Human prostate carcinoma cell lines PC-3 and LNCaP were a gift from Dr Dan Mercola (The SKCC, La Jolla, CA, USA). The cells were cultured in RPMI-1640 medium supplemented with 100 ml/l fetal bovine serum (FBS), 8×10⁵ U/l penicillin and 0.1 g/l streptomycin in a humidified incubator containing 50 ml/l CO₂ at 37°C (30–32). Tris-borate-EDTA and acrylamide:bisacrylamide (29:1) were obtained from Bio-Rad (Richmond, CA, USA). Egr-1 antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). STI571 was kindly provided by Novartis Pharma AG (Basel, Switzerland). Lipofectamine was obtained from Life Technologies, Inc., USA, and TNF-α was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Complete Mini-EDTA-Free Protease Inhibitor Cocktail Tablets and Annexin-V-Fluos were purchased from Roche Diagnostics GmbH (Mannheim, Germany). All other reagents were from Sigma Chemical Co. (Taufkirchen, Germany).

To generate siRNA/Egr-1, a 19-bp sequence was identified as unique to Egr-1, as determined by BLAST searches of the non-redundant National Center for Biotechnology Information (NCBI) nucleotide databases, and was flanked with 2 adenine ribonucleotides and 2 deoxythymidines. Double-stranded siRNA sequences were as follows: siEgr-1, 5′-AACGCAAGAGGCAUACCAAGAdTdT-3′, and 5′-AAUCUUGGUAUGCCUCUUGCGdTdT-3′. Cells were treated in parallel with siRNA-scrambled (5′-AACUCUUCGCCGGUCAUAUCCdTdT-3′ and 5′-AAGGAUAUGACCGGCGAAGAGdTdT-3′) as the control, and were synthesized by Shanghai GeneChem Co. These cells were cultured in medium without antibiotics and 24 h before transfection resulting in a confluence of the cell monolayer by 60–80%. Egr-1-siRNA or non-silencing siRNA (70 nmol) were mixed with Lipofectamine™ 2000 (Invitrogen Life Technologies) according to the manufacturer’s recommendation and added to the cells. After 6 h at 37°C, the medium was replaced, and the cells were cultivated in RPMI-1640 supplemented with 10% heat-inactivated FBS (8,26).

The prostate carcinoma cell lines PC-3 and LNCaP transfected with the empty vector (pCMV) or with an expression plasmid for Egr-1 (pCMV-Egr-1) (5×10⁵) were seeded onto 6-well plates. Forty-eight hours after transfection, 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 phenylmethylsulfonyl fluoride, 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 μg) were separated on a 15% SDS polyacrylamide gel and transferred to nitrocellulose membranes (Hybond C; Amersham, Freiburg, Germany). Membranes were blocked in 5% nonfat dry milk in TBS for 1 h at room temperature and probed with the rabbit anti-Egr-1 antibody (dilution 1:500; Santa Cruz Biotechnology, Inc.) overnight at 4°C. After 3 washing 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 luminol-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 (8,9).

Protein cell extracts were prepared form PC-3 and LNCaP cells treated with either siRNA-Egr-1 or STI-571 in CE buffer (25 mM PIPES pH 7.0, 25 mM KCl, 5 mM EGTA, 1 mM DTT, 10 μM cytochalasin B, 0.5% NP-40 and protease inhibitor) and cleared by centrifugation at 20,000 × g for 30 min. Protein (10–20 μg) were diluted in a total volume of 500 ml of caspase buffer (50 mM HEPES pH 7.4, 100 mM NaCl, 1 mM EDTA, 5 mM DTT, 0.1% CHAPS and 10% sucrose) containing 100 μM Ac-DEVD-AFC. Reactions were incubated at 37°C, and the fluorescence at 400 nm Ex/505 nm Em was measured every 5 min for 30–60 min. Caspase activity was calculated from the initial slope of a standard curve with known concentrations of free AFC. Caspase inhibitors were purchased from Enzyme Systems Products (Livermore, CA, USA) (26).

The effect of blocking c-Abl and/or Egr-1 activity in human LNCaP and PC-3 prostate carcinoma cells was determined by the MTT survival assay, or using a commercial MTT assay kit (CellTiter 96^® AQueous One Solution Cell Proliferation Assay; Promega Corporation, Madison, WI, USA) according to the manufacturer’s instructions. The MTT survival assay was performed as described previously (Yu et al, 2000). The MTT assay is a commonly used method for the evaluation of cell survival, based on the ability of viable cells to convert MTT, a soluble tetrazolium salt [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT)], into an insoluble formazan precipitate, which is quantitated by spectrophotometry following solubilization in dimethyl sulfoxide (DMSO). Briefly, LNCaP and PC-3 cells untreated and treated with TNF-α alone, or in combination with siRNA-Egr-1 or STI-571, were cultured in 96-well tissue culture dishes and incubated with MTT (2 mg/ml) for 4 h. The cells were then solubilized in 125 ml of DMSO, and absorbance readings were taken using a 96-well Opsys MR™ microplate reader (Thermo Labsystems, Chantilly, VA, USA). The amount of MTT dye reduction was calculated based on the difference between absorbance at 570 and 630 nm. Cell viability in treated cells was expressed as the amount of dye reduction relative to that of untreated control cells. The wells which contained only medium and 10 ml of MTT were used as blanks for the plate reader.

Cell death was assessed as previously described (27). Annexin V-fluorescein isothiocyanate (Annexin V-FITC) and propidium iodide (PI) labeling was performed using the Apoptosis/Necrosis Detection kit (Abcam, UK) according to the manufacturer’s instructions. After 24 h of treatment, the cells were harvested and Annexin V-FITC was added to a final concentration of 2.5 mg/ml. To detect necrotic cells, PI was added at a concentration of 5 mg/ml. The Annexin V-FITC and PI-labeled cells were analyzed by FACS (FACSCanto; BD Biosciences, San Jose, CA, USA). Using flow cytometry, dot plots of Annexin V-FITC on the x-axis against PI on the y-axis were used to distinguish viable cells (negative for both PI and Annexin V-FITC), early apoptotic cells (Annexin V-positive and PI-negative) and late apoptotic or necrotic cells (positive for both PI and Annexin V-FITC staining). Unstained cells and untreated cells were used as negative controls. The data were analyzed using Cyflogic software (non-commercial version, CyFlo Ltd.).

The expression of Egr-1 and c-Abl protein and mRNA in the PC-3 and LNCaP cells were evaluated and compared. Proteins from cells were extracted and fractionated by SDS-PAGE. The results revealed that TNF-α caused Egr-1 activation and protein expression in both the PC-3 and LNCaP cells, while the activation of c-Abl was only marginal, when compared to the control cells, suggesting that the effect of TNF-α does not involve the c-Abl pathway (Fig. 1).

The involvement of Egr-1 and c-Abl was further examined by pre-treatment of cells with siRNA-Egr-1 and Abl inhibitor STI-571, respectively. Egr-1 and c-Abl responses were suppressed in PC-3 and LNCaP prostate carcinoma cell lines (Figs. 2 and 3). As shown by western blot assay, siRNA-Egr-1 was able to strongly suppress the expression of EGR-1 and to moderately suppress the expression of c-Abl in PC-3 (Fig. 2A) and LNCaP cells (Fig. 3A), while inhibition of c-Abl expression by STI-571 treatment did not affect the expression of EGR-1 (Figs. 2A and 3A). To gain further insight into the role of the overexpression of EGR-1 in prostate cells treated either with siRNA-Egr-1 or STI-571, we overexpressed EGR-1 in PC-3 and LNCaP cells and investigated the effect of this overexpression on cell proliferation in cells treated either with siRNA-Egr-1 or STI-571. Figs. 2B and 3B show the results obtained in PC-3 and LNCaP cells after 1, 2 and 3 days of incubation in the presence of siRNA-Egr-1 or STI-571. Cell growth was measured using an MTT assay. A strong anti-proliferative activity was noted following treatment with siRNA-Egr-1, while STI-571 showed only a moderate anti-proliferative effect (Figs. 2B and 3B). In addition, application of TNF-α was unable to increase the cell number in the presence of siRNA-Egr-1 (Figs. 2B and 3B). Notably, cells treated with STI-571 also showed a significant increase in the number of cells, but this number was lower than that in the cells treated with siRNA-Egr-1 both in PC-3 (Fig. 2B) and LNCaP cells (Fig. 3B). The results suggest that overexpression of EGR-1 partially decreased the c-Abl activity in a p53-independent manner since both p53-deficient (PC-3) and wild-type p53 (LNCaP) cells were similarly affected by overexpression of EGR-1. The results seem to contradict previous studies, which found that Abl activation in situ, and elevated phospho-c-Abl are correlated with increased local expression of Egr-1. Collectively, these results position Egr-1 downstream of c-Abl in the fibrotic response.

We investigated the effect of siRNA-Egr-1 and STI-571 on the ability to induce caspase activity in PC-3 and LNCaP cells. Cells overexpressing EGR-1 were treated or not with either siRNA-Egr-1 or STI-571 at the indicated periods of time, and caspase activity was subsequently assessed using DEVD-AFC as a substrate. The induction of DEVDase activity by blocking EGR-1 expression was stronger than that induced by inhibition of c-Abl in prostate carcinoma cell lines PC-3 and LNCaP (Fig. 4A and B). Externalization of phosphatidylserine was also investigated as a parameter for the induction of apoptosis by blocking EGR-1 and c-Abl expression in prostate cancer cells. The appearance of Annexin V-positive (apoptotic) cells was evident after only 1 h of incubation with siRNA-Egr-1 and STI-571 and reached a maximum after 3–4 h (Fig. 4C). As expected, the percentage of apoptotic cells was higher in the presence of cells treated with siRNA than the percentage in cells treated with STI-571.