Lentiviral Vector-Mediated PAX6 Overexpression Promotes Growth and Inhibits Apoptosis of Human Retinoblastoma Cells

Abstract

Purpose.: The cancer-associated gene PAX6 is a key regulator in the embryological development of the retina. The authors assessed whether PAX6 was associated with the development of retinoblastoma.

Methods.: Two human retinoblastoma cell lines (Y79 and SO-Rb50) were transfected with PAX6-GFP recombinant lentiviral vectors and were compared with cells undergoing transfection with GFP lentiviral vectors and cells without any intervention. Overexpression of PAX6 was assessed by quantitative real-time polymerase chain reaction analysis and Western blot analysis. Cell proliferation assays were evaluated by colorimetric cell counting kit-8. The cell cycle was analyzed by fluorescence-activated cell sorting (FACS). Apoptosis rates were assessed by TUNEL assay followed by FACS analysis. Using Western blot analysis, the authors measured levels of proteins p53, p21, p27, cdc2, proliferating cell nuclear antigen, and cleaved caspase-3.

Results.: Three days after transfection, both cell lines showed a statistically significant (P < 0.001) overexpression of PAX6, parallel to significantly (P < 0.001) increased cell proliferation. At 7 days after transfection, cell cycle analysis showed a significant (P < 0.001) reduction of G0/G1 arrest and a significant induction of G2/M arrest (P < 0.01). Parallel to a reduction in caspase-3 levels, the apoptosis rate significantly (P < 0.001) decreased. Levels of p53, p21, and p27 were reduced, and the levels of cdc2 were increased.

Conclusions.: Lentiviral vector-mediated overexpression of PAX6 in human retinoblastoma cells was associated with increased cell proliferation parallel to a reduced caspase-3–dependent apoptotic rate and a change in the p53 regulated cell cycle. PAX6 may be further explored for the diagnosis of and therapy for retinoblastomas.

Previous studies have shown that retinoblastoma is initiated by the loss of both alleles of the prototypic tumor suppressor gene RB1. ¹ A large number of cytogenetic studies have additionally suggested that these Knudson's M1 and M2 mutational events, though necessary for initiation, were not the only genomic changes occurring in the oncogenesis of retinoblastoma. ² The cell of origin of retinoblastoma has been the subject of intense debate for many years. ³ Studies have suggested that retinal stem cells or retinal progenitor cells can be cells of origin of retinoblastomas. ⁴ Zhong et al. ⁵ reported that retinal stem–like cells were present in human retinoblastomas and that cultured human retinoblastoma cells expressed retinal development-related genes, including PAX6 in vitro. PAX6 is a homeobox transcription factor in the PAX gene family containing a total of nine members in vertebrates. Many studies have shown that PAX proteins play an essential role in organogenesis by inhibiting terminal differentiation and apoptosis. These functional characteristics of PAX6 may facilitate the development and progression of specific cancers in association with PAX6. ^{6 –8} As one of the master regulators of ocular development, the PAX6 gene is involved in the development of the retina, including the control of cell proliferation, maintenance of retinal progenitor cell potency, and cell fate determination. ^9,10 Moreover, PAX6 positively and directly regulates cohorts of genes that promote neural stem cell self-renewal, progenitor cell genesis, and neurogenesis in a highly dosage-dependent manner. Removing PAX6 may reduce stem cell self-renewal by decreasing the expression of key cell cycle regulators. ^11,12 However, little is known about whether the PAX6 gene is involved in the progression of human retinoblastomas and how the effects occur. 

In a previous study, ¹³ we found that transient silencing of the PAX6 gene with small interfering RNA (siRNA) resulted in the inhibition of proliferation and the increased apoptosis of human retinoblastoma cells. The molecular mechanisms underlying siRNA-mediated PAX6 depletion-induced cell cycle changes and increased apoptosis have remained unclear. ¹³ We, therefore, conducted the present study in which we first constructed recombinant PAX6-GFP lentiviral vectors to induce the overexpression of PAX6 in human retinoblastoma cells. It was complementary to our previous study of silencing of the PAX6 gene. We then confirmed the overexpression of PAX6 by quantitative real-time polymerase chain reaction (Q-PCR) analysis and Western blot analysis. Finally, we examined the effects of PAX6 overexpression on cell proliferation by cell proliferation assays, on the cell cycle by fluorescence-activated cell sorting (FACS), on the rate of apoptosis by TUNEL assay followed by FACS analysis, and on the levels of cell cycle and apoptosis-related proteins p53, p21, p27, cdc2, proliferating cell nuclear antigen (PCNA), and cleaved caspase-3 by Western blot analysis. 

