-2 or IL-3 in the cells can faithfully monitor the activity of these kinases. To assess whether this cell line can be used as a cellular model for the identification of low-molecular-weight inhibitors specific for JAK3 over JAK2, we examined IL-2- or IL-3-induced STAT5 activity in the cells treated with the selective JAK3 inhibitor CP-690,550, or the pan-JAK inhibitor Curcumin. The cells were incubated with various concentrations of CP-690,550 for 16 hours in the absence of WEHI-3 cell-conditioned medium and then stimulated by recombinant IL-2 or IL-3. While phospho-STAT5 was barely detectable in the cells without cytokine stimulation, as assessed by its reactivity to an antibody specific for Tyr694, its levels were increased in response to IL-2 or IL-3 treatment (Fig. 1A & B). CP-690,550 efficiently blocked the phosphorylation of STAT5 by IL-2, which is JAK3-dependent, in a dose-dependent manner (Fig. 1A). In fact, phospho-STAT5 levels were decreased by more than 50% at 25 nmol/L of CP-690,550, as compared with those of control, and were undetectable at 50 nmol/L. As expected, this reagent potently inhibited IL-2-induced JAK3 phosphorylation (Fig. 1A). This demonstrates that CP-690,550 inhibited IL-2-induced activation of JAK3, which subsequently blocked the activation of STAT5. Importantly, CP-690,550 failed to affect the levels of phospho-STAT5 following treatment of cells with IL-3, which activates JAK2, at the concentrations up to 100 nmol/L (Fig. 1B). Consistent with this result, treatment of 32D/IL-2Rβ cells with CP-690,550 did not show any inhibitory effect on the levels of phospho-JAK2 following IL-3 treatment at the concentrations up to 100 nmol/L (Fig. 1B). We next examined the effect of Curcumin, which inhibits the activation of all JAK kinases by cytokines, on IL-2-induced phospho-JAK3/STAT5 and IL-3-induced phospho-JAK2/STAT5 activity. As expected, treatment of 32D/IL-2Rβ cells with this pan-JAK inhibitor resulted in a decrease in both IL-2-induced JAK3/STAT5 and IL-3-induced JAK2/STAT5 phosphorylation (Fig. 1C & D).

Fig. 1
CP-690,550 inhibits the phosphorylation of STAT5 by IL-2/JAK3 but not by IL-3/JAK2. Effect of the selective JAK3 inhibitor CP-690,550 on JAK3/STAT5 phosphorylation induced by IL-2 (A) and on JAK2/STAT5 phosphorylation induced by IL-3 (B) in 32D/IL-2Rβ ...

To confirm the selective inhibitory effect of CP-690,550 on JAK3, we next examined the cellular distribution of phospho-STAT5 in the 32D/IL-2Rβ cells treated with CP-690,550 and either IL-2 or IL-3. In resting cells, STAT5 resides mainly in the cytoplasm. However, in response to cytokine or growth factor, it becomes phosphorylated on Tyr694 and rapidly translocates to the nucleus, where activated STAT5 dimers alter the expression of numerous target genes. Since CP-690,550 selectively blocked STAT5 phosphorylation induced by IL-2 but not by IL-3 (Fig. 1A & B), we hypothesized that treatment of 32D/IL-2Rβ cells with CP-690,550 would interfere with the nuclear distribution of phospho-STAT5 only in cells treated with IL-2. In untreated 32D/IL-2Rβ cells, only basal levels of phosho-STAT5 were observed and signal was confined to the cytoplasm (Fig. 2A-A′). By contrast, significant accumulation of phosphorylated STAT5 was observed in the nuclei of the cells treated with either IL-2 or IL-3 (Fig. 2B-B′ & D-D′). Importantly, the nuclear accumulation of phospho-STAT5 in cells treated with IL-2 was almost completely abolished by co-administration of CP-690,550 (100 nmol/L) (Fig. 2C-C′). By contrast, this compound failed to affect the distribution of nuclear phospho-STAT5 in cells treated with IL-3 (Fig. 2E-E′). These data are consistent with the findings that CP-690,550 selectively decreases STAT5 activation by JAK3 but not by JAK2 (Fig. 1A and B). Taken together, these results suggest that 32D/IL-2Rβ cells can serve as a useful tool for the evaluation and discovery of small molecule inhibitors preferentially inhibit JAK3 and not JAK2.

Fig. 2
CP-690,550 disrupts the nuclear translocation of phospho-STAT5 induced by IL-2 but not by IL-3 in 32D/IL-2Rβ cells. The cells were deprived of WEHI-3 cell-conditioned medium for 6 hours. The cells were then seeded into 48-well plates and incubated ...

