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

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

Liang Li,1 Bin Li,1 Hao Zhang,1 Shuwei Bai,1 Yichen Wang,1 Bowen Zhao,1 and Jost B. Jonas1,2

PURPOSE. The cancer-associated gene PAX6 is a key regulator in ies have additionally suggested that these Knudson’s M1 and the embryological development of the retina. The authors M2 mutational events, though necessary for initiation, were assessed whether PAX6 was associated with the development not the only genomic changes occurring in the oncogenesis of retinoblastoma. of retinoblastoma.2 The cell of origin of retinoblastoma has 3 METHODS. Two human retinoblastoma cell lines (Y79 and been the subject of intense debate for many years. Studies SO-Rb50) were transfected with PAX6-GFP recombinant len- have suggested that retinal stem cells or retinal progenitor tiviral vectors and were compared with cells undergoing cells can be cells of origin of retinoblastomas.4 Zhong et al.5 transfection with GFP lentiviral vectors and cells without reported that retinal stem–like cells were present in human any intervention. Overexpression of PAX6 was assessed by retinoblastomas and that cultured human retinoblastoma quantitative real-time polymerase chain reaction analysis cells expressed retinal development-related genes, including and Western blot analysis. Cell proliferation assays were PAX6 in vitro. PAX6 is a homeobox transcription factor in evaluated by colorimetric cell counting kit-8. The cell cycle the PAX gene family containing a total of nine members in was analyzed by fluorescence-activated cell sorting (FACS). vertebrates. Many studies have shown that PAX proteins Apoptosis rates were assessed by TUNEL assay followed by play an essential role in organogenesis by inhibiting terminal FACS analysis. Using Western blot analysis, the authors mea- differentiation and apoptosis. These functional characteris- sured levels of proteins p53, p21, p27, cdc2, proliferating tics of PAX6 may facilitate the development and progression cell nuclear antigen, and cleaved caspase-3. of specific cancers in association with PAX6.6–8 As one of RESULTS. Three days after transfection, both cell lines showed a the master regulators of ocular development, the PAX6 gene Ͻ statistically significant (P 0.001) overexpression of PAX6, is involved in the development of the retina, including the Ͻ parallel to significantly (P 0.001) increased cell proliferation. control of cell proliferation, maintenance of retinal progen- At 7 days after transfection, cell cycle analysis showed a signif- itor cell potency, and cell fate determination.9,10 Moreover, icant (P Ͻ 0.001) reduction of G0/G1 arrest and a significant Ͻ PAX6 positively and directly regulates cohorts of genes that induction of G2/M arrest (P 0.01). Parallel to a reduction in promote neural stem cell self-renewal, progenitor cell gen- caspase-3 levels, the apoptosis rate significantly (P Ͻ 0.001) esis, and neurogenesis in a highly dosage-dependent man- decreased. Levels of p53, p21, and p27 were reduced, and the ner. Removing PAX6 may reduce stem cell self-renewal by levels of cdc2 were increased. decreasing the expression of key cell cycle regulators.11,12 CONCLUSIONS. Lentiviral vector-mediated overexpression of However, little is known about whether the PAX6 gene is PAX6 in human retinoblastoma cells was associated with in- involved in the progression of human retinoblastomas and creased cell proliferation parallel to a reduced caspase-3–de- how the effects occur. pendent apoptotic rate and a change in the p53 regulated cell In a previous study,13 we found that transient silencing of cycle. PAX6 may be further explored for the diagnosis of and the PAX6 gene with small interfering RNA (siRNA) resulted therapy for retinoblastomas. (Invest Ophthalmol Vis Sci. 2011; 52:8393–8400) DOI:10.1167/iovs.11-8139 in the inhibition of proliferation and the increased apoptosis of human retinoblastoma cells. The molecular mechanisms revious studies have shown that retinoblastoma is initi- underlying siRNA-mediated PAX6 depletion-induced cell cy- ated by the loss of both alleles of the prototypic tumor cle changes and increased apoptosis have remained un- P 13 suppressor gene RB1.1 A large number of cytogenetic stud- clear. 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 1 From the Beijing Institute of Ophthalmology, Beijing Tongren study of silencing of the PAX6 gene. We then confirmed the Hospital, Capital Medical University, Beijing, China; and the 2Depart- ment of Ophthalmology, Medical Faculty Mannheim, Ruprecht-Karls- overexpression of PAX6 by quantitative real-time polymer- University of Heidelberg, Mannheim, Germany. ase chain reaction (Q-PCR) analysis and Western blot analy- Supported by National Natural Science Foundation of China Grant sis. Finally, we examined the effects of PAX6 overexpres- 30772377. sion on cell proliferation by cell proliferation assays, on the Submitted for publication June 28, 2011; revised August 9 and cell cycle by fluorescence-activated cell sorting (FACS), on September 13, 2011; accepted September 14, 2011. the rate of apoptosis by TUNEL assay followed by FACS Disclosure: L. Li, None; B. Li, None; H. Zhang, None; S. Bai, analysis, and on the levels of cell cycle and apoptosis-related None; Y. Wang, None; B. Zhao, None; J.B. Jonas, None Corresponding author: Bin Li, Beijing Institute of Ophthalmology, proteins p53, p21, p27, cdc2, proliferating cell nuclear an- Beijing Tongren Hospital, Capital Medical University, 17 Hougou Lane, tigen (PCNA), and cleaved caspase-3 by Western blot analy- Chongnei Street, Beijing 100005, China; [email protected]. sis.