Methods

Cell Culture

The human retinoblastoma cell lines Y79 and SO-Rb50 were provided by the Department of Pathology of the Zhongshan Ophthalmic Center, Sun Yat-sen University, and the Chinese University of Hong Kong. The cells were cultured in RPMI-1640 medium (Hyclone Laboratories, Logan, UT) supplemented with 10% fetal bovine serum, 100 U/L penicillin, and 100 U/L streptomycin at 37°C in a humidified atmosphere of 95% air/5% CO₂. The culture medium was replaced every 3 days. 

Production of PAX6 Recombinant Lentiviral Vectors

A third generation of the self-inactivating lentiviral vector containing the cytomegalovirus promoter-driven enhanced green fluorescence protein (eGFP) reporter was purchased from Shanghai Genechem Co., Ltd. (Shanghai, China). The lentiviral vector system had three parts before packaging, including the pGCL-GFP vector, the pHelper 1.0 (gag/pol element) vector, and the pHelper 2.0 (VSVG element) vector. The pGCL-GFP vector encoding the full length of the human PAX6 gene (NCBI reference sequence ID, NM_000280.3) was constructed by Shanghai Genechem Co., Ltd. PCR and DNA sequencing confirmed the accurate insertion of the PAX6 cDNA. For the preparation of the recombinant lentiviral vectors, the three-plasmid transient transfection method was used. ¹⁴ The pHelper 1.0 plasmid (15 μg), the pHelper 2.0 plasmid (10 μg), and the pGCL-GFP-PAX6 or the pGCL-GFP plasmid (20 μg) were cotransfected into subconfluent 293T cells in serum-free medium using the cationic liposome based transfection reagent (Lipofectamine 2000; Invitrogen Co., Carlsbad, CA). After 8 hours of incubation, the medium was completely exchanged. High-titer recombinant lentiviral vectors with PAX6 were harvested 48 hours later. 

Establishment of Stable PAX6 Overexpression in Human Retinoblastoma Cells

For transfection, Y79 and SO-Rb50 human retinoblastoma cells were plated at a concentration of 5 × 10³ cells/well in 96-well plates. The cells were transfected either with PAX6-GFP lentiviral vectors (PAX6 overexpression group, study group) or with the GFP lentiviral vectors (negative GFP control group) in serum-free medium for 12 hours. Polybrene (5 μg/mL) (an enhancer reagent of retrovirus-mediated gene transduction) was added to increase the infection efficiency. A third group of cells without any intervention served as a second control group (negative control group without transfected cells). The cells were then washed and embedded into complete medium. Three days after transfection, reporter gene expression was examined using fluorescence microscopy, after which the experimental procedures were performed. 

Quantitative Real-time Polymerase Chain Reaction Analysis

Five days after lentiviral vector transfection, a Q-PCR assay was performed to measure the PAX6 mRNA levels in both retinoblastoma cell lines. Total RNA was extracted from the cells using the extraction reagent (TRIzol; Invitrogen). Reverse transcription and PCR-amplification reactions were performed according to the M-MLV reverse transcriptase protocol (Promega Biotech Co. Ltd, Beijing, China). Primers for Q-PCR were as follows: human β-actin (202 bp)—5-GGCGGCACCACCATGTACCCT-3 forward, 5-AGGGGCCGGACTCGTCATACT-3 reverse; human PAX6 (232 bp)—5-CGGCAGAAGATTGTAGAGC-3 forward, 5-GGATTTCCCAAGCAAAGAT-3 reverse. 

Q-PCR was performed using a sequence detection system (ABI prism 7900; Applied Biosystems, Foster City, CA). The 25-μL reaction mixture contained 12.5 μL SYBR green PCR master mix (TaKaRa Biotechnology Co. Ltd., Dalian, China), 2 μL cDNA template, 0.5 μL PCR forward primer, and 0.5 μL PCR reverse primer. PCR running conditions were 15 seconds at 95°C for the initial denaturation, 40 cycles of 5 seconds at 95°C and 30 seconds at 60°C, 1 minute at 95°C for annealing, and an additional incubation of 5 minutes at 72°C after completion of the last cycle. The threshold cycle (Ct values), which was the cycle number at which the amount of amplified gene of interest reached a fixed threshold, was subsequently determined. Relative quantification of the PAX6 mRNA levels was normalized to human β-actin levels and calculated with the 2^-ΔΔCt method. ¹⁵  

