Three patients with unilateral group E retinoblastoma, the most extensive stage according to the International Classification of Retinoblastoma (ICRB) (11), and one patient with bilateral retinoblastoma (group B and E by ICRB) were included in this study. The patients had no evidence of metastasis and underwent enucleation of eyes with group E retinoblastoma without prior systemic chemotherapy or local treatments at 8.3±3.3 months of age. All human retinoblastoma tissue samples were obtained with informed consent and approval by the Institutional Review Board for Clinical Research at the Seoul National University Hospital complying with the tenets of the Declaration of Helsinki.

Human retinoblastoma cell line Y79 (American Type Culture Collection, Rockville, MD, USA) and SNUOT-Rb1, which was established by our group and is distinguished from Y79 by adherent growth and rapid proliferation (16), were incubated in RPMI-1640 medium (WelGENE Inc., Daegu, Korea), supplemented with 10% fetal bovine serum (Gibco-BRL, Rockville, MD, USA) and 100 μg/ml streptomycin (Invitrogen Life Technologies, Carlsbad, CA, USA) at 37°C in a moist atmosphere of 95% air and 5% CO₂. We replaced the medium every third day and checked the cultured tumor cells daily under a phase-contrast microscope (Carl Zeiss, Chester, VA, USA). If needed, 5 μg/ml of clusterin (provided by B.H. Min, Korea University, Seoul, Korea) and/or 1 μg/ml of cisplatin (Sigma-Aldrich, St. Louis, MO, USA) were administered.

The enucleated eyes were fixed in formalin, embedded in paraffin, and then sectioned (4 μm). The slides were de-paraffinized and incubated with proteinase K at 37°C. After blocking endogenous peroxidase activity with hydrogen peroxide and blocking non-specific binding with a blocking kit (Zymed Laboratories Inc., South San Francisco, CA, USA), slides were incubated overnight with rabbit anti-clusterin antibody (1:100; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at 4°C, followed by a biotinylated goat anti-rabbit antibody (Dako, Glostrup, Denmark), revealed by the avidin-biotin complex (Vectastain kit; Vector Laboratories, Burlingame, CA, USA) and the 3-amino-9-ethyl-carbazole chromogen (AEC; Dako). Nuclei were then counterstained with methyl green. After being mounted with Faramount aqueous mounting medium (Dako), slides were observed under a light microscope (Carl Zeiss).

Clusterin was purified from fresh normal human plasma as described in our previous studies, in compliance with the Declaration of Helsinki (14,17,20). Human plasma supplemented with 0.5 mM phenylmethylsulfonyl fluoride (PMSF) was precipitated by adding 12–23% polyethylene glycol (PEG, MW 3350; Sigma-Aldrich) overnight at 4°C. The precipitate was dissolved and subjected to dimethylamino ethanol-Sepharose column chromatography (GE Healthcare Life Sciences, Buckinghamshire, UK). Fractions containing clusterin were subjected to heparin-Sepharose column chromatography (GE Healthcare Life Sciences). The obtained clusterin was finally purified by a clusterin monoclonal antibody affinity chromatography (cyanogen bromide-activated Sepharose 4B; Sigma-Aldrich). The eluted protein was dialyzed and lyophilized before being stored at −80°C.

For transfection, the Lipofectamine Plus™ reagent (Invitrogen Life Technologies) was used following the manufacturer's instructions. Briefly, 8 μl of Plus™ reagent and 5 μg of plasmid DNA (pcDNA-CLU; provided by B.H. Min) were suspended with 487 μl of RPMI-1640 medium without serum and antibiotics [RPMI(−)], and incubated for 15 min at room temperature. Lipofectamine (12 μl) was mixed with 488 μl of RPMI(−). The Lipofectamine suspension was then mixed with the Plus-pcDNA mixture, and incubated for 15 min at room temperature. The mixtures of 1 ml RPMI(−) containing 5 μg of pcDNA were added to 2.5×10⁵ cells and incubated for 3 h at 37°C. Then, the medium was replaced with RPMI-1640 medium. The time when pcDNA was added to cells was defined as time 0. After 24 h of induction, cisplatin treatment was carried out.

Cell viability was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. SNUOT-Rb1 cells were seeded into each well of 48-well plates at a concentration of 2×10⁴ cells/well. After incubation for 24 h, cells were treated with various concentrations of cisplatin. After incubation for the following 24 h, MTT solution was added to each well at a final concentration of 0.2 mg/ml. After incubation at 37°C for 2 h, the medium was carefully removed, and DMSO was added to solubilize the formazan produced by the viable cells. Optical density values at 540 nm were measured by a microplate spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). Three independent experiments were performed for each experimental condition.

Trypan blue dye exclusion was also used to assess the viability of cells following clusterin treatment. Viable cells were counted on a Luna™ automated cell counter (Logos Biosystems, Gyunggi-Do, Korea). Three independent experiments were performed for each experimental condition.

