Analysis of cytokine-induced NO-dependent apoptosis using RNA interference or inhibition by 1400W

N. Beeharry, J.A. Chambers, Richard Faragher, K.E. Garnett, I.C. Green

Research output: Contribution to journalArticle

Abstract

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.
Original languageEnglish
Pages (from-to)112-118
Number of pages7
JournalNitric Oxide: Biology and Chemistry
Volume10
Issue number2
DOIs
Publication statusPublished - Mar 2004

Keywords

  • siRNA
  • Insulin
  • RINm5F cells
  • Nitric oxide synthase 2
  • 1400W
  • Cytokines

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