Western Blot Analysis

Seven days after lentiviral vector transfection, 2 × 10⁶ human retinoblastoma cells per cell line were washed twice with cold PBS, resuspended in 200 μL ice-cold cell lysis buffer (20 mM Tris [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, 1 μg/mL leupeptin, 1 mM phenylmethylsulfonyl fluoride) and incubated at 4°C for 30 minutes. The lysates were centrifuged at 12,000 rpm for 10 minutes at 4°C. The protein concentration was determined by the bicinchoninic acid protein assay kit (Pierce Chemical Co., Rockford, IL). Samples (30 μg protein) were separated by SDS-PAGE using 5% stacking and 10% separating gels and subsequently were electroblotted onto polyvinylidene difluoride membranes (Immobilon-P; Millipore, Billerica, MA). After blocking in Tris buffer (50 mM Tris, pH 7.5) containing 5% skim milk, the membranes were incubated overnight at 4°C with primary antibodies, including mouse anti-human PAX6 (diluted 500; Millipore), mouse anti-human p53 (diluted 100; BD Biosciences PharMingen, San Diego, CA), rabbit anti-human p21 (diluted 1000; Cell Signaling Technology, Danvers, MA), rabbit anti-human p27 (diluted 1000; Cell Signaling Technology), mouse anti-human cdc2 (diluted 1000; Cell Signaling Technology), rabbit anti-human cleaved caspase-3 (diluted 1000; Cell Signaling Technology), and rabbit anti-human PCNA (diluted 50; Abgent, San Diego, CA). The secondary goat anti-rabbit IgG antibody (diluted 5000; Santa Cruz Biotechnology, Santa Cruz, CA) or goat anti-mouse IgG antibody (diluted 5000; Santa Cruz Biotechnology) conjugated with horseradish peroxidase was applied after washing in PBST (PBS solution with the detergent Tween 20) and incubated for 1 hour at 37°C. Equal amounts of protein loading were confirmed by reprobing the membranes with the rabbit anti-human GAPDH antibody (diluted 1000; Santa Cruz Biotechnology). Protein expression was visualized after extensive washing using an enhanced chemiluminescence advanced detection kit (GE Healthcare, Buckinghamshire, UK) and quantified with analysis software (Quantity One; Bio-Rad Laboratories Inc., Hercules, CA). 

Cell Proliferation Analysis

The standard colorimetric cell counting kit-8 (CCK8; Dojindo Laboratories, Kumamoto, Japan) was used for the determination of the number of viable cells in cell proliferation assays. The two human retinoblastoma cell lines were seeded with a volume of 90 μL cell suspension (3000 cells/well) into 96-well plates. After transfection, as described, the cells were incubated for 72 hours. After that, 10 μL CCK8 was added to each well and incubated for 4 hours at 37°C. Optical densities were read by a microplate reader scanning at 450 nm. This procedure was repeated every 24 hours during a 6-day period. Cell survival rates were measured at three time points of the cell growth curve through the log phase of growth for each cell line. 

Cell Cycle Analysis

Both retinoblastoma cell lines were seeded on a 24-well cell culture plate. Seven days after transfection, as described, approximately 5 × 10⁵ cells were washed in cold PBS, fixed with ice-cold 70% ethanol at 4°C for 12 hours, and then stained with 1 mL cold PBS containing 50 μg/mL propidium iodide (a DNA stain; Sigma-Aldrich Co., St. Louis, MO), 100 μg/mL nuclease (RNase A; Fermentas Inc., Hanover, MD), and 0.2% Triton X-100 at 4°C for 30 minutes in the dark. The cells were then passed through a flow cytometer (FACSCalibur; BD Biosciences, San Diego, CA) equipped with a 488-nm argon laser to measure the DNA content. Data analysis was performed with the appropriate software (Cell Quest [BD Biosciences]; ModFit LT [Verity Software House Inc, Topsham, ME]). Results were presented as percentages of the total cell counts in different phases of the cell cycle, namely the G0/G1 phase (diploid cells), S phase (diploid and tetraploid cells), and G2/M phase (tetraploid cells). 

Cell Apoptosis Analysis

The cell apoptotic rate was determined by the TUNEL assay (In Situ Cell Death Detection Kit, TMR red; Roche Applied Science, Basel, Switzerland). The two retinoblastoma cell lines were seeded onto a 24-well cell culture plate. On the seventh day after transfection, as described, approximately 5 × 10⁵ cells were washed with PBS and fixed with 4% paraformaldehyde in PBS (pH 7.4) at room temperature for 1 hour and then resuspended in 0.1% sodium citrate containing 0.1% Triton X-100 for 2 minutes on ice. In accordance with the manufacturer's protocol, the cells were treated with TUNEL reaction mixture containing terminal deoxynucleotidyl transferase (TdT) and fluorescein-dUTP, and the cells were incubated at 37°C in a humidified atmosphere in the dark for 1 hour. The TdT could catalyze the binding of fluorescein-dUTP to free 3′-OH ends in the nicked DNA. After washing with PBS, the cells were analyzed with a flow cytometer (FACSCalibur; BD Biosciences) equipped with a 540-nm excitation laser. Data analysis was performed with the software (Cell Quest). Results were shown as percentages of red fluorescence-emitting cells (apoptotic cells) in the Y79 and SO-Rb50 retinoblastoma cell lines. 

