Induction of stress proteins in SV-40 transformed human RPE-derived cells by organic oxidants.
The expression and induction of heat shock proteins (HSPs) were examined in cultured SV-40-transformed human retinal pigment epithelial (RPE)-derived cells following exposure to chemical oxidants. Concentrations of hydrogen peroxide and the organic oxidants tert-butyl hydroperoxide, cumene hydroperoxide and linoleic acid hydroperoxide were used under conditions where cell viability was between 75% and 90% as determined by the trypan blue exclusion tests. The types of HSPs that are either induced and/or elevated from constitutive levels in cultured transformed cells were separated both by SDS-PAGE and by two-dimensional gel electrophoresis. Subsequent immunoblotting was performed with both a monoclonal antibody (MAb C92) specific for only the stress-inducible form of HSP 70, namely HSP 72, and with a monoclonal antibody (MAb N27) specific for both the stress-inducible HSP 72 and its constitutive form, HSP 73. As positive controls for comparison, other types of stressing agents were used that included heat shock at 41 degrees C for various times and exposure to the proline analogue, azetidine-2-carboxylic acid (AzC). Protein immunoblotting analysis demonstrate that: (a) Stress-inducible HSP 72 is present at low levels in non-stressed cultured transformed RPE-derived cells and in fresh bovine retina and RPE, but is not detectable in non-stressed cultured lung fibroblasts until induction with heat shock; and (b) Stress-inducible HSP 72 is elevated from constitutive levels in RPE-derived cultured cells after exposure to various oxidants. After cellular exposure to both organic oxidants and to AzC in the presence of L-[35-S]-methionine, two-dimensional gel electrophoresis confirmed the elevation of newly synthesized HSP 72. Thus, these results indicate that cultured human SV-40-transformed RPE-derived cells are capable of elevated HSP 72 biosynthesis under conditions of oxidative stress produced by organic oxidants.
Kerendian
J
J
Department of Ophthalmology, University of California, College of Medicine, Irvine 92717.
Enomoto
H
H
Wong
C G
CG
D001383:Azetidinecarboxylic Acid / Q000494:pharmacology; D001555:Benzene Derivatives / Q000494:pharmacology; D002460:Cell Line; D002470:Cell Survival; D002472:Cell Transformation, Viral; D015180:Electrophoresis, Gel, Two-Dimensional; D006360:Heat-Shock Proteins / Q000096:biosynthesis*; D006801:Humans; D015151:Immunoblotting; D016877:Oxidants / Q000494:pharmacology*; D010545:Peroxides / Q000494:pharmacology*; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D013539:Simian virus 40; D020122:tert-Butylhydroperoxide
D001555:Benzene Derivatives; D006360:Heat-Shock Proteins; D016877:Oxidants; D010545:Peroxides; D001383:Azetidinecarboxylic Acid; D020122:tert-Butylhydroperoxide; C007164:cumene hydroperoxide