The protease inhibitors phenylmethylsulfonyl fluoride (PMSF), leupeptin, pepstatin, aprotinin, EDTA and bestatin were purchased from Roche (USA); T4 polynucleotide kinase and poly(dI-dC)2 were obtained from Amersham Pharmacia Biotech (Piscataway, NJ, USA). Tris-borate-EDTA buffer and acrylamide-bisacrylamide (29:1) were obtained from Bio-Rad (Richmond, CA, USA). Luciferase assay reagent, lysis buffer and the pGL-3 luciferase vector were obtained from Promega (Madison, WI, USA). All-trans-retinoic acid (ATRA), dimethyl sulphoxide (DMSO), Triton X-100, 1,4-dithiotreitol (DTT), paraformaldehyde, protease inhibitor cocktail, phosphatase inhibitor cocktail, Tween-20, Temed, 30% acrylamide, TPA and ionomycin were purchased from Sigma (USA). Anti JNK-1, -JNKp, -EGR-1, -β-actin, -HTLV-1 Tax (sc-34096) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cell Death Detection ELISA was purchased from Roche (Indianapolis IN, USA).

Jurkat T cells and Jurkat T cells transfected to express Tax protein, were grown in RPMI-1640 medium containing 10% heat-inactivated FBS, 200 mM glutamine, non-essential amino acids, penicillin, and streptomycin sulfate. Before treatment, the cells were grown overnight (16–20 h) in medium containing 0.5% heat-inactivated FBS and subsequently stimulated in the presence of the same medium with low concentrations of serum. ATRA was diluted in culture medium containing 0.5% FBS, keeping the DMSO concentration below 0.5%. Appropriate controls containing the same amount of solvent were included in each experiment. Intermittent passage in G418-containing medium was performed to ensure retention of the plasmid (28).

The plasmid expressing the wild-type of the Tax protein and the Tax-inducible cell line, were a gift from Dr Warner Greene (Gladstone Institute of Virology and Immunology, University of California, San Francisco, San Francisco, CA, USA). The human IL-2 promoter-enhancer fragment (−500 to +60) was subcloned from plasmid SV-IL-2-CAT into the luciferase vector pGL-2 (Promega) (28). The AP-1-luciferase reporter plasmid driven by the rat prolactin minimal promoter (−36 to +37) under the control of four copies of the human AP-1 site (28) was kindly provided by M. Rincón and R.A. Flavell (Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA). Plasmids containing multimers of the recognition sites for NF-κB and AP-1 were constructed and linked to the pLuc-prolactin minimal promoter plasmid (28). The orientation for each element was confirmed by restriction enzyme cleavage. The tandem sequences used to construct the different multimer plasmids were as follows: i) four copies of the AP-1-responsive element (the 12-O-tetradecanoylphorbol-13-acetate [TPA]-responsive element of the human collagenase promoter), (5′-TCGATTGAGTCAGGGTAA-3′), and ii) two copies of the NF-κB-binding site of the human Igκ light chain enhancer (5′-GGGACTTTCC-3′) (28).

The siRNAs for Egr-1 and JNK-1 were obtained as ready-annealed, purified duplex probes, and the scrambled control siRNAs were purchased from Shanghai Genechem Co.: siRNA for Egr-1 (sense, 5′-CAGCAGCAGCAGCAGCAGCTT-3′ and antisense, 5′-AAGCTGCTGCGCTGCTGCTG-3′); siRNA oligonucleotides for JNK-1 (sense, 5′-AAGCCCAGTA ATATAGTAGTA-3′ and antisense, 5′-TACTACTATATTACTGGGCTT-3′). The cells were cultured in medium without antibiotics and 24 h before transfection resulting in confluency of the cell monolayer by 50–70%. Specific EGR-1 and JNK-1 siRNAs or non-silencing siRNA (70 nmol) were mixed with Lipofectamine™ 2000 (Invitrogen) according to the manufacturer's recommendation and added to the cells. After 6 h at 37°C, the medium was changed, and the cells were cultivated in RPMI-1640 medium supplemented with 10% heat-inactivated FBS.