Statistical Analysis

Statistical analysis was performed using a commercially available software package (SPSS 19.0 for Windows; IBM-SPSS Inc., Chicago, IL). Data were presented as the mean ± SD of three independent experiments. Statistical analysis of differences was carried out by a one-way ANOVA. P < 0.05 was considered to indicate statistical significance. 

Results

Infection Efficiency of Lentivirus Vectors

To investigate PAX6 function in both retinoblastoma cell lines, we tried to overexpress this gene by transfecting the retinoblastoma cells with PAX6-GFP lentiviral vectors. The transfected cells expressed eGFP after transfection with the lentiviral vectors. The cells of cell lines Y79 and SO-Rb50 were transfected by the lentiviral vectors at different multiplicities of infection (MOIs). GFP expression was examined 3 days after transfection using fluorescence microscopy. The efficiency of the infection (averaged proportion of GFP-expressing cells on the total cell count) was approximately 80% at a MOI of 20 (Fig. 1). Consequently, a MOI of 20 was chosen for the next steps of this study. 

Figure 1.

 

Monitoring transfection efficiency of human retinoblastoma cells (cell lines SO-Rb50 and Y79) by GFP detection. Lentiviral vector-mediated PAX6 expression was visualized by fluorescence microscopy three days after transfection. Comparing assessment in bright field with the assessment in fluorescent conditions revealed a transfection efficiency of >80%. Mock, negative control group without transfected cells; pGCL-GFP, negative GFP control group with cells transfected with GFP lentiviral vectors alone; pGCL-GFP-PAX6, Study group (PAX6 overexpression group) with cells transfected with PAX6-GFP lentiviral vectors. b, bright field; f, fluorescent field. Scale bars, 50 μm.

Stable Overexpression of PAX6 in the Human Retinoblastoma Cell Lines

Q-PCR analysis demonstrated that the relative levels of PAX6 mRNA were markedly increased in the study groups of the Y79 and SO-RB50 cell lines (667.8 ± 12.2 and 10.4 ± 1.0, respectively) compared with the levels in the negative GFP control groups (cells transfected with GFP vectors alone; 1.00 ± 0.06 and 1.00 ± 0.07, respectively) and compared with the levels in the nontransfected control cells (0.10 ± 0.05 and 1.08 ± 0.07, respectively) (ANOVA; F = 9036, P < 0.001 in Y79 groups; F = 254, P < 0.001 in SO-Rb50 groups) (Figs. 2a, 2b). The effectiveness of the PAX6-GFP lentiviral vectors was also confirmed by examination of the levels of the PAX6 protein by Western blot analysis. The protein expression levels of PAX6 in the Y79 and SO-RB50 cell lines were significantly higher in the study groups than in the control groups (Fig. 2c). In comparison, the GAPDH protein expression levels did not vary significantly between all the groups (Fig. 2c). 

Figure 2.

 

Q-PCR analysis (a, b) and Western blot analysis (c) for the assessment of the PAX6 overexpression in two human retinoblastoma cell lines, Y79 and SO-Rb50, after transfection by lentiviral vectors. (a, b) Five days after transfection, total RNA was extracted from the cells of the study groups (transfected with PAX6-GFP lentiviral vectors) (pGCL-GFP-PAX6), cells of the negative GFP control groups (transfected with GFP lentiviral vectors) (pGCL-GFP), and cells of the negative control groups without transfection (Mock). Q-PCR was performed to measure the relative PAX6 mRNA levels. The amount of each product was normalized to human β-actin mRNA levels. Each value represented the mean ± SD of three replicates. PAX6 mRNA levels were significantly higher in the study groups (**P < 0.001). (c) Seven days after transfection, protein expression levels of PAX6 were assessed by Western blot analysis using the anti-human PAX6 antibody, applying the enhanced chemiluminescence method and quantification with the analysis software. The levels of the PAX6 protein were significantly higher in the study groups (pGCL-GFP-PAX6) than in the control groups (pGCL-GFP and Mock). In comparison, the GAPDH protein did not vary markedly among the groups. (Note: the protein band at 46 kDa represents the PAX6 protein, and the protein band at 74 kDa represents the PAX6-GFP fusion protein.)

