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Fig. 2. Plasmid map of PNF-AT3. The plasmid PNF-AT3 contains the luciferase gene upstream of three tandem NF-AT/AP-1 murine IL-2 promoter sequences. Restriction endonuclease recognition sites, the transcription start site (arrowhead), the ampicillin resistance gene (AmpR), the luciferase open reading frame, and pBR322- and simian virus 40 (SV40)-derived sequences are labeled. The murine NF-AT/AP-1 promoter sequence used in PNF-AT3 is shown below the plasmid map.

sequence upstream of the luciferase gene is transfected into the human T lymphocyte cell line Jurkat, a cell line that expresses both endogenous calcineurin and NF-AT. Treatment of the transfected cells with tetradecanoylphorbol acetate (TPA, a phorbol ester) activates AP-1 (Fos-Jun heterodimer) whereas ionomycin, a calcium ionophore, mobilizes Ca2+, thereby activating calcineurin (Fig. 1). Activated cells can be treated with a redox reagent and harvested for luciferase measurements to provide a specific and sensitive assay for calcineurin activity in intact Jurkat cells.

Reagents

Plasmids

The luciferase reporter plasmid pNF-AT3-luc (pNF-AT3) contains three copies of the murine NF-AT/AP-1 mixed promoter sequence at approximately -287 bp of the murine IL-2 promoter upstream of the luciferase gene.21 The reporter plasmid pAPlD3-luc (pAP-luc) contains three tandem copies of the distal AP-1 sequence at approximately —180 bp of the murine IL-2 promoter placed upstream of the luciferase gene. Both luciferase reporter plasmids are constructed from pT81Luc (ATCC 37584; American Type Culture Collection, Rockville, MD), a derivative of p232AL-A5'.22 The plasmid pcDNA3.1/His is purchased from Invitrogen (Carlsbad, CA). All plasmids are prepared by CsCl density centrifugation at 340,000g in a Beckman (Fullerton, CA) NVT65 rotor.

Other Materials

2-Mercaptoethanol (2-ME), TPA, ionomycin, and hydrogen peroxide (H2O2) are purchased from Sigma (St. Louis, MO). Fetal calf serum (FCS) is purchased from HyClone Laboratories (Logan, UT). RPMI medium and L-glutamine are purchased from GIBCO-BRL Life Technologies (Frederick, MD). The Promega luciferase assay system is purchased from Promega (Madison, WI).

Buffers and Media

Phosphate-buffered saline (PBS): Dissolve 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2P04, and 0.24 g of KH2P04 in 800 ml of H20. Adjust to pH 7.4 with HCl and bring the final volume to 1 liter Tris-EDTA (TE) buffer: Prepare 10 mMTris-1 mM EDTA buffer and adjust to pH 7.4 with hydrochloric acid Sodium acetate (3 M): Dissolve solid sodium acetate in H20 and adjust to pH 4.8 with glacial acetic acid. Bring to final volume with distilled H20

21 K. E. Hedin, M. P. Bell, K. R. Kalli, C. J. Huntoon, B. M. Sharp, and D. J. McKean,./. Immunol. 159,5431 (1997).

22 J. R. de Wet, K. V. Wood, M. DeLuca, D. R. Helinski, and S. Subramani, Mol. Cell. Biol. 7, 725 (1987).

RPMI-10% (v/v) FCS: Sterile fetal calf serum (FCS, 55 ml) is heated at 55° for 30 min and added to 500 ml of sterile RPMI medium (without glutamine) under sterile conditions in a biosafety hood. Using sterile pipettes, 5 ml of 1 M HEPES, 200 mM L-glutamine, and 2 \i\ of 14.3 M 2-ME are added. The medium is filtered through sterile 0.2-/im pore size filters and stored at 4°

RPMI-0.5% (v/v) FCS: RPMI-10% FCS is diluted 20-fold in sterile RPMI medium to create a final FCS concentration of 0.5% (v/v). The medium is stored at 4°

Trypan blue cell viability dye: Dissolve trypan blue in 0.15 M NaCl to give a final concentration of 0.2% (w/v). Store the dye solution at room temperature

Ionomycin stock solution: A 2.64 mM ionomycin stock solution is prepared by dissolving ionomycin in ethanol-dimethyl sulfoxide (DMSO) (1:1, v/v). Additions to culture medium are made so that ethanol-DMSO <0.1% (v/v) of the final volume. The stock solution is stored at —20°

TPA stock solution: A 100-^g/ml stock solution of TPA is prepared in ethanol and stored at -20°. Dilutions of TPA are made in RPMI-10% (v/v) FCS before addition to cell culture

Procedure

Cell Culture

The human T lymphocyte cell line Jurkat is cultured in RPMI-10% (v/v) FCS at 37° and 5% C02 in a humidified incubator. Cells are passaged every 48 hr and are not allowed to exceed a cell density of 1 x 106 cells/ml. Cells are counted periodically with a cytometer and viability is determined by staining with trypan blue. To count cells, 1 ml of medium is removed from a culture flask and briefly vortexed, and 100 /il is removed and diluted with an equal volume of trypan blue dye. After another brief vortex, 10 ß 1 of the mixture is placed on a cytometer and the cells are counted under a light microscope (x 100 magnification). Viable cells are clear but stain dark blue if nonviable.