Cell lysates were obtained by resuspending cells in radioimmunoprecipitation assay (RIPA) buffer (Tris 50 mM pH 7.4; NaCl 150 mM; SDS 0.1%; Na deoxycholate 0.5%; Triton X-100 1%; Cell Signaling Technology, Inc., Danvers, MA, USA) with a complete protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN, USA), incubated on ice for 1 h and centrifuged at 20,000 × g for 30 min at 4°C. After measurement of the protein concentration using a BCA protein assay kit (Pierce Biotechnology Inc., Rockford, IL, USA), equal amounts of protein from the supernatant were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 7% Tris-Tricine gel (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and transferred to nitrocellulose membranes (Amersham Hybond ECL; GE Healthcare, Piscataway, NJ, USA). After being blocked with diluted 5% dry skim milk for 1 h, the membranes were rinsed and incubated with specific antibodies against clusterin and β-actin in PBS-T (PBS containing 0.1% Tween-20; Bio-Rad Laboratories, Inc.) overnight at 4°C. The primary antibody was removed by washing the membranes with PBS-T and incubation for 1 h with horseradish peroxidase-conjugated secondary antibodies.

We performed 4,6-diamidino-2-phenolindole (DAPI; Sigma-Aldrich) staining for cell viability analysis. SNUOT-Rb1 cells (1×10⁶ cells) were plated in 6-well plates and cultured for 24 h. The cells were treated with cisplatin (1 μg/ml) and/or clusterin (5 μg/ml). After 24 h of incubation, cells were fixed and stained with 10 μg/ml of DAPI (Sigma-Aldrich). After incubation for 5 min in the dark, the cells were washed and observed under a fluorescence microscope (Leica, Wetzlar, Germany).

Statistical analyses were performed using SPSS software version 18.0 (SPSS Inc., Chicago, IL, USA). A P-value <0.05 was considered to indicate a statistically significant result. Values are expressed as means ± SD.

To investigate the expression of clusterin in clinical samples, we performed immunohistochemical staining with an anti-clusterin antibody on tissues from enucleated human eyeballs with retinoblastoma. Microscopic examination showed that all the retinoblastoma tissues consisted of viable cells around blood vessels and zones of necrosis relatively far from the vessels. Clusterin was highly expressed in the retinoblastoma particularly at the peripheral areas of viable cells, adjacent to the necrotic zones, while barely detectable in the area with numerous Flexner-Wintersteiner rosettes marked by central lumen with surrounding cells showing cytoplasmic extensions (Fig. 1A). Counterstaining with methyl green revealed that clusterin was mainly stained in the cytoplasm.

Furthermore, we assessed the expression of clusterin in human retinoblastoma cell lines. As shown in Fig. 1B, clusterin was detected in SNUOT-Rb1 and Y79 cells by western blot analysis. Following evidence of clusterin expression in human retinoblastoma tissues and SNUOT-Rb1 cells, we further evaluated the effect of clusterin on cisplatin-induced cell death in SNUOT-Rb1 cells.

To investigate the effect of cisplatin on retinoblastoma cell death, we performed a cell viability assay under a condition of gradually increasing concentrations of cisplatin for 24 h. As shown in Fig. 2A, at least 1 μg/ml of cisplatin was required to significantly affect the cell viability of SNUOT-Rb1 cells (P=0.001). We determined the concentration with enough chemotherapeutic activity as 1 μg/ml for further experiments.

As cisplatin is known to impair proper DNA replication and activate apoptotic pathways (7,8), apoptosis was evaluated in SNOT-Rb1 cells. After 24 h of cisplatin treatment, cleaved caspase-3 was increased (Fig. 2B). Thus, cisplatin induced the apoptotic death of retinoblastoma cells.

To determine the effect of exogenous clusterin on cisplatin-induced apoptotic cell death, trypan blue dye exclusion was carried out following treatment with cisplatin and/or exogenous clusterin. Clusterin was administered 4 h prior to the cisplatin treatment. The viability of SNUOT-Rb1 cells was not affected by 5 μg/ml of exogenous clusterin (Fig. 3A). Cisplatin (1 μg/ml) significantly decreased the viability, but the exogenous supplement of clusterin effectively prevented cisplatin-induced cell death (Fig. 3A). The results were confirmed by DAPI staining of the culture plates. Condensated and fragmented nuclei (arrow) with decreased cell numbers in the cisplatin-treated group reflected cell death by cisplatin, which was completely abrogated by the supplement of exogenous clusterin (Fig. 3B).

To evaluate the mechanism by which clusterin protects retinoblastoma cells from cisplatin-induced cell death, the inhibitory effect of clusterin on caspase-3 activity was assessed by western blot analysis. As demonstrated in Fig. 3C, increased activity of caspase-3 by cisplatin treatment was dramatically attenuated by cotreatment with 5 μg/ml of clusterin. These data suggest that exogenous clusterin inhibits apoptosis induced by cisplatin in retinoblastoma cells.

Given that clusterin is overexpressed in human retinoblastoma tissues and cells as determined in the present study, we evaluated the effect of clusterin overexpression on cisplatin-induced cell death. SNUOT-Rb1 cells were transfected with pcDNA-CLU using empty vectors as a control, and underwent a cell viability assay. Cells transfected with pcDNA-CLU exhibited increased expression of clusterin (Fig. 4A). The cell viability assay demonstrated that transfected cells exhibited significantly less cell death following cisplatin treatment (Fig. 4B). The effect of clusterin overexpression on apoptosis was also evaluated by western blot analysis of cleaved caspase-3. As shown in Fig. 4C, increased activity of cleaved caspase-3 following cisplatin treatment was attenuated in cells transfected with pcDNA-CLU. Thus, clusterin overexpression inhibits cisplatin-induced apoptotic cell death similar to that of exogenous clusterin.