Promotion of Cell Proliferation Induced by PAX6 Overexpression In Vitro

To test the pGCL-GFP-PAX6 lentiviral vectors for their ability to promote the proliferation of the Y79 and SO-Rb50 retinoblastoma cells, we performed a cell proliferation assay using the standard colorimetric cell counting technique (CCK8). Cell survival rates were measured at three time points of the cell growth curve through the log phase of growth for each cell line. A significant increase of cell survival rates was found in the study group of the Y79 cells. The percentages of surviving Y79 cells increased sharply from 103.5% ± 0.9%, 106.0% ± 0.5%, and 106.7% ± 0.5% in the negative GFP control group at 48 hours, 72 hours, and 96 hours after cell plating, respectively, to 143.2% ± 0.6%, 159.1% ± 0.5%, and 151.8% ± 1.2% in the study group, respectively (ANOVA; F = 4689, 6941, and 4864, respectively; all P < 0.001) (Fig. 3a). In a parallel manner, the percentages of surviving SO-Rb50 cells increased from 107.9% ± 1.3%, 101.3% ± 1.3%, and 100.7% ± 1.7% in the negative GFP control group at 96 hours, 120 hours, and 144 hours after cell plating, respectively, to 117.1% ± 1.7%, 124.2% ± 1.6%, and 126.6% ± 1.1% in the study group, respectively (ANOVA; F = 156, 412, and 623, respectively; all P < 0.001) (Fig. 3b). 

Figure 3.

 

Three days after transfection, cell survival rates of cells of the human retinoblastoma cell line Y79 (a) and human retinoblastoma cell line SO-Rb50 (b) were evaluated by colorimetric cell counting kit-8 (CCK8) after treatment with either PAX6-GFP lentiviral vectors (study groups) (pGCL-GFP-PAX6) or with GFP lentiviral vectors (negative GFP control groups) (pGCL-GFP). Cell proliferation was significantly (P < 0.001) higher in the study groups than in the negative GFP control groups (pGCL-GFP) and in the negative control groups without transfection of cells (Mock) at three time points of the cell growth curve through the log phase of growth for each cell line, particularly for the Y79 cell line. The negative GFP control groups and the negative control groups without transfection did not differ significantly. Each value represented the mean ± SD of four replicates. (**P < 0.001).

Changes of the Cell Cycle Induced by PAX6 Overexpression In Vitro

To study the causes of the promotion of cell proliferation induced by PAX6 overexpression, we analyzed the cell cycle by flow cytometry. In both retinoblastoma cell lines Y79 and SO-RB50, the percentages of cell counts in the G2/M phase in relation to the total cell counts were significantly higher in the study groups (22.9% ± 0.3% and 12.1% ± 0.7%, respectively) than in the negative GFP control groups (13.4% ± 0.5% and 8.7% ± 0.7%, respectively) and the negative control groups without transfected cells (12.8% ± 0.1% and 9.2% ± 0.6%, respectively) (ANOVA; F = 857, P < 0.001 in Y79 groups; F = 23, P = 0.002 in SO-Rb50 groups). Correspondingly, the percentages of the cell counts in the G0/G1 phase in relation to the total cell counts were significantly lower in the study groups (47.6% ± 0.7% and 60.2% ± 0.3%, respectively) than in the negative GFP control groups (57.1% ± 0.3% and 70.1% ± 0.5%, respectively) and the negative control groups without transfected cells (57.5% ± 0.6% and 69.8% ± 0.7%, respectively) (ANOVA; F = 286, P < 0.001 in Y79 groups; F = 354, P < 0.001 in SO-Rb50 groups). In a similar manner, the percentage of the count of the SO-Rb50 cells in the S phase in relation to the total SO-Rb50 cell count was significantly higher in the study group (27.7% ± 0.5%) than in the negative GFP control group (21.2% ± 1.1%) and in the negative control group without transfected cells (21.1% ± 0.3%) (ANOVA; F = 87, P < 0.001). The percentage of Y79 cell count in the S phase in relation to the total Y79 cell count did not vary significantly between the study group (29.5% ± 1.0%), the negative GFP control group (29.5% ± 0.3%), and the negative control group without transfected cells (29.7% ± 0.7%) (ANOVA; F = 0.08, P = 0.93) (Figs. 4a–c). 

Figure 4.