Development of a cell line 32D/IL-2Rβ/6×STAT5 stably expressing a STAT5 reporter gene

The identification of specific JAK3 antagonists is essential both for basic research and for developing therapeutic strategies for the treatment of various human disorders, including immune-related diseases and hematopoietic neoplasms. Notably, many of the newly developed JAK3 inhibitors that have significant off-target effects on JAK2 were identified by structure-based inhibitor designs or in vitro biochemical JAK3 enzymatic assays. Small molecules identified from these cell-free assays may not elicit the same effects in cellular assays, due to issues of membrane permeability, off-target effects, and cytotoxicity. By contrast, results obtained from small molecule screening in cell-based assays reflect the physiological effect of a compound in the context of a normal cellular milieu. In particular, luciferase reporter gene assays provide simple, quantitative, sensitive, and cost-effective means for monitoring the activity of target molecules in a cellular context. To develop a tool that affords quantitative measurement of STAT5 activity in 32D/IL-2Rβ cells, we established a 32D/IL-2Rβ cell line stably expressing a STAT5 reporter. We placed six tandem repeats of the STAT5 response element of β-casein gene promoter upstream of a TATA-box minimal promoter and a firefly luciferase gene. 32D/IL-2Rβ cells stably expressing this reporter are hereafter referred to as 32D/IL-2Rβ/6×STAT5 cells (see also Materials and Methods). We first assessed if this reporter gene is responsive to JAK/STAT signaling by treating these cells with IL-2 or IL-3. Specifically, 32D/IL-2Rβ/6×STAT5 cells were deprived of WEHI-3 cell-conditioned medium for 6 hours and then treated with IL-2 or IL-3 at various concentrations for an additional 16 hours. We observed that both IL-2 and IL-3 treatment led to an increase in STAT5 reporter activity in a dose-dependent manner and that activity of the reporter was saturated at higher cytokine concentrations (Fig. 3A & B). In fact, treatment of 32D/IL-2Rβ/6×STAT5 cells with 1 μg/mL of IL-2 or 5 ng/mL of IL-3 resulted in almost 17- or 50-fold increase in the reporter activity, respectively, compared with those of controls. We next determined the time course of activation of the reporter after treatment of 32D/IL-2Rβ/6×STAT5 cells with IL-2 or IL-3. Both cytokines increased the reporter activity in a time-dependent manner, with approximately 40- and 100-fold increase in maximum at 24 hours for IL-2 and IL-3, respectively (Fig. 3C & D). These observations indicate that the STAT5 reporter activity in 32D/IL-2Rβ/6×STAT5 cells is robust and sensitive to cytokine stimulation.

Fig. 3
Establishment of a cell line 32D/IL-2Rβ/6×STAT5 that stably expresses a STAT5 reporter gene. (A-D) Dose and time effects of IL-2 or IL-3 on STAT5 reporter activity in 32D/IL-2Rβ/6×STAT5 cells. (A, B) The cells were deprived ...

JAK3 inhibitors selectively block the activation of the STAT5 reporter by IL-2/JAK3 in 32D/IL-2Rβ/6×STAT5 cells in a high-throughput format

