title: Pax6 overexpression suppresses cell proliferation and retards the cell cycle in corneal epithelial cells.
abstract: OBJECTIVE
  The gene encoding transcription factor Pax6 resides at the top of a genetic hierarchy controlling development and morphogenesis of the eye. Pax6 continues to be expressed in the ocular surface epithelia of the postnatal eye. The goal of this study was to investigate a possible role for Pax6 in controlling dynamics of the ocular surface epithelia.
  
  
  METHODS
  Full-length mouse Pax6 (mPax) cDNA, or truncated mPax6Delta286 lacking the transcriptional activation domain was inserted into a tetracycline-inducible vector (Tet-on). A rabbit corneal epithelial cell line SIRC was used to establish stable transformants. Induction of Pax6 or truncated Pax6Delta286 proteins by doxycycline (DOX) was examined by Western blot and immunohistochemistry. The effects of Pax6 overexpression on cell cycle progression were assessed by the cell proliferation index, cell growth curve, and cell cycle assay. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay was performed to detect apoptotic cells. Recombinant adenovirus-carrying mPax6 or mPax6Delta286 transgenes were used for transient transduction of primary rabbit corneal epithelial cells, and the effect on cell cycle progression was assayed.
  
  
  RESULTS
  The level of Pax6 or truncated Pax6 was tightly regulated by DOX. Overexpression of full-length Pax6 retarded the rate of cell proliferation, whereas the truncated form had no effect. Full-length Pax6 affected the rate at which individual cells traversed the cell cycle and induced caspase-3-independent apoptosis in a small percentage of cells. Transient transduction of cells with recombinant mPax6 adenovirus also inhibited cell proliferation.
  
  
  CONCLUSIONS
  Inhibition of cell proliferation in Pax6-overexpressing corneal epithelial cell lines and primary cell culture is consistent with the notion that Pax6 plays a role in controlling corneal epithelial cell dynamics in vivo.
authors.affiliation: Bascom Palmer Eye Institute, Miller School of Medicine, University of Miami, FL 33136, USA, and Department of Ophthalmology, Veterans General Hospital, Taichung, Taiwan.
authors.forename: Jie
authors.initials: J
authors.lastname: Ouyang
authors.forename: Ying-Cheng
authors.initials: YC
authors.lastname: Shen
authors.forename: Lung-Kun
authors.initials: LK
authors.lastname: Yeh
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.forename: Brad M
authors.initials: BM
authors.lastname: Coyle
authors.forename: Chia-Yang
authors.initials: CY
authors.lastname: Liu
authors.forename: M Elizabeth
authors.initials: ME
authors.lastname: Fini
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000502:physiology*; D002460:Cell Line; D049109:Cell Proliferation; D019573:Epithelium, Corneal / Q000166:cytology* / Q000378:metabolism; D005136:Eye Proteins / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology*; D005822:Genetic Vectors; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D007120:Immunochemistry; D020287:In Situ Nick-End Labeling; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000235:genetics* / Q000378:metabolism; D011817:Rabbits; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
chemical_list: D005136:Eye Proteins; D018398:Homeodomain Proteins; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; C000605732:Pax6 protein, mouse; D012097:Repressor Proteins; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases