ATRA was obtained from Sigma Chemical Co. (St. Louis, MO, USA). The protease inhibitors phenylmethylsulfonyl fluoride (PMSF), leupeptin, pepstatin, aprotinin 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-2 luciferase vector were obtained from Promega (Madison, WI, USA). TPA and ionomycin were purchased from Sigma Chemical Co. Anti-JNK, p38 and ERK antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). MBP was purchased from Stratagene (La Jolla, CA, USA).

Jurkat T cells and Jurkat T cells expressing the Tax protein, were grown in RPMI-1640 containing 10% heat-inactivated fetal bovine serum (FBS), 200 mM glutamine, nonessential amino acids, penicillin and streptomycin sulfate. Before ATRA treatment, 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 a culture medium containing 0.5% FBS, with 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.

The plasmid expressing the wild-type 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, CA, USA). The human IL-2 promoter-enhancer fragment (−500 to +60) was subcloned from plasmid SV-IL-2-CAT into the luciferase vector pGL2 (35). 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 (36) 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 (35,36). The orientation for each element was confirmed by the restriction enzyme cleavage. The tandem sequences used to construct the different plasmid multimers were as follows: i) four copies of the AP-1, 12-O-tetradecanoylphorbol-13-acetate (TPA), 5′-TCGATTGAGTCAGG-GTAA3′; ii) two copies of the NF-κB-binding site of the human Ig κ light-chain enhancer 5′-GGGACTTTCC-3′; iii) IL-2 promoter construct bearing a −500 to +30 (35).

Transfection of cells was conducted by electroporation, using an Electro Cell Manipulator 600 (BTX, San Diego, CA, USA) using 130 V/1700 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 or SP600125 for another 8 h. Cells were harvested 32 h post-transfection, washed twice in phosphate-buffered saline (PBS) and treated with lysis buffer (luciferase assay; Promega) for 5–10 min on ice. Lysates were spun down for 1 min, and the total supernatants were analyzed using luciferase reagent 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 were expressed either as recorded light units of luciferase activity or as relative activity compared with extracts from unstimulated cells (35,36).

Nuclear extracts, were prepared as previously described (34). The cells were grown at 37°C in a humidified atmosphere of 10% CO₂. Cells (2×10⁷), incubated with 0.1% DMSO (control) or 30 nM TPA, or 1 mM ionomycin, were collected and washed with ice-cold PBS, washed again in buffer A [10 mM HEPES (pH 7.9), 15 mM KCl, 2 mM MgCl₂, 6 mM DTT, 0.1 mM EDTA and 1 mM PMSF] and lysed in buffer A with 0.2% Nonidet P-40. The pelleted nuclei were re-suspended in buffer B [50 mM HEPES (pH 7.6), 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 1 mM PMSF and 10% glycerol], in the presence of 0.3 M (NH₄)₂SO₄ (pH 7.9) and rocked for 30 min at 4°C. The broken nuclei were centrifuged for 10 min at 100,000 × g. A 125 μl aliquot of supernatant was transferred to a second tube and additional (NH₄)₂SO₄ was added to a final concentration of 1.5 M followed by a second centrifugation of 50,000 × g for 5 min. The supernatant was removed and the pellet was re-suspended in 50 μl of buffer B and stored at 70°C. Protein concentration was estimated using the Bio-Rad stain protein assay kit with bovine albumin as the standard.

Jurkat cells (5×10⁷) were 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 and 0.1% SDS], and supplemented with protease inhibitors (1 mmol/l phenylmethylsulfonyl fluoride, 10 mg/l pepstatin, 10 mg/l aprotinin and 5 mg/l leupeptin) (all from Sigma Chemical Co.). 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 onto a nitrocellulose membrane (Hybond C; Amersham, Freiburg, Germany). Membranes were blocked in 5% non-fat dry milk in TBS for 1 h at room temperature and probed with rabbit anti-JNK-1 (SC-1648) antibody (dilution 1:500; Santa Cruz Biotechnology, Inc.) overnight at 4°C. After washing 3 times with TBS containing 0.1% Tween-20, membranes were incubated with anti-rabbit IgG horseradish peroxidase (1:5,000; Santa Cruz Biotechnology, Inc.) and developed by luminal mediated chemiluminescence (Appylgen Technologies, Inc., China). To confirm equal protein loading, membranes were reprobed with a 1:1,000 dilution of an anti-actin antibody (Santa Cruz Biotechnology, Inc.). Densitometric analyses were performed using Scion Image software (39,40).

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

Jurkat leukemia T cells stably transfected with the Tax protein were cultured under a condition identical to that described for proliferation assay. ATRA was added at a final 10⁻⁷ M concentration 36 h after the initiation of the culture in two independent pulses (every 12 h). After 60 h of culture, supernatants were collected and the different cytokines were measured in triplicate by using an enzyme-linked immunosorbent assay (ELISA) system: IL-2, IL-4, IL-6 and IL-10 (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s protocols.