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METHODS 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 Cell Culture completion of the last cycle. The threshold cycle (Ct values), which was the cycle number at which the amount of amplified gene of The human retinoblastoma cell lines Y79 and SO-Rb50 were provided interest reached a fixed threshold, was subsequently determined. Rel- by the Department of Pathology of the Zhongshan Ophthalmic Center, ative quantification of the PAX6 mRNA levels was normalized to Sun Yat-sen University, and the Chinese University of Hong Kong. The ⌬⌬ human ␤-actin levels and calculated with the 2- Ct method.15 cells were cultured in RPMI-1640 medium (Hyclone Laboratories, Lo- gan, UT) supplemented with 10% fetal bovine serum, 100 U/L penicil- Western Blot Analysis lin, and 100 U/L streptomycin at 37°C in a humidified atmosphere of Seven days after lentiviral vector transfection, 2 ϫ 106 human retino- 95% air/5% CO2. The culture medium was replaced every 3 days. blastoma cells per cell line were washed twice with cold PBS, resus- Production of PAX6 Recombinant pended in 200 ␮L ice-cold cell lysis buffer (20 mM Tris [pH 7.5], 150 Lentiviral Vectors mM NaCl, 1 mM EDTA, 1% Triton X-100, 2.5 mM sodium pyrophos- ␤ ␮ phate, 1 mM -glycerophosphate, 1 mM Na3VO4,1 g/mL leupeptin, A third generation of the self-inactivating lentiviral vector containing 1 mM phenylmethylsulfonyl fluoride) and incubated at 4°C for 30 the cytomegalovirus promoter-driven enhanced green fluorescence minutes. The lysates were centrifuged at 12,000 rpm for 10 minutes at protein (eGFP) reporter was purchased from Shanghai Genechem Co., 4°C. The protein concentration was determined by the bicinchoninic Ltd. (Shanghai, China). The lentiviral vector system had three parts acid protein assay kit (Pierce Chemical Co., Rockford, IL). Samples (30 before packaging, including the pGCL-GFP vector, the pHelper 1.0 ␮g protein) were separated by SDS-PAGE using 5% stacking and 10% (gag/pol element) vector, and the pHelper 2.0 (VSVG element) vector. separating gels and subsequently were electroblotted onto polyvi- The pGCL-GFP vector encoding the full length of the human PAX6 nylidene difluoride membranes (Immobilon-P; Millipore, Billerica, MA). gene (NCBI reference sequence ID, NM_000280.3) was constructed by After blocking in Tris buffer (50 mM Tris, pH 7.5) containing 5% skim Shanghai Genechem Co., Ltd. PCR and DNA sequencing confirmed the milk, the membranes were incubated overnight at 4°C with primary accurate insertion of the PAX6 cDNA. For the preparation of the antibodies, including mouse anti-human PAX6 (diluted 500; Millipore), recombinant lentiviral vectors, the three-plasmid transient transfection mouse anti-human p53 (diluted 100; BD Biosciences PharMingen, San 14 ␮ method was used. The pHelper 1.0 plasmid (15 g), the pHelper 2.0 Diego, CA), rabbit anti-human p21 (diluted 1000; Cell Signaling Tech- ␮ plasmid (10 g), and the pGCL-GFP-PAX6 or the pGCL-GFP plasmid nology, Danvers, MA), rabbit anti-human p27 (diluted 1000; Cell Sig- ␮ (20 g) were cotransfected into subconfluent 293T cells in serum-free naling Technology), mouse anti-human cdc2 (diluted 1000; Cell Signal- medium using the cationic liposome based transfection reagent (Lipo- ing Technology), rabbit anti-human cleaved caspase-3 (diluted 1000; fectamine 2000; Invitrogen Co., Carlsbad, CA). After 8 hours of incu- Cell Signaling Technology), and rabbit anti-human PCNA (diluted 50; bation, the medium was completely exchanged. High-titer recombi- Abgent, San Diego, CA). The secondary goat anti-rabbit IgG antibody nant lentiviral vectors with PAX6 were harvested 48 hours later. (diluted 5000; Santa Cruz Biotechnology, Santa Cruz, CA) or goat anti-mouse IgG antibody (diluted 5000; Santa Cruz Biotechnology) Establishment of Stable PAX6 Overexpression in conjugated with horseradish peroxidase was applied after washing in Human Retinoblastoma Cells PBST (PBS solution with the detergent Tween 20) and incubated for 1 For transfection, Y79 and SO-Rb50 human retinoblastoma cells were hour at 37°C. Equal amounts of protein loading were confirmed by plated at a concentration of 5 ϫ 103 cells/well in 96-well plates. The reprobing the membranes with the rabbit anti-human GAPDH antibody cells were transfected either with PAX6-GFP lentiviral vectors (PAX6 (diluted 1000; Santa Cruz Biotechnology). Protein expression was overexpression group, study group) or with the GFP lentiviral vectors visualized after extensive washing using an enhanced chemilumines- (negative GFP control group) in serum-free medium for 12 hours. cence advanced detection kit (GE Healthcare, Buckinghamshire, UK) Polybrene (5 ␮g/mL) (an enhancer reagent of retrovirus-mediated gene and quantified with analysis software (Quantity One; Bio-Rad Labora- transduction) was added to increase the infection efficiency. A third tories Inc., Hercules, CA). group of cells without any intervention served as a second control group (negative control group without transfected cells). The cells Cell Proliferation Analysis were then washed and embedded into complete medium. Three days The standard colorimetric cell counting kit-8 (CCK8; Dojindo Labora- after transfection, reporter gene expression was examined using fluo- tories, Kumamoto, Japan) was used for the determination of the num- rescence microscopy, after which the experimental procedures were ber of viable cells in cell proliferation assays. The two human retino- performed. blastoma cell lines were seeded with a volume of 90 ␮L cell suspension (3000 cells/well) into 96-well plates. After transfection, as described, Quantitative Real-time Polymerase Chain the cells were incubated for 72 hours. After that, 10 ␮L CCK8 was Reaction Analysis added to each well and incubated for 4 hours at 37°C. Optical densities Five days after lentiviral vector transfection, a Q-PCR assay was per- were read by a microplate reader scanning at 450 nm. This procedure formed to measure the PAX6 mRNA levels in both retinoblastoma cell was repeated every 24 hours during a 6-day period. Cell survival rates lines. Total RNA was extracted from the cells using the extraction were measured at three time points of the cell growth curve through reagent (TRIzol; Invitrogen). Reverse transcription and PCR-amplifica- the log phase of growth for each cell line. tion reactions were performed according to the M-MLV reverse tran- scriptase protocol (Promega Biotech Co. Ltd, Beijing, China). Primers Cell Cycle Analysis for Q-PCR were as follows: human ␤-actin (202 bp)—5-GGCGGCAC- Both retinoblastoma cell lines were seeded on a 24-well cell culture CACCATGTACCCT-3 forward, 5-AGGGGCCGGACTCGTCATACT-3 re- plate. Seven days after transfection, as described, approximately 5 ϫ verse; human PAX6 (232 bp)—5-CGGCAGAAGATTGTAGAGC-3 for- 105 cells were washed in cold PBS, fixed with ice-cold 70% ethanol at ward, 5-GGATTTCCCAAGCAAAGAT-3 reverse. 4°C for 12 hours, and then stained with 1 mL cold PBS containing 50 Q-PCR was performed using a sequence detection system (ABI ␮g/mL propidium iodide (a DNA stain; Sigma-Aldrich Co., St. Louis, prism 7900; Applied Biosystems, Foster City, CA). The 25-␮L reaction MO), 100 ␮g/mL nuclease (RNase A; Fermentas Inc., Hanover, MD), mixture contained 12.5 ␮L SYBR green PCR master mix (TaKaRa and 0.2% Triton X-100 at 4°C for 30 minutes in the dark. The cells were Biotechnology Co. Ltd., Dalian, China), 2 ␮L cDNA template, 0.5 ␮L then passed through a flow cytometer (FACSCalibur; BD Biosciences, PCR forward primer, and 0.5 ␮L PCR reverse primer. PCR running San Diego, CA) equipped with a 488-nm argon laser to measure the conditions were 15 seconds at 95°C for the initial denaturation, 40 DNA content. Data analysis was performed with the appropriate soft-

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ware (Cell Quest [BD Biosciences]; ModFit LT [Verity Software House (Fig. 1). Consequently, a MOI of 20 was chosen for the next Inc, Topsham, ME]). Results were presented as percentages of the total steps of this study. 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 Stable Overexpression of PAX6 in the Human phase (tetraploid cells). Retinoblastoma Cell Lines Cell Apoptosis Analysis Q-PCR analysis demonstrated that the relative levels of PAX6 The cell apoptotic rate was determined by the TUNEL assay (In Situ mRNA were markedly increased in the study groups of the Y79 Cell Death Detection Kit, TMR red; Roche Applied Science, Basel, and SO-RB50 cell lines (667.8 Ϯ 12.2 and 10.4 Ϯ 1.0, respec- Switzerland). The two retinoblastoma cell lines were seeded onto a tively) compared with the levels in the negative GFP control 24-well cell culture plate. On the seventh day after transfection, as groups (cells transfected with GFP vectors alone; 1.00 Ϯ 0.06 described, approximately 5 ϫ 105 cells were washed with PBS and and 1.00 Ϯ 0.07, respectively) and compared with the levels in fixed with 4% paraformaldehyde in PBS (pH 7.4) at room temperature the nontransfected control cells (0.10 Ϯ 0.05 and 1.08 Ϯ 0.07, for 1 hour and then resuspended in 0.1% sodium citrate containing respectively) (ANOVA; F ϭ 9036, P Ͻ 0.001 in Y79 groups; 0.1% Triton X-100 for 2 minutes on ice. In accordance with the F ϭ 254, P Ͻ 0.001 in SO-Rb50 groups) (Figs. 2a, 2b). The manufacturer’s protocol, the cells were treated with TUNEL reaction effectiveness of the PAX6-GFP lentiviral vectors was also con- mixture containing terminal deoxynucleotidyl transferase (TdT) and firmed by examination of the levels of the PAX6 protein by fluorescein-dUTP, and the cells were incubated at 37°C in a humidified Western blot analysis. The protein expression levels of PAX6 in atmosphere in the dark for 1 hour. The TdT could catalyze the binding the Y79 and SO-RB50 cell lines were significantly higher in the of fluorescein-dUTP to free 3Ј-OH ends in the nicked DNA. After study groups than in the control groups (Fig. 2c). In compar- washing with PBS, the cells were analyzed with a flow cytometer ison, the GAPDH protein expression levels did not vary signif- (FACSCalibur; BD Biosciences) equipped with a 540-nm excitation icantly between all the groups (Fig. 2c). laser. Data analysis was performed with the software (Cell Quest). Results were shown as percentages of red fluorescence-emitting cells Promotion of Cell Proliferation Induced by PAX6 (apoptotic cells) in the Y79 and SO-Rb50 retinoblastoma cell lines. Overexpression In Vitro Statistical Analysis To test the pGCL-GFP-PAX6 lentiviral vectors for their ability to Statistical analysis was performed using a commercially available soft- promote the proliferation of the Y79 and SO-Rb50 retinoblas- ware package (SPSS 19.0 for Windows; IBM-SPSS Inc., Chicago, IL). toma cells, we performed a cell proliferation assay using the Data were presented as the mean Ϯ SD of three independent experi- standard colorimetric cell counting technique (CCK8). Cell ments. Statistical analysis of differences was carried out by a one-way survival rates were measured at three time points of the cell ANOVA. P Ͻ 0.05 was considered to indicate statistical significance. 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 surviv- RESULTS ing Y79 cells increased sharply from 103.5% Ϯ 0.9%, 106.0% Ϯ 0.5%, and 106.7% Ϯ 0.5% in the negative GFP control group at Infection Efficiency of Lentivirus Vectors 48 hours, 72 hours, and 96 hours after cell plating, respec- To investigate PAX6 function in both retinoblastoma cell lines, tively, to 143.2% Ϯ 0.6%, 159.1% Ϯ 0.5%, and 151.8% Ϯ 1.2% we tried to overexpress this gene by transfecting the retino- in the study group, respectively (ANOVA; F ϭ 4689, 6941, and blastoma cells with PAX6-GFP lentiviral vectors. The trans- 4864, respectively; all P Ͻ 0.001) (Fig. 3a). In a parallel fected cells expressed eGFP after transfection with the lentivi- manner, the percentages of surviving SO-Rb50 cells in- ral vectors. The cells of cell lines Y79 and SO-Rb50 were creased from 107.9% Ϯ 1.3%, 101.3% Ϯ 1.3%, and 100.7% Ϯ transfected by the lentiviral vectors at different multiplicities of 1.7% in the negative GFP control group at 96 hours, 120 infection (MOIs). GFP expression was examined 3 days after hours, and 144 hours after cell plating, respectively, to transfection using fluorescence microscopy. The efficiency of 117.1% Ϯ 1.7%, 124.2% Ϯ 1.6%, and 126.6% Ϯ 1.1% in the the infection (averaged proportion of GFP-expressing cells on study group, respectively (ANOVA; F ϭ 156, 412, and 623, the total cell count) was approximately 80% at a MOI of 20 respectively; all P Ͻ 0.001) (Fig. 3b).

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 fluo- rescent conditions revealed a transfection efficiency of Ͼ80%. 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, fluo- rescent field. Scale bars, 50 ␮m.

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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 ␤-actin mRNA levels. Each value represented the mean Ϯ SD of three replicates. PAX6 mRNA levels were significantly higher in the study groups (**P Ͻ 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.)

Changes of the Cell Cycle Induced by PAX6 with the negative GFP control groups and the negative control Overexpression In Vitro groups without transfected cells (Fig. 4d). The levels of the p27 protein were slightly downregulated, and the levels of the cdc2 To study the causes of the promotion of cell proliferation protein were upregulated in both retinoblastoma cell lines. induced by PAX6 overexpression, we analyzed the cell cycle Differences in the levels of the PCNA protein were not detect- by flow cytometry. In both retinoblastoma cell lines Y79 and able. In a similar manner, the GAPDH protein did not differ SO-RB50, the percentages of cell counts in the G2/M phase in between the groups. relation to the total cell counts were significantly higher in the study groups (22.9% Ϯ 0.3% and 12.1% Ϯ 0.7%, respectively) Inhibition of Cell Apoptosis Induced by PAX6 than in the negative GFP control groups (13.4% Ϯ 0.5% and Overexpression In Vitro 8.7% Ϯ 0.7%, respectively) and the negative control groups without transfected cells (12.8% Ϯ 0.1% and 9.2% Ϯ 0.6%, To confirm the cell apoptosis secondary to PAX6 overex- respectively) (ANOVA; F ϭ 857, P Ͻ 0.001 in Y79 groups; F ϭ pression, we determined the extent of DNA fragmentation 23, P ϭ 0.002 in SO-Rb50 groups). Correspondingly, the per- seven days after transfection using TUNEL assay followed by centages of the cell counts in the G0/G1 phase in relation to FACS analysis. Flow cytometric analysis revealed a reduced the total cell counts were significantly lower in the study TUNEL fluorescence signal in the study groups compared groups (47.6% Ϯ 0.7% and 60.2% Ϯ 0.3%, respectively) than in with the negative GFP control groups and the negative the negative GFP control groups (57.1% Ϯ 0.3% and 70.1% Ϯ control groups without transfected cells of Y79 and SO- 0.5%, respectively) and the negative control groups without RB50 cell lines. The percentages of TUNEL-positive apopto- transfected cells (57.5% Ϯ 0.6% and 69.8% Ϯ 0.7%, respec- tic cell counts to total cell counts were significantly lower in tively) (ANOVA; F ϭ 286, P Ͻ 0.001 in Y79 groups; F ϭ 354, the study groups (0.04% Ϯ 0.03% and 0.0% Ϯ 0.0%, respec- P Ͻ 0.001 in SO-Rb50 groups). In a similar manner, the per- tively) than in the negative GFP control groups (0.60% Ϯ centage of the count of the SO-Rb50 cells in the S phase in 0.03% and 0.40% Ϯ 0.04%, respectively) and in the negative relation to the total SO-Rb50 cell count was significantly higher control groups without transfected cells (0.26% Ϯ 0.05% in the study group (27.7% Ϯ 0.5%) than in the negative GFP and 0.37% Ϯ 0.02%, respectively) (ANOVA; F ϭ 187, P Ͻ control group (21.2% Ϯ 1.1%) and in the negative control 0.001 in Y79 groups; F ϭ 671, P Ͻ 0.001 in SO-Rb50 groups) group without transfected cells (21.1% Ϯ 0.3%) (ANOVA; F ϭ (Figs. 5a–c). Correspondingly, Western blot analysis re- 87, P Ͻ 0.001). The percentage of Y79 cell count in the S phase vealed that the levels of the apoptosis-related protein, in relation to the total Y79 cell count did not vary significantly cleaved caspase-3 (the active form of caspase-3), were down- between the study group (29.5% Ϯ 1.0%), the negative GFP regulated in the study groups of both retinoblastoma cell control group (29.5% Ϯ 0.3%), and the negative control group lines (Fig. 5d). without transfected cells (29.7% Ϯ 0.7%) (ANOVA; F ϭ 0.08, ϭ P 0.93) (Figs. 4a–c). DISCUSSION To identify signaling pathways of the changes of the cell cycle induced by PAX6 overexpression, we performed a West- Using the technology of lentiviral vector–mediated stable ern blot analysis of the cell cycle regulatory proteins p53, p21, transfection, our study demonstrated that the overexpres- p27, cdc2, and PCNA. The levels of the p53 and p21 proteins sion of PAX6 in human retinoblastoma cells resulted in were downregulated in the study groups of both retinoblas- increased tumor cell proliferation in vitro paralleled by a toma cell lines (particularly for the Y79 cell line) compared downregulation of the p53, p21, and p27 proteins and an

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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. Corre- spondingly, 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 elevat- ing 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 FIGURE 3. Three days after transfection, cell survival rates of cells of mechanisms of the PAX6 gene in different cell types. the human retinoblastoma cell line Y79 (a) and human retinoblastoma To further explore how the PAX6 gene works to regulate cell line SO-Rb50 (b) were evaluated by colorimetric cell counting kit-8 cell cycle progression, the cell cycle regulatory proteins (CCK8) after treatment with either PAX6-GFP lentiviral vectors (study p53, p21, p27, cdc2, and PCNA were assessed by Western groups) (pGCL-GFP-PAX6) or with GFP lentiviral vectors (negative GFP blot analysis. The ability of p53 to promote cell cycle arrest control groups) (pGCL-GFP). Cell proliferation was significantly (P Ͻ has been connected with its ability to transactivate one of its 0.001) higher in the study groups than in the negative GFP control critical target genes, P21WAF1. At the G1/S checkpoint, the groups (pGCL-GFP) and in the negative control groups without trans- induction of p21, also known as cyclin-dependent kinase fection 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 inhibitor 1A, arrests cells in the G1 phase and prevents Y79 cell line. The negative GFP control groups and the negative S-phase entry by inhibiting the binding between cyclin- 26 control groups without transfection did not differ significantly. Each dependent protein kinase 2 (cdk2) and cyclin E. At the value represented the mean Ϯ SD of four replicates. (**P Ͻ 0.001). 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 upregulation of the cdc2 protein. Correspondingly, we ob- PCNA, a DNA polymerase accessory factor involved in DNA served a reduction in G0/G1 arrest and an induction in G2/M replication and repair during the S phase of the cell cycle, arrest in the cell cycle and an inhibition of caspase-3– and p21 may block the ability of PCNA to bind with dependent apoptosis. Gadd45.27,28 At the G2/M checkpoint, the cyclin-dependent Consistent with our results, Mascarenhas et al.16 reported kinase 1 (cdc2), which may control entry into the mitosis that PAX6 activated the expression of the MET gene and that (M) phase, is pivotal in regulating this transition. p21, as an the inhibition of PAX6 led to a decline in cell proliferation and inhibitor of cdc2, may be turned on by p53 and inactivate cell survival and resulted in a differentiation of pancreatic the cdc2/cyclin B complex.29,30 In our study, the levels of carcinoma cells.16,17 In contrast to our study, Mayes et al.18,19 p53 and p21 were downregulated and the level of the cdc2 found that PAX6 suppressed the tumorigenesis of glioblastoma was upregulated in both retinoblastoma cell lines trans- multiforme and inhibited its invasiveness by repressing the fected with the PAX6-GFP lentiviral vectors (Fig. 4d). These

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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 Ϯ SD of three replicates (*P Ͻ 0.01, **P Ͻ 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.

findings may be associated with the decreasing percentages The levels of PCNA were, however, not altered by the of tumor cells in the G0/G1 phase and the increasing per- overexpression of PAX6. This finding was partially consistent centages of tumor cells in the G2/M phase, induced by the with the finding that the percentage of the Y79 cell count in overexpression of PAX6. Because p53 and p21 dysregula- the S phase in relation to the total number of Y79 cells did not tion were important associations with the PAX6 overexpres- differ between the groups. It may indicate that other S-phase sion in our study, one may further conclude that the over- regulators were responsible for the control of the cell cycle. expression of PAX6 was associated with a reduction of a Previous studies have suggested that increased levels of the p53/p21-dependent G0/G1 arrest and an induction of p53- p27Kip1 protein typically caused cells to arrest in the G1 phase dependent G2/M arrest by a positive regulation of the cdc2 of the cell cycle.31,32 Kase et al.33 reported that functional loss gene activity in both retinoblastoma cell lines. of the retinoblastoma protein (Rb) led to a downregulation of

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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 Ϯ SD of three replicates (**P Ͻ 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.

p27 and an uncontrolled retinal tumor cell proliferation, and SO-Rb50 in vitro through the suppression of p53 and the whereas a lack of p27 degradation resulted in the suppres- caspase-3 protein. It is noteworthy that the activation of sion of cdc2 activity and the consequent inhibition of entry PAX6 may inhibit p53-mediated caspase-3–dependent apo- into the M phase.34 In our study, p27 protein levels were ptosis in human retinoblastoma cells. These partially contra- downregulated, whereas the levels of the cdc2 protein were dicting results may be explained by differences between upregulated after PAX6 was overexpressed in both retino- rabbit corneal epithelial cells and human retinoblastoma blastoma cell lines. Thus, our results of cell cycle analysis cells. agreed with the findings of Kase et al.33 and with those of Potential limitations of our study should be mentioned. previous studies. First, recovery tests by RNA interference in retinoblastoma Previous studies have suggested that the ability of p53 to cells overexpressing PAX6 will be necessary to further con- control apoptosis in response to abnormal proliferative sig- solidate the results and conclusions of our study. Second, nals and stress, including DNA damage, is important for its our investigation was a cell culture study in which the cells tumor suppression function.35 Caspase 9 and its cofactor were in an artificial environment and unnatural living con- Apaf-1 were found to be essential downstream components ditions. It has always been questionable how far results from of p53 in Myc-induced apoptosis. Once activated, caspase-9, cell culture studies can be transferred to clinical conditions. as one of the initiator caspases, cleaves and activates down- Third, because the apoptotic rate in the immortalized reti- stream effector caspases, including caspase-3, which in turn noblastoma cell lines of our study were considerably lower executes apoptosis.36,37 Ouyang et al.38 reported that the than the apoptotic rate in retinoblastoma tumors in loco, the overexpression of mouse full-length PAX6 reduced the rate importance of our results in terms of a significant reduction of cell proliferation and induced caspase-3–independent ap- in the apoptotic rate of cultured retinoblastoma cells after optosis of rabbit corneal epithelial cell lines. In our study, the overexpression of PAX6 for the pathophysiology of the we demonstrated that overexpression of the PAX6 protein tumors and for the potential clinical implications have re- inhibited apoptosis in human retinoblastoma cell lines Y79 mained unclear thus far.

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