Docetaxel (Taxotere) was obtained from Aventis Pharmaceuticals (Bridgewater, NJ, USA). Hoechst 33258, RNase A and propidium iodide (PI) were from Sigma Chemical Co. (St. Louis, MO, USA). 3,3′-Dihexyloxacarbocyanine iodide [DiOC₆(3)] was from Invitrogen Molecular Probes (Carlsbad, CA, USA).

The antibodies used included rabbit anti-p53 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); rabbit anti-cleaved caspase-3, -7, -8 and -9 (Cell Signaling Technology, Inc., Beverly, MA, USA); rabbit anti-tBID (p15) cleavage site-specific antibody (BioSource International, Camarillo, CA, USA); mouse anti-PARP and rabbit anti-phosphorylated p53 (Calbiochem, San Diego, CA, USA); mouse anti-β-actin (Sigma); mouse anti-XIAP (BD Pharmingen, San Diego, CA, USA). Horseradish peroxidase-conjugated anti-rabbit IgG and anti-mouse IgG (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and FITC-conjugated anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA) were also used.

LNCaP, DU145 and PC3 (human prostate carcinoma cell lines; American Type Culture Collection, Rockville, MD, USA) were maintained in Dulbecco’s modified Eagle’s medium/ Nutrient Mixture F-12 HAM (Sigma) containing 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen) and 1.2 g/l sodium bicarbonate supplemented with 10 μg/ml penicillin-streptomycin (Invitrogen). HCT-116 p53^+/+ and p53^−/− cells were provided by Dr Bert Vogelstein (Howard Hughes Medical Institute, Baltimore, MD, USA) and maintained in RPMI-1640 (Invitrogen) containing 10% heat-inactivated FBS and 2 g/l sodium bicarbonate supplemented with 10 μg/ml penicillin-streptomycin. Cells were incubated in a humidified incubator at 37°C with 5% CO₂. Doc (10 mg) was dissolved in 7.8 ml of 1X PBS (pH 7.4), 7.8 ml of DMSO and 15.6 ml of absolute ethanol as 375 μM stock, and it was added to cells at 50% confluency.

For transfection, PC3 cells were seeded in 6-well plates and grown until they attained 50% confluency, and then 1.5 μg of p53 plasmid was introduced into the cells using Transfectin (Bio-Rad, Richmond, CA, USA) according to the manufacturer’s instructions. After 24 h, the cells were treated with 5 nM Doc and then incubated at 37°C for an additional 24 h.

For transfection, cells were seeded in 6-well plates and grown until they attained 50% confluency, after which 6 μl of 10 μM stock siRNA (SignalSilence^® p53 siRNA; Cell Signaling Technology, Inc.) was introduced into the cells using RNAiFect (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions.

Cells were harvested for cell cycle analysis, fixed with 95% ethanol (with 0.5% Tween-20) for 24 h, incubated with 0.05 mg/ml PI and 1 μg/ml RNase A at 37°C for 30 min, and analyzed by flow cytometry using an Epics XL flow cytometer with analysis software (EXPO32™; Beckman Coulter, Miami, FL, USA). The cells in the sub-G₁ population were considered apoptotic.

Cells were stained with Annexin V-FITC and PI using the Annexin V-FITC Apoptosis Detection kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s protocol. Briefly, 1×10⁵ cells were pelleted and resuspended in 100 μl of binding buffer. Subsequently, 5 μl of Annexin V-FITC and PI (0.05 mg/ml) were added to the cells, and cells were incubated for 15 min at room temperature (RT). After incubation, 400 μl of the binding buffer was added to the stained cells, and the cells were analyzed by flow cytometry.

Cells (5×10⁵) were incubated with 100 nM DiOC₆ at 37°C for 30 min and then washed and resuspended with PBS; thereafter, the fluorescence was measured by flow cytometry.

For western blot analysis, whole cell lysates were prepared by incubating cell pellets in lysis buffer [30 mM NaCl, 0.5% Triton X-100, 50 mM Tris-HCl (pH 7.4), 1 mM Na₃VO₄, 25 mM NaF, 10 mM Na₄P₂O₇] for 30 min on ice. After insoluble fractions were removed by centrifugation at 14,000 rpm at 4°C for 40 min, the supernatants were collected, and the protein concentration was determined using a BCA protein assay kit (Pierce Biotechnology, Inc., Woburn, MA, USA). Cell lysates (50 μg) were subjected to SDS-PAGE and transferred onto a nitrocellulose membrane (Amersham Pharmacia Biotech). Membranes were incubated for 1 h at RT with a primary antibody in Tris-buffered saline containing 0.05% Tween-20 (TBS-T; pH 7.4) in the presence of 5% nonfat dry milk. After the membranes were washed in TBS-T, secondary antibody reactions were performed with an appropriate source of antibody conjugated with horseradish peroxidase. The signals were detected with an Enhanced-Chemiluminescence Detection kit (Amersham Pharmacia Biotech) in the LAS-3000 detector (Fuji, Japan). Immunoblotting for β-actin was performed in every experiment as an internal control.