Cell survival was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay. Cells (10,000/well) were seeded in 96-well plates in 100 μl of medium containing the appropriate amount of serum. Jurkat and Jurkat-Tax cells were transfected or not with siRNA-control or siRNA-Egr-1 and were then treated with ATRA 8 μM or solvent (DMSO) for 1, 2, 4 or 6 days, when 10 μl/well of 5 mg/ml MTT was added. After 4 h of incubation at 37°C, the tetrazolium crystals were solubilized overnight at 37°C with 10% SDS and 10 mM HCl, and the optical density (OD) at 595 nm was measured.

Transfection of cells was conducted by electroporation, using an Electro Cell Manipulator 600 (BTX, San Diego, CA, USA) using 130 V/1,700 μF capacitance. Briefly, 8×10⁶ cells were transfected with 10 μg of luciferase reporter plasmid and 5 μg of each expression plasmid, and the mixture was incubated for 24 h. Transfected cells were cultured in complete medium for 24 h and stimulated with ATRA for another 8 h. Cells were harvested 32 h post-transfection, washed twice in PBS, and treated with lysis buffer (Luciferase assay; Promega) for 5 to 10 min on ice. Lysates were spun down for 1 min, and the total supernatants were analyzed using luciferase reagent (Promega) and measured as a duplicate in a luminometer (MicroLumat LB 96 P; Berthold) for 5 sec. Background measurement was subtracted from each duplicate, and experimental values are expressed either as recorded light units of luciferase activity or as relative activity compared with extracts from unstimulated cells (29). Nuclear extracts were prepared as previously described (29,30).

The Jurkat cell line (5×10⁷) was seeded onto 6-well plates. Forty-eight hours after transfection, the cells were collected and washed twice by cold PBS, and each well was treated with 50 ml lysis buffer (2 mmol/l Tris-HCl, pH 7.4, 50 mmol/l NaCl, 25 mmol/l EDTA, 50 mmol/l NaF, 1.5 mmol/l Na₃VO₄, 1% Triton X-100, 0.1% SDS, supplemented with protease inhibitors 1 mmol/l phenylmethylsulfonylfluoride, 10 mg/l pepstatin, 10 mg/l aprotinin and 5 mg/l leupeptin) (all from Sigma). Protein concentrations were determined using the Bradford protein assay. Equal amounts of protein (50 mg) were separated on a 15% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane (Hybond-C; Amersham, Freiburg, Germany). The membranes were blocked in 5% non-fat dry milk in TBS for 1 h at room temperature and probed with anti-HTLV-1 Tax (sc-34096), anti-JNK-1 (sc-1648) or with anti-EGR-1 (sc-110) antibody (dilution 1:500; Santa Cruz Biotechnology) overnight at 4°C. After 3 washings with TBS containing 0.1% Tween-20, the membranes were incubated with anti-rabbit IgG-horseradish peroxidase (1:5,000; Santa Cruz Biotechnology), and developed by luminal-mediated chemiluminescence (Appylgen Technologies, Inc., China). To confirm equal protein loading, the membranes were reprobed with a 1:1,000 dilution of an anti-actin antibody (Santa Cruz Biotechnology). Densitometric analyses were performed using Scion Image software (31).

DNA fragmentation was determined using a Cell Death Detection ELISA (Roche). Jurkat and Jurkat-Tax cells were treated with apoptotic compounds for the indicated periods of time. Cells were harvested by centrifugation and lysed in 0.5 ml of the lysis buffer provided with the kit. Two milliliters of the extract was used for the ELISA, which was performed as instructed by the manufacturer. The OD at 405 nm was measured and the fold induction of apoptosis was calculated using untreated cells as control.

Jurkat cells (500,000) were treated with RA as indicated. The cells were washed with PBS and stained with Annexin V-FITC (Pharmingen) and PI in binding buffer (10 mM HEPES (pH 7.4, 140 mM NaCl 2.5 mM CaCl₂) for 15 min at room temperature in the dark. Cells were subsequently analyzed by flow cytometry (FACSCalibur) for apoptosis (FITC) and viability (PI).

Jurkat cells (wt) and Jurkat cells expressing the Tax protein were grown with increasing concentrations of ATRA for 1, 2, or 3 days in the presence of a low (0.5%) or high (10%) concentration of serum. Cell growth was measured using the MTT assay. Fig. 1A shows the results obtained in Jurkat and Jurkat-Tax cells after 24 h of incubation.