Transfection

In a single transfection, 107 Jurkat cells are centrifuged at 100g for 5 min, the supernatant is discarded, and the cell pellet is resuspended in 250 /¿I of RPMI-10% (v/v) FCS (final concentration of 40 x 106 cells/ml). The cells are kept at room temperature while the plasmids are prepared. A total of 30 fig of plasmid DNA is prepared for each transfection. In a single transfection, 10 ßg of either pNF-AT3 or pAP-luc and 20 ßg of pcDNA3.1/His are aliquoted into sterile microcentrifuge tubes and brought to a 200-/il final volume with TE buffer. The plasmid DNA

is precipitated by addition of 20 of 3 M sodium acetate (pH 4.8) and 500 /A of cold ethanol and centrifuged at 16,000g at 4°. The supernatant is removed by gentle aspiration and the DNA pellets are washed once with 100 ¡A of 70% (v/v) ethanol. The microcentrifuge tubes are then placed in the hood and opened for 5 min to allow complete evaporation of ethanol from the pellets. The pellets are resuspended in 50 ¡A of serum-free RPMI and placed in a 37° water bath for 10 min. After incubation the tubes are vortexed briefly and 250 /il of the cell suspension prepared above (10 x 106 cells) is added to each. The plasmid DNA and cells are allowed to incubate at room temperature for 10 min with no agitation, after which the cell-DNA suspensions are transferred to sterile 4-mm gap cuvettes and electroporated at 300 V for 10 msec (Electro Square Porator T820; BTX, San Diego, C A). After electroporation, the cells are allowed to rest for 10 min in the cuvettes, transferred to 21 ml of RPMI-10% (v/v) FCS (for pNF-AT3 transfections) or RPMI-0.5% (v/v) FCS (for pAP-luc transfections), and incubated overnight (16-18 hr) at 37° and 5% C02. Cells are then aliquoted into six-well tissue culture plates (5 ml per well) and stimulated by the addition of ionomycin (2 ¡iM final concentration) and TPA (2-ng/ml final concentration) for PNF-AT3 transfections or with TPA (2 ng/ml) alone for pAP-luc transfections. The transfected cells are treated with H2O2 or a preferred reagent coincident with ionomycin and/or TPA addition. Cells are incubated for a total of 6 hr (at 37° and 5% CO2) and harvested for luciferase assays as described below.

Luciferase Assays

The content (5 ml) of each well is removed and centrifuged in a 15-ml Falcon tube (Becton Dickinson Labware, Lincoln Park, NJ) at lOOg for 5 min at room temperature. The supernatant is carefully aspirated and discarded. Each pellet is resuspended in 1 ml of cold PBS and transferred to a new microcentrifuge tube and centrifuged at lOOg for 5 min at room temperature. The supernatant is removed and 50 ¡A of lx lysis buffer [25 mM phosphate (pH 7.8), 2 mM DTT, 2 mM EDTA, 10% (v/v) glycerol, 1% (v/v) Triton X-100] from the Promega luciferase assay system is added to each cell pellet. The suspensions are incubated at room temperature for 10 min and subsequently centrifuged at 16,000g for 5 min at 4°. The supernatant (50 1) of each sample is then carefully removed and placed in a luminometer tube (Starstedt, Newton, NC), and the light emission is measured with an EG&G Berthold (Pforzheim, Germany) LB 9507 luminometer after automated addition of 100 /i\ of Promega luciferin substrate.

Discussion and Example Results

Our reporter plasmid assay of calcineurin activity is based on the T cell receptor signal transduction pathway that regulates interleukin 2 (IL-2) gene expression

Fig. 3. Time course of luciferase production from stimulated pNF-Aiytransfected cells. Jurkat cells transfected with PNF-AT3 were stimulated at t = 0 hr with TPA (2 ng/ml) and 2 pM ionomycin. Cells were harvested at various time points after stimulation (40 min to 20 hr) and cell lysate was measured for luciferase activity. Data are represented in units of luciferase activity (RLU). [Reprinted with permission from T. A. Reiter, R. T. Abraham, M. Choi, and F. Rusnak, J. Biol, lnorg. Chem. 4, 632 (1999), copyright © 1999, Society of Biological Inorganic Chemistry.]

Time (Hours)

Fig. 3. Time course of luciferase production from stimulated pNF-Aiytransfected cells. Jurkat cells transfected with PNF-AT3 were stimulated at t = 0 hr with TPA (2 ng/ml) and 2 pM ionomycin. Cells were harvested at various time points after stimulation (40 min to 20 hr) and cell lysate was measured for luciferase activity. Data are represented in units of luciferase activity (RLU). [Reprinted with permission from T. A. Reiter, R. T. Abraham, M. Choi, and F. Rusnak, J. Biol, lnorg. Chem. 4, 632 (1999), copyright © 1999, Society of Biological Inorganic Chemistry.]

(Fig. 1). Although in this pathway several other proteins are involved upstream of calcineurin and protein kinase C (PKC) activation, treatment with ionomycin and TPA bypasses these upstream signaling components, resulting in calcineurin and PKC activation. Calcineurin dephosphorylates NF-AT, which migrates to the nucleus and activates IL-2 transcription. Nuclear translocation of NF-AT is blocked when calcineurin is inhibited with the drugs cyclosporin A and FK506. Thus, luciferase activity, which is under the control of NF-AT in pNF-AT3-luc, provides an indirect measurement of calcineurin activation in intact cells.

Positive and negative controls must be included for each experiment performed. The negative control measures luciferase activity in unstimulated (by TPA and ionomycin), transfected cells. The positive control measures luciferase activity in transfected cells stimulated by ionomycin and TPA for 6 hr. Transfected cells are harvested after 6 hr of ionomycin-TPA treatment because luciferase production is maximized after this time point (Fig. 3). Relative light unit (RLU) readings from the luminometer for negetive and positive controls, using a single plasmid preparation, are 2.55(±0.63) x 104 RLU and 1.92(±0.04) x 107 RLU, respectively, representing a 590-fold increase in NF-AT activity on treatment of the cells

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