We next examined whether JAK inhibitors can block the activation of the STAT5 reporter by IL-2 or IL-3 in 32D/IL-2Rβ/6×STAT5 cells. The cells were incubated in the absence of WEHI-3 cell-conditioned medium for 6 hours. The cells were then re-suspended without WEHI-3 cell-conditioned medium (4×105 cells/mL), mixed with IL-2 (20 ng/mL) or IL-3 (1 ng/mL) to activate JAK3 and JAK2, respectively, seeded into 96-well plates (~2.2×104 cells/well) containing varying concentrations of JAK inhibitors, and incubated for 16 hours. We showed that IL-2 or IL-3 enhances the reporter activity in a time-dependent manner (Fig. 3C & D), but we optimized the assay condition by incubating the reporter cells for 16 hours in the presence of a compound to obtain relatively robust reporter activity and eliminate non-specific effects of the compound. Consistent with our earlier results, CP-690,550 selectively decreased in a dose-dependent manner activation of the STAT5 reporter in cells treated with IL-2 but not in those treated with IL-3 (Fig. 4A & B). In fact, 25 nmol/L of CP-690,550 reduced the reporter activity by almost 70%. By contrast, the same compound failed to inhibit IL-3-induced reporter activity, which is JAK2-dependent, at the concentrations up to 100 nmol/L. We next generated the full dose response curve of CP-690.550 in IL-2- and IL-3-induced reporter activity. The IC50 value of CP-690.550 in IL-2- and IL-3-induced reporter activity was 18.4 nmol/L and 562.2 nmol/L, respectively, indicating the selectivity of CP-690.550 for JAK3 over JAK2 (Fig. 4C). Interestingly, these results are consistent with the report that CP-690,550 shows 20-fold greater selectivity for JAK3 over JAK2 in ex vivo JAK3 kinase assay,11 suggesting that our cell-based reporter assay is as efficacious as in vitro enzymatic assays. To further demonstrate that JAK inhibitors can inhibit the activation of the STAT5 reporter by IL-2 or IL-3 in 32D/IL-2Rβ/6×STAT5 cells, we tested the effect of JAK3 inhibitor VI, which can block JAK3 kinase activity at nanomolar levels and exhibits much higher selectivity for JAK3 than JAK2,18 on IL-2-induced JAK3 and IL-3-induced JAK2 activity. As expected, while 1 μmol/L JAK3 inhibitor VI reduced the IL-2-induced reporter activity by almost 50%, the same compound showed no inhibitory effect on IL-3-induced JAK2 at the concentrations up to 10 μmol/L (Fig. 4D & E). We next generated the full dose response curve of JAK3 inhibitor VI in IL-2- and IL-3-induced reporter activity. The IC50 value of JAK3 inhibitor VI in IL-2- and IL-3-induced reporter activity was 0.9 μmol/L and 40 μmol/L, respectively (data not shown). This result is comparable to the reported observation that JAK3 inhibitor VI exhibits ~16-fold greater selectivity for JAK3 over JAK2. On the other hand, the pan-JAK inhibitor Curcumin at the concentrations between 15 and 20 μmol/L reduced both IL-2- and IL-3-induced STAT5 reporter activity by 50% (Fig. 4F & G). We found that other pan-JAK inhibitors, such as AG490 and WP1066, also non-selectively inhibited both IL-2- and IL-3-induced STAT5 reporter activity (data not shown). These data suggest that luciferase activity in the reporter cells can reflect JAK2 or JAK3 activity induced by IL-3 or IL-2, and that JAK3 inhibitors show high specificity for IL-2-induced JAK3 over IL-3-induced JAK2 in our reporter assay.

Fig. 4
CP-690,550 selectively inhibits STAT5 reporter activity induced by IL-2 but not IL-3 in 32D/IL-2Rβ/6×STAT5 cells. (A, B) Effect of CP-690,550 on STAT5 reporter activity induced by IL-2 or IL-3. CP-690,550 decreased IL-2-induced STAT5 reporter ...

We next determined Z′ values to assess whether our assay is suitable for high-throughput screening. The Z′ value for the assay performed in 96-well plates was 0.738±0.03 (Supplementary Fig. 1A). In addition, we found no significant day-to-day variation in Z′ values over a one week period (Z′=0.715±0.031) (Supplementary Fig. 1B). Furthermore, we could not detect any significant edge effects in our assay as the entire incubation time after the treatment of compound is a mere 16 hours (data not shown). Taken together, these results demonstrate that our assay using 32D/IL-2Rβ/6×STAT5 cell line is very likely to be suitable and reliable for use in a full-scale, high-throughput screen of chemical libraries to identify new JAK3 inhibitors.

It is worth noting that 32D/IL-2Rβ/6×STAT5 cells were designed to test the selectivity of a compound for JAK3 over JAK2. Therefore, compounds identified through this reporter assay will be needed to examine their effects on JAK1 and TYK2, by assessing, for example, if a lead compound can inhibit the phosphorylation levels of STAT1 in U266 myeloma cells following treatment of interferon-α (IFN-α), which activates JAK1 and TYK2. In sum, our findings suggest that the 32D/IL-2Rβ/6×STAT5 cell line represents a powerful cellular tool for large-scale primary chemical screens to identify specific low-molecular-weight inhibitors of JAK3.

Supplementary Material

Supplementary Fig. 1. Suitability of 32D/IL-2Rβ/6×STAT5 cell line for use in a high-throughput screen. (A) Z′ value for the 96-well plate assay using the reporter cells treated with IL-2 in the presence of either DMSO alone or CP-690.550 (Z′-factor=0.738±0.03). (B) Determination of day-to-day variation in the reporter assay by calculated Z′ values ranging from day 1 to day 7. No significant day-to-day variation in the assay was observed (Z′-factor=0.715±0.031). These results demonstrate that the reporter assay using 32D/IL-2Rβ/6×STAT5 cell line is suitable for use in a full-scale, high-throughput screen of chemical libraries to identify JAK3 inhibitors.


We thank Dr. R. A. Kirken for pGL3-3xSTAT5-Luc plasmid, and Drs. A. D. Keegan and W. J. Leonard for 32D/IL-2Rβ cell line. We also thank Dr. R. DasGupta (NYU-RNAi facility) for statistical data analysis. This work was supported by the Children's Cancer Fund (Millwood, NY).


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