Nrf2 is an inhibitor of the Fas pathway as identified by Achilles' Heel Method, a new function-based approach to gene identification in human cells

Kumar U. Kotlo, Furma Yehiely, Elena Efimova, Heather Harasty, Bahar Hesabi, Ksenya Shchors, Paz Einat, Ada Rozen, Eva Berent, Louis P. Deiss*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

65 Scopus citations

Abstract

Here we describe the Achilles' Heel Method (AHM), a new function-based approach for identification of inhibitors of signaling pathways, optimized for human cells. The principle of AHM is the identification of 'sensitizing' cDNAs based on their decreased abundance following selection. As a proof of principle, we have employed AHM for the identification of Fas/CD95/APO-1 pathway inhibitors. HeLa cells were transfected with an antisense cDNA expression library in an episomal vector followed by selection with a suboptimal dose of the apoptotic inducer. Antisense inactivation of Fas inhibitors rendered the cells more sensitive to apoptosis resulting in their preferential death and consequent loss of their sensitizing episomes that were identified by subtraction. We show that the resulting products were enriched for sensitizing cDNAs as seven out of eight candidates tested were confirmed as inhibitors of Fas-induced killing either by transfection or by pharmacological inhibition. Furthermore, we demonstrate by multiple approaches that one candidate, NF-E2 related factor 2 (Nrf2), is an inhibitor of Fas-induced apoptosis. Inactivation of Nrf2 by antisense or by a membrane permeable dominant-negative polypeptide sensitized cells while overexpression of Nrf2 protected cells from Fas-induced apoptosis. In addition, dicumarol, an inhibitor of the phase II detoxifying enzyme NQO1, a downstream target of Nrf2, sensitized cells. Nrf2 induces the production of Glutathione (GSH) and we demonstrated that N-acetyl L-cysteine (NAC), a precursor to GSH, protected cells from Fas-mediated killing. Taken together, AHM is a powerful approach for the identification of inhibitors of a signaling pathway with a low rate of false positives that opens new avenues for function profiling of human genes and discovery of new drug targets.

Original languageEnglish (US)
Pages (from-to)797-806
Number of pages10
JournalOncogene
Volume22
Issue number6
DOIs
StatePublished - Feb 13 2003

Keywords

  • Apoptosis
  • FaS
  • Function profiling
  • Functional genomics
  • Nrf2
  • Phase II detoxifying enzymes

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cancer Research

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