EGR-1 is not required for the expression of Tax protein induced by certain stimuli. We investigated whether the siRNA-EGR-1 induced or blocked Tax activity, compared with the induction of EGR-1 and JNK-1 proteins. Cells were incubated for 16 h in medium containing 0.5% FBS prior to ionomycin or TPA stimulation as positive control, or treated with siRNA-control and siRNA against EGR-1 (Fig. 2). A significant activation of Tax (top), EGR-1 (middle) and JNK-1 (bottom) was observed after 120 min of exposure to the stimuli. However, the treatment of cells with siRNA against EGR-1 did not affect the expression of either Tax or JNK-1 proteins (Fig. 2 top and bottom, respectively).

We transfected cells with a reporter plasmid containing multiple consensus sequences of 4xAP-1 (Fig. 3A) and/or 4xNF-κB (Fig. 3B) response elements linked to a minimal promoter controlling transcription of the firefly luciferase cDNA. Both nuclear factors, AP-1 and NF-κB, play important roles in inflammation, immune responses, cell growth and apoptosis. PMA and ionomycin are potent activators of AP-1 and/or NF-κB responsive genes. In the present study, we demonstrated that transiently transfected cells with the 4xAP-1 (Fig. 3A) exhibited stronger and consistent luciferase expression in response to PMA and ionomycin stimulation compared with cells transfected with the luciferase vector carrying the 4xNF-κB (Fig. 3B) response element. At the same time, the expression of luciferase activity in Jurkat-Tax cells was not affected by treatment of the cells with either the siRNA-control or siRNA-Egr-1 (Fig. 3).

To determine whether the silencing of Egr-1 expression is correlated with the increase or reduction in ATRA-induced Jurkat-Tax cell apoptosis, RNA interference was used. As shown in Fig. 4, transient transfection of a specific siRNA against Egr-1 (siRNA-Egr-1) slightly attenuated ATRA-induced apoptosis in the Jurkat and Jurkat-Tax-cells. However, these results were clearly dependent on the concentration of FBS and on the time of incubation at a given ATRA concentration. As shown in Fig. 4, Jurkat and Jurkat-Tax cells were incubated with either PBS 0.5% (left columns) or with PBS 10% (right columns) for the indicated periods of time, and apoptosis was determined by double staining with Annexin V-FITC and PI followed by cytometric analysis. The percentages of Annexin V-positive/PI-negative cells (indicative of early apoptosis) (black columns) and double-positive cells (late apoptotic and/or necrotic) (white columns) are shown. Cells incubated with vehicle for 1 h were used as control. The induction of DEVDase activity was higher when a low concentration of serum was present in the culture medium. The appearance of Annexin V-positive (apoptotic) cells was evident after only 1 h of incubation with ATRA, and reached a maximum after 4–6 h (Fig. 4). We showed that the percentage of apoptotic cells was higher in the presence of a low serum concentration (0.5% FBS) compared with a high serum concentration (10% FBS).

Several studies have demonstrated the inhibitory effect of ATRA on the proliferation of Jurkat cells (31). We examined the ability of ATRA to induce apoptosis in Jurkat and Jurkat-Tax cells by analysis of DEVDase activity (Fig. 5A) and DNA fragmentation (Fig. 5B). To examine whether EGR-1 and/or JNK-1 activities are required for the induction of apoptosis by ATRA in Jurkat-Tax cells, we examined the effect of small interfering RNA inhibitors against Egr-1 and JNK-1 on the induction of apoptosis by ATRA. Jurkat cells were treated with siRNA-Egr-1 or siRNA-JNK-1 or non-silencing siRNA as the control, prior to treatment with 8 μM ATRA for 2 h. Fig. 5A shows that the specific siRNA-Egr-1 had no effect on the apoptosis induced by ATRA, as determined by DEVDase activity (Fig. 5A) and by DNA fragmentation (Fig. 5B). In contrast, inhibition of JNK-1 by a specific siRNA reduced ATRA-induced apoptosis (Fig. 5). The results demonstrated that the induction of apoptosis by ATRA in Jurkat-Tax cells was clearly dependent on the presence of JNK kinase activity (Fig. 5).