RNAi-mediated knockdown of inducible nitric oxide synthase mRNA in murine lymphoma cells

At low concentrations nitric oxide (NO^.) is a signalling molecule involved in processes such as angiogenesis, while at higher concentration it functions as a reactive nitrogen species, mediating apoptosis. thus tumour growth and death are both influenced by NO^..  Studies with nitric oxide synthase (NOS)-knockout mice, in which NOS inhibitors were used to eliminate tumour-derived NO^., demonstrated that thymoma tumour immune rejection was mediated by tumour-derived NO^., and was independent of host-derived NO^.. The use of NOS inhibitors is sub-optimal however, as it inevitably leads to questions of specificity. Therefore these data were confirmed by selective knock down of iNOS mRNA in EL4 lymphoma cells. this was achieved by short hairpin RNA (shRNA)-mediated RNA interference. Two anti-iNOS and one control anti-luciferase shRNAs have been stably expressed in EL4 cells and the resultant phenotypes characterised. Depletion of iNOS caused improved mitochondrial function and provided protection from genotoxic stress-induced apoptosis. However, evidence was found for both antioxidant and pro-growth (at low cell density) functions for NO^.. Expression of the shRNAs was lost during passaging of the cell lines, resulting in loss of these phenotypes. to circumvent this, shRNAs were transferred to a HIV-1-based lentiviral vector, under control of a tetracycline-inducible promoter. Proof-of-function for this system has been demonstrated in the human adenocarcinoma line MDA-MB-231. However, induction of shRNA-expression has so far proved fatal in retrovirus-transduced EL4 cells.