We examined the effect of ATRA (4 μM) treatment on JNK kinase activity in Jurkat and Jurkat Tax-expressing cells. JNK-1 and JNK-2 activity was detected in both Jurkat and Jurkat Tax-expressing cells (Fig. 1A). The strong inhibition of JNK by ATRA, in Jurkat Tax-expressing cells occurred in a dose-dependent manner, ranging from 2 to 6 μM, compared with the untreated Jurkat cells (Fig. 1B). However, the effect of ATRA in the Jurkat cells was lower than that observed in the untransfected Jurkat cells (Fig. 1B). The results obtained in the Jurkat and Jurkat Tax-expressing cells after 24 h of incubation are displayed in Fig. 1. Non-stimulated cells and cells treated with sorbitol were used as the control.

We investigated the effect of ATRA on the ability to induce caspase activity. Cells were pre-incubated in 10% FCS for 16 h and were later treated with 8 μM ATRA for different incubation times (Fig. 2A) and with increasing concentrations of ATRA (1–12 μM) for 4 h (Fig. 2B). Cytosol extracts were prepared at the indicated periods of time, and caspase activity was subsequently measured using DEVD-AFC as a substrate. We demonstrated that the induction of DEVDase activity was clearly dependent on the time of incubation and the concentration level of ATRA. The increase in DEVDase activity was observed with concentrations of ATRA >2 μM in the Jurkat Tax-expressing cells. The presence of Annexin V-positive (apoptotic) cells was evident after 1 h of incubation with ATRA (8 μM), and the number reached a maximum after 4 h (Fig. 2A). The apoptotic cells, dependent on ATRA concentration, as determined by Annexin V labeling, were evident following 2 μM of ATRA treatment and the number of apoptotic cells reached a maximum level following 10 μM ATRA treatment (Fig. 2B).

We demonstrated that ATRA, at concentration levels of 2–6 μM, was able to decrease JNK expression in Jurkat and Jurkat Tax-expressing cells (Fig. 1B). To further study the inhibitory effect of ATRA on the expression of JNK, we performed a combination assay using ATRA and SP600125 in untransfected Jurkat cells (Fig. 3A) and Jurkat cells transfected with a plasmid expressing the Tax protein (Fig. 3B). The effect of increasing concentrations of SP600125 on ATRA-mediated inhibition of JNK expression in Jurkat Tax-expressing cells was further investigated. Jurkat and Jurkat Tax-expressing cells were pre-incubated with increasing concentrations of SP600125 (2.5, 5.0, 7.5, 10 and 15 μM) for 120 min. Then, 10 μM ATRA was added and cells were incubated for an additional 180 min. Cell extracts were prepared and subsequently assayed using a western blot assay for JNK-1 and JNK-2 protein expression. We increased the ATRA concentration to 10 μM, to reach a major inhibitory effect of JNK-1 expression. The combination treatment of ATRA with SP600125 further decreased the JNK expression protein. Both ATRA and SP600125 treatments reduced the activity of JNK-1 and JNK-2 (Fig. 3A and B). ATRA-mediated inhibition of JNK-1/JNK-2 expression in untransfected Jurkat cells was lower (Fig. 3A) compared to that observed in Jurkat cells transfected with the Tax protein expression plasmid (Fig. 3B). The expression of JNK-1 was almost completely inhibited with increasing concentrations of SP600125 (15 μM) and ATRA (10 μM) (Fig. 3B).

To further understand the effect of ATRA, cytokine induction and secretion experiments were performed in Jurkat and Jurkat Tax-expressing cells for IL-2, IL-4, IFN-γ and IL-10. A strong induction of IL-2 (Fig. 4A), IFN-γ (Fig. 4B) and IL-4 (Fig. 4C) production in Jurkat-Tax cells, compared with Jurkat cells were observed, but not for IL-10 (Fig. 4D). The mean secretion of IL-2 was statistically decreased in Jurkat Tax-expressing cells, but not for IFN-γ, IL-4 and IL-10 when ATRA was added to the Jurkat Tax-expressing stimulated cell culture.

Jurkat and Jurkat T cells expressing the Tax protein were transiently co-transfected with luciferase plasmid reporter constructs containing the inducible region of the IL-2 enhancer/promoter (−500 to +60) (Fig. 5A) or an AP-1 (Fig. 5B), or an NF-κB (Fig. 5C) reporter construct. Tax expression was required to induce high transcription of these constructs. The effect observed after Tax expression was 3-to 4-fold higher in all three constructs, the IL-2 promoter, the AP-1- and the NF-κB-driven transcription, compared with untransfected Jurkat cells which showed a moderate transcriptional activity. However, after ATRA treatment the activity of the IL-2 promoter (Fig. 5A) and AP-1 reporter construct (Fig. 5B) was strongly affected decreasing the transcriptional activity of both the IL-2 promoter and the AP-1 reporter, but failed to affect and slightly decreased the transcriptional activity of the NF-κB reporter construct promoter (Fig. 5C). Noteworthy, NF-κB-driven transcription in the Tax-transfected and untransfected Jurkat cells was not affected by ATRA (Fig. 5C). Apparently, NF-κB was not a target for ATRA, since no activity was noted in Jurkat cells transfected or not with the Tax-expression plasmid.