title: The role of cell cycle on polyplex-mediated gene transfer into a retinal pigment epithelial cell line.
abstract: BACKGROUND
  Retinal pigment epithelium (RPE) maintains the function of photoreceptors and eyesight and is an important target for gene delivery. Since in some diseases RPE cells proliferate uncontrollably, we investigated the role of cell cycle in non-viral gene delivery into a RPE cell line (D407).
  
  
  METHODS
  D407 (human) cells were transfected with cationic DNA complexes. Cells were synchronized into different phases of the cell cycle and transfected using poly-L-lysine (PLL) or polyethyleneimine (PEI) carriers for 3 h. The effects of different reporters (beta-galactosidase, luciferase) or promoters (CMV, SV40, tk, PDE-beta) on gene expression were evaluated 43 h later. Cellular uptake of ethidium monoazide/DNA complexes with PLL or PEI was determined by flow cytometry. Fluorescent DNA and the complexes were localized with a confocal microscope. The role of cell cycle in transcription was evaluated by stable luciferase-expressing cells.
  
  
  RESULTS
  PLL showed lower transfection levels than PEI in synchronized cells and only slight dependence on cell cycle. PEI showed minimal efficiency at G1 phase and maximum level at S phase. All promoters and reporter genes showed dependence on cell cycle. Cellular uptake of polyplexes was highest at S phase (80-90%) and lowest at G1 phase (5-30%). Confocal microscopy showed minor differences of free DNA between groups in the nucleus, where it was largely carrier-bound. Cell cycle effects on luciferase expression were clear in stable cell line
  
  
  CONCLUSIONS
  Transfection by polyplexes in the RPE cell line is influenced by cellular uptake and transcription, and both processes are cell-cycle-dependent. The results have implications in retinal gene therapy.
authors.affiliation: Department of Pharmaceutics, University of Kuopio, P.O. Box 1627, 70211 Kuopio, Finland. Marjo.Mannisto@uku.fi
authors.forename: Marjo
authors.initials: M
authors.lastname: Männistö
authors.forename: Seppo
authors.initials: S
authors.lastname: Rönkkö
authors.forename: Mikko
authors.initials: M
authors.lastname: Mättö
authors.forename: Paavo
authors.initials: P
authors.lastname: Honkakoski
authors.forename: Mika
authors.initials: M
authors.lastname: Hyttinen
authors.forename: Jukka
authors.initials: J
authors.lastname: Pelkonen
authors.forename: Arto
authors.initials: A
authors.lastname: Urtti
mesh_terms: D002453:Cell Cycle; D002460:Cell Line; D018014:Gene Transfer Techniques; D006801:Humans; D008156:Luciferases / Q000235:genetics; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism*; D010957:Plasmids; D019076:Transgenes
chemical_list: D008156:Luciferases