title: Lentiviral vector-mediated PAX6 overexpression promotes growth and inhibits apoptosis of human retinoblastoma cells.
abstract: OBJECTIVE
  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.
authors.affiliation: Beijing Institute of Ophthalmology, Beijing Tongren Hospital, Capital Medical University, Beijing, China.
authors.forename: Liang
authors.initials: L
authors.lastname: Li
authors.forename: Bin
authors.initials: B
authors.lastname: Li
authors.forename: Hao
authors.initials: H
authors.lastname: Zhang
authors.forename: Shuwei
authors.initials: S
authors.lastname: Bai
authors.forename: Yichen
authors.initials: Y
authors.lastname: Wang
authors.forename: Bowen
authors.initials: B
authors.lastname: Zhao
authors.forename: Jost B
authors.initials: JB
authors.lastname: Jonas
mesh_terms: D017209:Apoptosis; D015153:Blotting, Western; D016203:CDC2 Protein Kinase; D002452:Cell Count; D049109:Cell Proliferation; D019926:Cyclin B / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism; D018844:Cyclin-Dependent Kinases; D005136:Eye Proteins / Q000235:genetics*; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology*; D005822:Genetic Vectors; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D020287:In Situ Nick-End Labeling; D016086:Lentivirus / Q000235:genetics*; D008856:Microscopy, Fluorescence; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000235:genetics*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D012097:Repressor Proteins / Q000235:genetics*; D019572:Retinal Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D012175:Retinoblastoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D014162:Transfection; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
chemical_list: C493956:CDKN1A protein, human; D019926:Cyclin B; D050759:Cyclin-Dependent Kinase Inhibitor p21; D005136:Eye Proteins; D018398:Homeodomain Proteins; D000071841:PAX6 Transcription Factor; C000605730:PAX6 protein, human; D051761:Paired Box Transcription Factors; D018809:Proliferating Cell Nuclear Antigen; D012097:Repressor Proteins; D016159:Tumor Suppressor Protein p53; D050760:Cyclin-Dependent Kinase Inhibitor p27; D016203:CDC2 Protein Kinase; C528899:CDK1 protein, human; D018844:Cyclin-Dependent Kinases