Chaum Edward, Yang Huaitao
Department of Ophthalmology, University of Tennessee Health Science Center, Memphis 38163, USA.
Invest Ophthalmol Vis Sci. 2002 Dec;43(12):3758-64.
To induce the expression of insulin-like growth factor (IGF)-1, by using gene transfer methods, to modify the growth characteristics of human retinal pigment epithelial cells.
Human retinal pigment epithelial cells were transfected in vitro with plasmid vector pcDNA:IGF-1, which encodes an epitope-tagged human IGF-1 fusion protein and a selectable neomycin resistance gene. Transduced cells were cloned in G418 and expanded for analysis of IGF-1 transgene expression and its effect on the cell phenotype. The expression of the IGF-1 transgene in cloned cells was confirmed by reverse transcription-polymerase chain reaction and quantified by Western blot analysis. The growth characteristics of transduced clones were compared with the control by spectrophotometric and flow cytometric cell proliferation assays.
Cloned retinal pigment epithelial cells expressed the IGF-1 transgene and secreted the IGF-1 fusion protein into the tissue culture medium. Transduced clones demonstrated a dose-dependent, enhanced ability to proliferate in low serum conditions, compared with the control. Clones that expressed moderate and high levels of the IGF-1 fusion protein were isolated and grew at a significantly faster rate and showed a statistically significant increase in the number of cells after 6 days, compared with the control (P < 0.002, paired samples, t-test). Expression of the IGF-1 transgene in synchronized clones enhanced cell cycle kinetics by increasing recruitment of the G(0)-G(1)-phase cells into the proliferative phase of the cell cycle.
The human IGF-1 fusion protein encoded by the pcDNA:IGF-1 vector demonstrates paracrine biological activity in human retinal pigment epithelial cells in vitro. Expression and secretion of the IGF-1 transgene enhances growth characteristics in a dose-dependent manner and can modulate the proliferative potential of the retinal pigment epithelial cell.
