A Method for Isolating Prosurvival Targets of NF-κB/Rel Transcription Factors

  • Christian Kuntzen
  • Francesca Zazzeroni
  • Can G. Pham
  • Salvatore Papa
  • Concetta Bubici
  • James R. Knabb
  • Guido Franzoso
Part of the Methods in Molecular Biology book series (MIMB, volume 399)


NF-κB/Rel transcription factors are critical regulators of immunity, inflammation, development, and cell survival. Activation of NF-κB inhibits programmed cell death (PCD) triggered by tumor necrosis factor α (TNFα) and several other stimuli. The prosurvival activity of NF-κB is also crucial to lymphopoiesis, neuroprotection, tumorigenesis, and cancer chemoresistance. The characterization of the downstream targets that mediate the prosurvival activity of NF-κB 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-κB-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 TNFα-induced cytotoxicity and are then enriched in prosurvival genes upon selection with TNFα in NF-κB/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-κB-proficient and NF-κB-deficient cells.

Key Words

NF-κB RelA TNFα apoptosis programmed cell death library screen transcriptional regulation spheroplasts 


  1. 1.
    Kucharczak, J., Simmons, M. J., Fan, Y., and Gelinas, C. (2003) To be, or not to be: NF-κB is the answer-role of Rel/NF-κB in the regulation of apoptosis. Oncogene 22, 8961–82.CrossRefPubMedGoogle Scholar
  2. 2.
    Papa, S., Zazzeroni, F., Pham, C. G., Bubici, C., and Franzoso, G. (2004) Linking JNK signaling to NF-κB: a key to survival. J. Cell Sci. 117, 5197–208.CrossRefPubMedGoogle Scholar
  3. 3.
    Hayden, M. S. and Ghosh, S. (2004) Signaling to NF-κB. Genes Dev. 18, 2195–224.CrossRefPubMedGoogle Scholar
  4. 4.
    Karin, M., Yamamoto, Y., and Wang, Q. M. (2004) The IKK NF-κB system: a treasure trove for drug development. Nat. Rev. Drug Discov. 3, 17–26.CrossRefPubMedGoogle Scholar
  5. 5.
    Alizadeh, A. A., Eisen, M. B., Davis, R. E., Ma, C., Lossos, I. S., Rosenwald, A., et al. (2000) Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature 403, 503–11.CrossRefPubMedGoogle Scholar
  6. 6.
    Grumont, R. J., Rourke, I. J., and Gerondakis, S. (1999) Rel-dependent induction of A1 transcription is required to protect B cells from antigen receptor ligationinduced apoptosis. Genes Dev. 13, 400–11.CrossRefPubMedGoogle Scholar
  7. 7.
    Hinz, M., Lemke, P., Anagnostopoulos, I., Hacker, C., Krappmann, D., Mathas, S., et al. (2002) Nuclear factor κB-dependent gene expression profiling of Hodgkin’s disease tumor cells, pathogenetic significance, and link to constitutive signal transducer and activator of transcription 5a activity. J. Exp. Med. 196, 605–17.CrossRefPubMedGoogle Scholar
  8. 8.
    Khoshnan, A., Tindell, C., Laux, I., Bae, D., Bennett, B., and Nel, A. E. (2000) The NF-κB cascade is important in Bcl-xL expression and for the anti-apoptotic effects of the CD28 receptor in primary human CD4+ lymphocytes. J. Immunol. 165, 1743–54.PubMedGoogle Scholar
  9. 9.
    Wang, C. Y., Mayo, M. W., Korneluk, R. G., Goeddel, D. V., and Baldwin, A. S., Jr. (1998) NF-κB antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science 281, 1680–3.CrossRefPubMedGoogle Scholar
  10. 10.
