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

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....

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Bibliographic Details
Published in:	Oncogene Vol. 22; no. 6; pp. 797 - 806
Main Authors:	KOTLO, Kumar U, YEHIELY, Furma, EFIMOVA, Elena, HARASTY, Heather, HESABI, Bahar, SHCHORS, Ksenya, EINAT, Paz, ROZEN, Ada, BERENT, Eva, DEISS, Louis P
Format:	Journal Article
Language:	English
Published:	Basingstoke Nature Publishing 13-02-2003 Nature Publishing Group
Subjects:	Acetylcysteine Antisense DNA Antisense therapy Apoptosis Apoptosis - physiology Biological and medical sciences CD95 antigen Cell physiology Cell transformation and carcinogenesis. Action of oncogenes and antioncogenes Diverse techniques DNA-Binding Proteins - biosynthesis DNA-Binding Proteins - genetics DNA-Binding Proteins - metabolism fas Receptor - metabolism Fundamental and applied biological sciences. Psychology Gene Expression Profiling - methods Genes Genetics Genomics Glutathione HeLa Cells Humans Molecular and cellular biology NF-E2-Related Factor 2 NRF2 protein Signal transduction Signal Transduction - genetics Therapeutic targets Trans-Activators - biosynthesis Trans-Activators - genetics Trans-Activators - metabolism Transfection Tumor necrosis factor-TNF United States > US Chicago Illinois Human Fas ligand Functional analysis Identification functional genomics Method Recombinant cell Hela cell line function profiling Signal transduction Cell line phase II detoxifying enzymes Nrf2 FaS Inhibitor Antisense DNA Apoptosis
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Summary:	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.
Bibliography:	ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2
ISSN:	0950-9232 1476-5594
DOI:	10.1038/sj.onc.1206077