Analysis of cytokine-induced NO-dependent apoptosis using RNA interference or inhibition by 1400W.
RNA interference has been used to silence gene expression and evaluate the contribution of a gene product to cell function. Here, we investigated conditions under which expression of an inducible protein, nitric oxide synthase 2 (NOS2), is decreased by RNA interference. Cytokine treatment of insulin-producing RINm5F cells results in NOS2 induction and cell death. Conditions used here favoured cytokine-induced apoptosis, for the first time--rather than necrosis, previously recorded. In RINm5F cells, transfected with NOS2-specific small interfering RNA followed by a 12 h exposure to cytokines, there was a significant reduction in NOS2 protein, nitrite, and apoptosis. There were no changes in these three parameters when experiments were carried out with unrelated vimentin siRNA. To interpret the NOS2-siRNA result further, we compared it with complete pharmacological inhibition of nitric oxide (NO) production by the NOS2 competitive inhibitor, 1400W, which lowered apoptosis by only 50% in the RINm5F cells. We conclude that the use of NOS2-specific siRNA has resulted in the subsequent lowering of expression of a cytokine-inducible protein whose function can be quantified. siRNA results have compared favourably with use of a pharmacological inhibitor of NOS2, in revealing the subtle, partial contribution of cytokine-induced NO to apoptosis induction in these insulin-producing cells.
Beeharry
Neil
N
School of Pharmacy and Biomolecular Sciences, University of Brighton, Cockcroft Building, Lewes Road, Brighton BN2 4GJ, UK. n.beeharry@brighton.ac.uk
Chambers
Julie A
JA
Faragher
Richard G A
RG
Garnett
Kay E
KE
Green
Irene C
IC
D000578:Amidines / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis; D001483:Base Sequence; D001596:Benzylamines / Q000494:pharmacology*; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology*; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism
D000578:Amidines; D001596:Benzylamines; D016207:Cytokines; D004791:Enzyme Inhibitors; C104820:N-(3-(aminomethyl)benzyl)acetamidine; D034741:RNA, Small Interfering; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II