Switching off HSP70 and i-NOS to study their role in normal and H2O2-stressed human fibroblasts.
i-NOS and HSP70 antisense oligonucleotides were used to study the role of the two well known stress-regulated molecules on cell survival of both untreated control, and H2O2-stressed human fibroblasts. Cell survival was assessed either by LDH release or by MTT assay. The levels of cytosolic i-NOS and HSP70 were tested by using immunoblotting analysis, and reactive oxygen species (ROS) production was quantified. Compared to the values observed in untreated control cells, anti HSP70-transfected human fibroblasts showed an increase in ROS production, i-NOS level and LDH release. The addition of 0.12 mM H2O2 for 20 min. to the HSP70-deprived fibroblasts did not modify the percentage of LDH release observed in H2O2 stressed cells, but reduced cell viability increasing both ROS production and i-NOS level. Anti i-NOS-transfected fibroblasts, compared to the control untreated cells, showed no modification in ROS production, while cell survival was improved. When treated with H2O2 the i-NOS depleted cells counteracted ROS formation as well as LDH release but negatively affected cell viability and HSP70 levels, compared to the results obtained with H2O2 alone-treated fibroblasts. The data indicates that the induced decrease in HSP70 level in oxidative stress conditions makes fibroblasts more prone to oxidative injury and also increases i-NOS level. Whereas in one way the forced decrease in i-NOS expression seems to counteract ROS production stimulated by the oxidative insult in the cells, in another way, since it causes a decrease in HSP70 expression as well as in cell viability, it seems to activate some unidentified pathways affecting cell demise.
Renis
Marcella
M
Department of Biological Chemistry, Medicinal Chemistry and Molecular Biology, University of Catalina, Viale A. Doria, 6, 95125 Catania, Italy. renis@unict.it
Cardile
Venera
V
Grasso
Salvatore
S
Palumbo
Maddalena
M
Scifo
Christian
C
D002461:Cell Line, Transformed; D002470:Cell Survival / Q000187:drug effects; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D005562:Formazans / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics* / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D018384:Oxidative Stress; D017382:Reactive Oxygen Species / Q000378:metabolism; D013778:Tetrazolium Salts / Q000378:metabolism; D014162:Transfection
D005562:Formazans; D018840:HSP70 Heat-Shock Proteins; D016376:Oligonucleotides, Antisense; D017382:Reactive Oxygen Species; D013778:Tetrazolium Salts; C079782:MTT formazan; D006861:Hydrogen Peroxide; D007770:L-Lactate Dehydrogenase; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II