    Zhou, A., Scoggin, S., Gaynor, R. B., and Williams, N. S. (2003) Identification of NF-κB-regulated genes induced by TNFα utilizing expression profiling and RNA interference. Oncogene 22, 2054–64.CrossRefPubMedGoogle Scholar
  11. 11.
    Zong, W. X., Edelstein, L. C., Chen, C., Bash, J., and Gelinas, C. (1999) The prosurvival Bcl-2 homolog Bfl-1/A1 is a direct transcriptional target of NF-κB that blocks TNFα-induced apoptosis. Genes Dev. 13, 382–7.CrossRefPubMedGoogle Scholar
  12. 12.
    De Smaele, E., Zazzeroni, F., Papa, S., Nguyen, D. U., Jin, R., Jones, J., et al. (2001) Induction of gadd45ß by NF-κB downregulates pro-apoptotic JNK signalling. Nature 414, 308–13.CrossRefPubMedGoogle Scholar
  13. 13.
    Pham, C. G., Bubici, C., Zazzeroni, F., Papa, S., Jones, J., Alvarez, K., et al. (2004) Ferritin heavy chain upregulation by NF-κB inhibits TNFα-induced apoptosis by suppressing reactive oxygen species. Cell 119, 529–42.CrossRefPubMedGoogle Scholar
  14. 14.
    Beg, A. A. and Baltimore, D. (1996) An essential role for NF-κB in preventing TNF-α-induced cell death. Science 274, 782–4.CrossRefPubMedGoogle Scholar
  15. 15.
    Vito, P., Lacana, E., and D’Adamio, L. (1996) Interfering with apoptosis: Ca(2+)-binding protein ALG-2 and Alzheimer’s disease gene ALG-3. Science 271, 521–5.CrossRefPubMedGoogle Scholar
  16. 16.
    Hoffman, R. M. (2005) The multiple uses of fluorescent proteins to visualize cancer in vivo. Nat. Rev. Cancer 5, 796–806.CrossRefPubMedGoogle Scholar
  17. 17.
    Gruber, C. E. (1995) Production of cDNA libraries by electroporation. Methods Mol. Biol. 47, 67–79.PubMedGoogle Scholar
  18. 18.
    Rassoulzadegan, M., Binetruy, B., and Cuzin, F. (1982) High frequency of gene transfer after fusion between bacteria and eukaryotic cells. Nature 295, 257–9.CrossRefPubMedGoogle Scholar
  19. 19.
    Sandri-Goldin, R. M., Goldin, A. L., Levine, M., and Glorioso, J. (1983) High-efficiency transfer of DNA into eukaryotic cells by protoplast fusion. Methods Enzymol. 101, 402–11.CrossRefPubMedGoogle Scholar
  20. 20.
    Schaffner, W. (1980) Direct transfer of cloned genes from bacteria to mammalian cells. Proc. Natl. Acad. Sci. U. S. A. 77, 2163–7.CrossRefPubMedGoogle Scholar
  21. 21.
    Belt, P. B., Groeneveld, H., Teubel, W. J., van de Putte, P., and Backendorf, C. (1989) Construction and properties of an Epstein-Barr-virus-derived cDNA expression vector for human cells. Gene 84, 407–17.CrossRefPubMedGoogle Scholar
  22. 22.
    Makrides, S. C. (1999) Components of vectors for gene transfer and expression in mammalian cells. Protein Expr. Purif. 17, 183–202.CrossRefPubMedGoogle Scholar
  23. 23.
    Barquinero, J., Eixarch, H., and Perez-Melgosa, M. (2004) Retroviral vectors: new applications for an old tool. Gene Ther. 11 (Suppl 1), S3–9.CrossRefPubMedGoogle Scholar
  24. 24.
    Verma, I. M. and Weitzman, M. D. (2005) Gene therapy: twenty-first century medicine. Annu. Rev. Biochem. 74, 711–38.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2007

Authors and Affiliations

  • Christian Kuntzen
    • 1
  • Francesca Zazzeroni
    • 2
  • Can G. Pham
    • 1
  • Salvatore Papa
    • 1
  • Concetta Bubici
    • 1
  • James R. Knabb
    • 1
  • Guido Franzoso
    • 1
  1. 1.The Ben May Institute for Cancer ResearchThe University of ChicagoChicago
  2. 2.Department of Experimental MedicineThe University of L’AquilaL’AquilaItaly

Personalised recommendations