Collected cells were attached on slide glass by cytospin centrifugation using a Cellspin™ (Hanil, Korea). Cells were fixed with 4% paraformaldehyde at RT for 30 min, washed with PBS for 10 min, and incubated with 0.2% Triton X-100 for 10 min. Then, cells were washed, incubated with the appropriate primary antibody in 1% bovine serum albumin at RT for 2 h. For the secondary antibody reaction, cells were incubated with the appropriate fluorescein (FITC)-conjugated secondary antibody at RT for 1 h. For counterstaining of the nucleus (when required), cells were incubated with PI (50 μg/ml) at RT for 15 min. Finally, cells were washed, mounted, and observed using a confocal microscope (LSM510; Carl Zeiss, Germany).

Data are expressed as the mean ± SD of 3 or 4 separate experiments and analyzed by the Student’s t-test. Mean values were considered statistically significant at P<0.05.

To monitor the role of p53 in Doc-induced cancer cell death, three types of prostate cancer cells [LNCaP (p53 wild-type), DU145 (p53 mutant) and PC3 (p53 null)] with a different p53 status were primarily employed in this study. According to our previous study (10), 5 nM Doc (for 48 h) was considered as the optimal condition for the induction of apoptosis in these prostate cancer cell lines. Flow cytometric sub-G₁ analysis showed that significant cell death occurred in DU145 and PC3 cells, but not in LNCaP cells (Fig. 1A). The type of cell death induced by Doc was mostly apoptotic, as evaluated by Annexin V binding assay (Fig. 1B). Moreover, Doc-induced apoptosis was accompanied by depolarization of mitochondrial membrane potential (MMP; ΔΨm) (Fig. 1C). These results indicate that Doc effectively induces apoptosis in prostate cancer cells that harbor defective p53.

Changes in total p53 levels and phosphorylated p53 (at serine 15) after exposure to Doc were detected only in LNCaP cells (Fig. 2). While significant activation of caspase-9, -8, -7 and -3 was not observed in LNCaP cells, all caspases were cleaved in both DU145 and PC3 cells (Fig. 2). Appearance of tBID supports the evidence that the caspase-8 pathway was also associated with Doc-induced apoptosis, in conjunction with the caspase-9 pathway (Fig. 2). Cleavage of PARP and decreased levels of XIAP protein indicate that Doc-induced apoptosis in prostate cancer cells was caspase-3 dependent (Fig. 2).

In order to examine whether the ablation of p53 in LNCaP cells increases their susceptibility to Doc-induced apoptosis, we used siRNA-mediated knockdown of p53. As shown in Fig. 3A and B, the expression of endogenous p53 was dramatically reduced by siRNA transfection. p53 knockdown synergistically increased LNCaP cell death induced by Doc (Fig. 3C), which was associated with a caspase-dependent pathway (Fig. 3D). These results indicate that p53 plays a crucial role in the suppression of Doc-dependent apoptosis in prostate cancer cells.

We next tested whether transient transfection of p53 into PC3 cells suppresses Doc-induced apoptosis. An increase in total and phospho-p53 was detected in PC3 cells following transfection (Fig. 4A and C) and was associated with a significant repression of Doc-induced apoptosis (Fig. 4B). The inhibitory role of p53 in Doc-induced apoptosis was confirmed by the inhibition of the cleavage of caspase-3 and -7, and PARP that are provoked by Doc (Fig. 4C). These results imply that p53 interferes with Doc-induced apoptosis in prostate cancer cells.

To determine whether p53 plays an inhibitory role in Doc-induced apoptosis in cells other than prostate cancer cells, an isogenic pair of colorectal carcinoma (HCT-116) cell lines differing only in their p53 status was investigated. Doc induced cell death in a higher number HCT-116 p53^−/− cells when compared to the number in the HCT-116 p53^+/+ cells (Fig. 5A). Apoptosis occurred in a caspase-dependent manner, as revealed by cleavage of caspase-3 and -7, and PARP (Fig. 5B). Furthermore, the functional knockdown of p53 in HCT-116 p53^+/+ cells by either p53 siRNA or pifithrin-α (PFT-α), a p53 inhibitor, sensitized cells to Doc-induced apoptosis, which was concomitant with caspase-3 and -7, and PARP cleavage (Fig. 5C). Taken together, these results indicate that p53 also inhibits colorectal cancer cell apoptosis induced by Doc.