Lens epithelium-derived growth factor: increased survival and decreased DNA breakage of human RPE cells induced by oxidative stress.
OBJECTIVE
Lens epithelium-derived growth factor (LEDGF) has been shown to be a growth and survival factor and to be present in a wide variety of cell types. The purpose of this study was to determine whether LEDGF enhances survival of human retinal pigment epithelial (RPE) cells when challenged by oxidative stress or by ultraviolet (UVB) irradiation in a culture system.


METHODS
Primary RPE cells were cultured in standard Dulbecco's modified Eagle's medium (DMEM) containing 15% fetal bovine serum. Protein blot analysis with antibodies to LEDGF was used to detect LEDGF in RPE cells. Initially, RPE cells were cultured in the standard medium for 1 day to allow attachment to the culture plates and then cultured in serum-free DMEM, with and without LEDGF. The trypan blue exclusion method was used to test RPE cell viability. Single-cell electrophoresis was used to evaluate single strand breaks of genomic DNA after exposure to H(2)O(2) or irradiation by UVB.


RESULTS
LEDGF was present in RPE cells, predominantly in the nucleus. RPE cells grew for 1 week and survived for 3 weeks in the presence of LEDGF. In the absence of LEDGF, they increased in number for the first week and gradually died in the following 2 weeks. LEDGF protected RPE cells against H(2)O(2) exposure and UVB irradiation. DNA damage induced by H(2)O(2) exposure or UVB irradiation was lower in the presence than in the absence of LEDGF. The expression of heat shock protein (Hsp)27 was elevated by LEDGF.


CONCLUSIONS
LEDGF enhanced survival of RPE cells in culture when challenged by oxidative stress and UVB irradiation. LEDGF protected DNA from single-strand breakage and upregulated the expression of Hsp27. These results suggest that LEDGF may be a potential agent for protecting RPE cells under various stress conditions.
Matsui
H
H
Kellogg Eye Center, Department of Ophthalmology and Visual Sciences, University of Michigan, Ann Arbor 48105, USA.
Lin
L R
LR
Singh
D P
DP
Shinohara
T
T
Reddy
V N
VN
D002455:Cell Division; D002470:Cell Survival / Q000187:drug effects* / Q000528:radiation effects; D002478:Cells, Cultured; D004249:DNA Damage / Q000187:drug effects* / Q000528:radiation effects; D004261:DNA Replication; D004591:Electrophoresis, Polyacrylamide Gel; D006133:Growth Substances / Q000494:pharmacology*; D055551:HSP27 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D007124:Immunoenzyme Techniques; D036341:Intercellular Signaling Peptides and Proteins; D018832:Molecular Chaperones; D009363:Neoplasm Proteins / Q000378:metabolism; D018384:Oxidative Stress; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D014343:Trypan Blue / Q000378:metabolism; D015854:Up-Regulation
D006133:Growth Substances; D055551:HSP27 Heat-Shock Proteins; C057725:HSPB1 protein, human; D006360:Heat-Shock Proteins; D036341:Intercellular Signaling Peptides and Proteins; D018832:Molecular Chaperones; D009363:Neoplasm Proteins; C119380:lens epithelium-derived growth factor; D006861:Hydrogen Peroxide; D014343:Trypan Blue