title: A method for isolating prosurvival targets of NF-kappaB/Rel transcription factors.
abstract: NF-KappaB/Rel transcription factors are critical regulators of immunity, inflammation, development, and cell survival. Activation of NF-KB inhibits programmed cell death (PCD) triggered by tumor necrosis factor alpha (TNFalpha) and several other stimuli. The prosurvival activity of NF-KB is also crucial to lymphopoiesis, neuroprotection, tumorigenesis, and cancer chemoresistance. The characterization of the downstream targets that mediate the prosurvival activity of NF-KB is therefore a topic of intense investigation. Early screens aimed at identifying these genes were mainly based on expression criteria and so were poised to only isolate genes already known to have protective effects. Here, we describe a new method for the identification of these genes, whereby expression libraries are screened for their ability to halt PCD in NF-KB-deficient cells. This complementation approach provides substantial advantages over other approaches, as it enables functional assessment of isolated genes without any preconceived notion about their sequence or presumed role. Expression libraries are generated from cells that are resistant to TNFalpha-induced cytotoxicity and are then enriched in prosurvival genes upon selection with TNFa in NF-kappaB/RelA-null cells, which are highly susceptible instead to this cytotoxicity. Upon enrichment, libraries are screened through a randomized two-step approach, whereby cDNAs are first tested for cytoprotective function and then for differential expression in NF-kappaB-proficient and NF-KappaB-deficient cells.
authors.affiliation: The Ben May Institute for Cancer Research, The University of Chicago, IL, USA.
authors.forename: Christian
authors.initials: C
authors.lastname: Kuntzen
authors.forename: Francesca
authors.initials: F
authors.lastname: Zazzeroni
authors.forename: Can G
authors.initials: CG
authors.lastname: Pham
authors.forename: Salvatore
authors.initials: S
authors.lastname: Papa
authors.forename: Concetta
authors.initials: C
authors.lastname: Bubici
authors.forename: James R
authors.initials: JR
authors.lastname: Knabb
authors.forename: Guido
authors.initials: G
authors.lastname: Franzoso
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D051017:Apoptosis Regulatory Proteins / Q000235:genetics*; D002470:Cell Survival / Q000235:genetics / Q000502:physiology; D002471:Cell Transformation, Neoplastic / Q000187:drug effects / Q000235:genetics; D019008:Drug Resistance, Neoplasm / Q000187:drug effects / Q000235:genetics; D015723:Gene Library; D038041:Lymphopoiesis / Q000187:drug effects / Q000235:genetics; D051379:Mice; D016328:NF-kappa B / Q000235:genetics*; D041681:NIH 3T3 Cells; D051996:Transcription Factor RelA / Q000235:genetics*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D051017:Apoptosis Regulatory Proteins; D016328:NF-kappa B; C498144:Rela protein, mouse; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha