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.