The role of cell cycle on polyplex-mediated gene transfer into a retinal pigment epithelial cell line.
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.
Männistö
Marjo
M
Department of Pharmaceutics, University of Kuopio, P.O. Box 1627, 70211 Kuopio, Finland. Marjo.Mannisto@uku.fi
Rönkkö
Seppo
S
Mättö
Mikko
M
Honkakoski
Paavo
P
Hyttinen
Mika
M
Pelkonen
Jukka
J
Urtti
Arto
A
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
D008156:Luciferases