 

Seven days after the transfection of human retinoblastoma cell lines Y79 and SO-Rb50 with either PAX6-GFP lentiviral vectors (study groups) (pGCL-GFP-PAX6) or with GFP lentiviral vectors (negative GFP control groups) (pGCL-GFP), the cell cycle was analyzed by FACS. A third group of cells without transfection served as the second negative control group without transfection (Mock). DNA was stained by propidium iodide. (a) Representative DNA content histograms of a flow cytometric analysis of human retinoblastoma cell lines Y79 and SO-Rb50. (b, c) The percentages of cell counts in the G0/G1 phase, S phase, and G2/M phase of the cell cycle in relation to the total cell counts were plotted in bar diagrams. In both retinoblastoma cell lines, the percentages of cell counts in the G2/M phase were significantly higher in the study groups, and the percentages of cell counts in the G0/G1 phase were significantly lower in the study groups than in the control groups. In addition, the percentage of SO-Rb50 retinoblastoma cell count in the S phase was significantly higher in the study group. The percentages of the retinoblastoma Y79 cell counts in the S phase did not vary significantly between the groups. Each value represented the mean ± SD of three replicates (*P < 0.01, **P < 0.001). (d) Western blot analysis was performed to detect the levels of cell cycle regulatory proteins p53, p21, p27, cdc2, and PCNA in retinoblastoma cell line Y79 (left) and cell line SO-Rb50 (right). Protein expression was visualized by the enhanced chemiluminescence method and was quantified with analysis software. In the study groups, the levels of the p53 protein and the p21 protein were downregulated, particularly for the Y79 cell line. The levels of the p27 protein were slightly downregulated, and the levels of the cdc2 protein were upregulated in the study groups. The levels of the PCNA protein did not differ between the groups. In comparison, the GAPDH protein did not vary markedly between the groups.

To identify signaling pathways of the changes of the cell cycle induced by PAX6 overexpression, we performed a Western blot analysis of the cell cycle regulatory proteins p53, p21, p27, cdc2, and PCNA. The levels of the p53 and p21 proteins were downregulated in the study groups of both retinoblastoma cell lines (particularly for the Y79 cell line) compared with the negative GFP control groups and the negative control groups without transfected cells (Fig. 4d). The levels of the p27 protein were slightly downregulated, and the levels of the cdc2 protein were upregulated in both retinoblastoma cell lines. Differences in the levels of the PCNA protein were not detectable. In a similar manner, the GAPDH protein did not differ between the groups. 

Inhibition of Cell Apoptosis Induced by PAX6 Overexpression In Vitro

To confirm the cell apoptosis secondary to PAX6 overexpression, we determined the extent of DNA fragmentation seven days after transfection using TUNEL assay followed by FACS analysis. Flow cytometric analysis revealed a reduced TUNEL fluorescence signal in the study groups compared with the negative GFP control groups and the negative control groups without transfected cells of Y79 and SO-RB50 cell lines. The percentages of TUNEL-positive apoptotic cell counts to total cell counts were significantly lower in the study groups (0.04% ± 0.03% and 0.0% ± 0.0%, respectively) than in the negative GFP control groups (0.60% ± 0.03% and 0.40% ± 0.04%, respectively) and in the negative control groups without transfected cells (0.26% ± 0.05% and 0.37% ± 0.02%, respectively) (ANOVA; F = 187, P < 0.001 in Y79 groups; F = 671, P < 0.001 in SO-Rb50 groups) (Figs. 5a–c). Correspondingly, Western blot analysis revealed that the levels of the apoptosis-related protein, cleaved caspase-3 (the active form of caspase-3), were downregulated in the study groups of both retinoblastoma cell lines (Fig. 5d). 

Figure 5.

 

Seven days after the transfection of human retinoblastoma cell lines Y79 and SO-Rb50 with either PAX6-GFP lentiviral vectors (study groups) (pGCL-GFP-PAX6) or with GFP lentiviral vectors (negative GFP control groups) (pGCL-GFP), TUNEL assay was performed followed by FACS analysis to assess cell apoptotic rates. A third group of cells without transfection served as the second negative control group without transfection (Mock). (a) Representative flow cytometric histograms of the percentages of TUNEL-positive apoptotic cell counts to total cell counts. The percentages of apoptotic cells was represented as M1 in the histograms. (b, c) The percentages of TUNEL-positive apoptotic cell counts in relation to the total cell counts were plotted in corresponding bar diagrams. In both retinoblastoma cell lines, the percentages of apoptotic cells were significantly lower in the study groups than in the negative GFP control groups and in the negative control groups without transfection. Each value represented the mean ± SD of three replicates (**P < 0.001). (d) Western blot analysis revealed that the levels of apoptosis-related protein, cleaved caspase-3 (the active form of caspase-3), were lower in the study groups of the Y79 cell line (left) and the SO-Rb50 cell line (right). In comparison, the GAPDH protein did not vary markedly between the groups. The protein expression was visualized by the enhanced chemiluminescence method and quantified with analysis software.

Discussion

Using the technology of lentiviral vector–mediated stable transfection, our study demonstrated that the overexpression of PAX6 in human retinoblastoma cells resulted in increased tumor cell proliferation in vitro paralleled by a downregulation of the p53, p21, and p27 proteins and an upregulation of the cdc2 protein. Correspondingly, we observed a reduction in G0/G1 arrest and an induction in G2/M arrest in the cell cycle and an inhibition of caspase-3–dependent apoptosis. 

Consistent with our results, Mascarenhas et al. ¹⁶ reported that PAX6 activated the expression of the MET gene and that the inhibition of PAX6 led to a decline in cell proliferation and cell survival and resulted in a differentiation of pancreatic carcinoma cells. ^16,17 In contrast to our study, Mayes et al. ^18,19 found that PAX6 suppressed the tumorigenesis of glioblastoma multiforme and inhibited its invasiveness by repressing the expression of MMP2. In addition, Shyr et al. ²⁰ reported that the overexpression of PAX6 suppressed cell proliferation and inhibited colony formation of human prostate adenocarcinoma cells in vitro. The studies cited indicate that PAX6 may have different tumorigenic functions in different tumor cells. 

One of our novel findings was that significantly increased tumor cell proliferation followed the overexpression of PAX6 in human retinoblastoma cells (Figs. 3a, 3b). Overexpression of PAX6 led to the accumulation of Y79 retinoblastoma cells in the G2/M cell cycle phase and to the accumulation of SO-Rb50 retinoblastoma cells in the S phase and G2/M phase. Correspondingly, the percentages of cells at the G0/G1 phase significantly decreased in both retinoblastoma cell lines compared with the control groups (Figs. 4b, 4c). These results suggested that the PAX6 gene could effectively be overexpressed by transfection with PAX6-GFP lentiviral vectors and that the overexpression of PAX6 might have caused a reduction in G0/G1 arrest and the induction of G2/M arrest. Given that many cells in vivo are in a quiescent state (i.e., the G0 phase), the cell proliferation rate may increase when cells are activated to reenter the cell cycle undergoing cell division during the G1 phase. ^21,22  

Previous studies have suggested that the PAX6 gene regulates cell proliferation by controlling the cell cycle in a cell type–specific manner. Xu et al. ²³ found that PAX6 was enriched in retinal stem cells and that the inactivation of PAX6 in adult retinal stem cells resulted in a proliferation defect. They also suggested that PAX6 was required for the proliferation of retinal stem cells. ²³ Hsieh et al. ²⁴ showed that PAX6 is essential for the proliferation and pluripotency of chicken retinal progenitor cells during the pre-neurogenic to neurogenic transition and that either elevating or knocking down PAX6 attenuated cell proliferation and resulted in cell death. They also demonstrated that maintaining a relatively low level of PAX6 was necessary for S phase reentry. ²⁴ On the contrary, Zhou et al. ¹⁸ showed that PAX6 suppressed glioblastoma tumor cell growth in vitro by increasing the number of cells in the G1 phase and by decreasing the number of cells in the S phase. The inhibition of tumor growth occurred through the PAX6-mediated suppression of vascular endothelial growth factor A. ^18,25 The reports cited here are partially in accordance with our study and possibly reflect distinct cell cycle control mechanisms of the PAX6 gene in different cell types. 

To further explore how the PAX6 gene works to regulate cell cycle progression, the cell cycle regulatory proteins p53, p21, p27, cdc2, and PCNA were assessed by Western blot analysis. The ability of p53 to promote cell cycle arrest has been connected with its ability to transactivate one of its critical target genes, P21^WAF1 . At the G1/S checkpoint, the induction of p21, also known as cyclin-dependent kinase inhibitor 1A, arrests cells in the G1 phase and prevents S-phase entry by inhibiting the binding between cyclin-dependent protein kinase 2 (cdk2) and cyclin E. ²⁶ At the intra-S-phase checkpoint, p53 may regulate the transcription of the growth arrest and DNA damage-inducible transcripts alpha and beta (Gadd45 alpha/beta). Gadd45 may bind to PCNA, a DNA polymerase accessory factor involved in DNA replication and repair during the S phase of the cell cycle, and p21 may block the ability of PCNA to bind with Gadd45. ^27,28 At the G2/M checkpoint, the cyclin-dependent kinase 1 (cdc2), which may control entry into the mitosis (M) phase, is pivotal in regulating this transition. p21, as an inhibitor of cdc2, may be turned on by p53 and inactivate the cdc2/cyclin B complex. ^29,30 In our study, the levels of p53 and p21 were downregulated and the level of the cdc2 was upregulated in both retinoblastoma cell lines transfected with the PAX6-GFP lentiviral vectors (Fig. 4d). These findings may be associated with the decreasing percentages of tumor cells in the G0/G1 phase and the increasing percentages of tumor cells in the G2/M phase, induced by the overexpression of PAX6. Because p53 and p21 dysregulation were important associations with the PAX6 overexpression in our study, one may further conclude that the overexpression of PAX6 was associated with a reduction of a p53/p21-dependent G0/G1 arrest and an induction of p53-dependent G2/M arrest by a positive regulation of the cdc2 gene activity in both retinoblastoma cell lines. 

The levels of PCNA were, however, not altered by the overexpression of PAX6. This finding was partially consistent with the finding that the percentage of the Y79 cell count in the S phase in relation to the total number of Y79 cells did not differ between the groups. It may indicate that other S-phase regulators were responsible for the control of the cell cycle. Previous studies have suggested that increased levels of the p27^Kip1 protein typically caused cells to arrest in the G1 phase of the cell cycle. ^31,32 Kase et al. ³³ reported that functional loss of the retinoblastoma protein (Rb) led to a downregulation of p27 and an uncontrolled retinal tumor cell proliferation, whereas a lack of p27 degradation resulted in the suppression of cdc2 activity and the consequent inhibition of entry into the M phase. ³⁴ In our study, p27 protein levels were downregulated, whereas the levels of the cdc2 protein were upregulated after PAX6 was overexpressed in both retinoblastoma cell lines. Thus, our results of cell cycle analysis agreed with the findings of Kase et al. ³³ and with those of previous studies. 

Previous studies have suggested that the ability of p53 to control apoptosis in response to abnormal proliferative signals and stress, including DNA damage, is important for its tumor suppression function. ³⁵ Caspase 9 and its cofactor Apaf-1 were found to be essential downstream components of p53 in Myc-induced apoptosis. Once activated, caspase-9, as one of the initiator caspases, cleaves and activates downstream effector caspases, including caspase-3, which in turn executes apoptosis. ^36,37 Ouyang et al. ³⁸ reported that the overexpression of mouse full-length PAX6 reduced the rate of cell proliferation and induced caspase-3–independent apoptosis of rabbit corneal epithelial cell lines. In our study, we demonstrated that overexpression of the PAX6 protein inhibited apoptosis in human retinoblastoma cell lines Y79 and SO-Rb50 in vitro through the suppression of p53 and the caspase-3 protein. It is noteworthy that the activation of PAX6 may inhibit p53-mediated caspase-3–dependent apoptosis in human retinoblastoma cells. These partially contradicting results may be explained by differences between rabbit corneal epithelial cells and human retinoblastoma cells. 

Potential limitations of our study should be mentioned. First, recovery tests by RNA interference in retinoblastoma cells overexpressing PAX6 will be necessary to further consolidate the results and conclusions of our study. Second, our investigation was a cell culture study in which the cells were in an artificial environment and unnatural living conditions. It has always been questionable how far results from cell culture studies can be transferred to clinical conditions. Third, because the apoptotic rate in the immortalized retinoblastoma cell lines of our study were considerably lower than the apoptotic rate in retinoblastoma tumors in loco, the importance of our results in terms of a significant reduction in the apoptotic rate of cultured retinoblastoma cells after the overexpression of PAX6 for the pathophysiology of the tumors and for the potential clinical implications have remained unclear thus far. 

In conclusion, lentiviral vector-mediated overexpression of PAX6 in human retinoblastoma cells was associated with increased cell proliferation parallel to a reduced apoptotic rate, reduced levels of some cell cycle and apoptosis-related proteins, and a change in the cell cycle that may be regulated by p53 in vitro. PAX6 may be further explored for the diagnosis of and therapy for retinoblastomas. 

Footnotes

 Supported by National Natural Science Foundation of China Grant 30772377.

Footnotes

 Disclosure: L. Li, None; B. Li, None; H. Zhang, None; S. Bai, None; Y. Wang, None; B. Zhao, None; J.B. Jonas, None

The authors thank Qingjun Lu (Beijing School of Ophthalmology, Beijing Institute of Ophthalmology, Beijing Tongren Hospital, Capital Medical University) for technical assistance.