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 H 2O 2-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 H 2O 2 for 20 min. to the HSP70-deprived fibroblasts did not modify the percentage of LDH release observed in H 2O 2 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 H 2O 2 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 H 2O 2 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.