Abstract
Transcription factor NFκB is a key regulator of genes involved in immune and inflammatory responses, as well as genes regulating cell proliferation and survival. In addition to many inflammatory disorders, NFκB is constitutively activated in a variety of human cancers and leukemia. Thus, inhibition of NFκB DNA binding activity represents an important therapeutic approach for disorders characterized by high levels of constitutive NFκB activity. We have previously shown that NFκB DNA binding activity is suppressed by the nuclear translocation and accumulation of IκBα, which is induced by inhibition of the 26S proteasome. In this chapter, we describe a protocol that uses small inhibitory RNA (si RNA) interference followed by electrophoretic mobility shift assay (EMSA) to analyze the regulation of NFκB DNA binding by nuclear IκBα induced by the proteasome inhibitor MG132. Using this protocol, we show that in human leukemia Hut-78 cells that exhibit high levels of NFκB DNA binding activity, MG132 induces nuclear translocation and accumulation of IκBα, which then specifically inhibits NFκB DNA binding. This protocol uses human leukemia Hut-78 cells; however, it can be easily adapted for other cells exhibiting high levels of constitutive NFκB DNA binding.
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References
Baeuerle, P, A. and Baltimore, D. (1996) NFκB: ten years after. Cell 87, 13–20.
Ghosh, S. and Karin, M. (2002) Missing pieces in the NFκB puzzle. Cell 109, S81–96.
Barnes, P. J. and Karin, M. (1997) NFκB – a pivotal transcription factor in chronic inflammatory diseases. New Engl. J. Med. 336, 1066–1071.
Baldwin, A. S. (2001) The transcription factor NFκB and human disease. J. Clin. Invest. 107, 3–6.
Yamamoto, Y. and Gaynor, R. B. (2001) Therapeutic potential of inhibition of the NFκB pathway in the treatment of inflammation and cancer. J. Clin Invest. 107, 135–142.
Hayden, M. S. and Ghosh, S. (2008) Shared principles in NFκB signaling. Cell 132, 344–362.
Prasad, S., Ravindran, J. and Aggarwal, B. B. (2010) NFκB and cancer: how intimate is this relationship. Mol. Cell. Biochem. 336, 25–37.
Bharti, A. C. and Aggarwal, B. B. (2002) NFκB and cancer: its role in prevention and therapy. Biochem. Pharmacol. 64, 883–888.
Dutta, J., Fan, Y., Gupta, N., Fan, G. and Gelinas, C. (2006) Current insights into the regulation of programmed cell death by NFκB. Oncogene 25, 6800–6816.
Karin, M. (2006) NFκB in cancer development and progression. Nature 441, 431–436.
Wan, F. and Lenardo, M. J. (2010) The nuclear signaling of NFκB: current knowledge, new insights, and future perspectives. Cell. Res. 20, 24–33.
Lenz, G. and Staudt, L. M. (2010) Aggressive lymphomas. N. Engl. J. Med. 362, 1417–1429.
Mancino, A. and Lawrence, T. (2010) NFκB and tumor-associated macrophages. Clin. Cancer Res. 16, 784–789.
Karin, M. and Neriah, Y. B. (2000) Phosphorylation meets ubiquitination: The control of NF-κB activity. Annu. Rev. Immunol. 18, 621–663.
McConkey, D.J. and Zhu, K. (2008) Mechanisms of proteasome inhibitor action and resistance in cancer. Drug Resist. Updat. 11, 164–179.
Shah, J. J. and Orlowski, R. Z. (2009) Proteasome inhibitors in the treatment of multiple myeloma. Leukemia 23, 1964–1979.
Izban, K. F., Ergin, M., Qin, J. Z., Martinez, R. L., Pooley, R. J., Saeed, S. and Alkan, S. (2000) Constitutive expression of NFκB is a characteristic feature of mycosis fungoides: implications for apoptosis resistance and pathogenesis. Hum. Pathol. 31, 1482–1490.
Sors, A., Jean-Louis, F., Pellet, C., Laroche, L., Dubertret, L., Courtois, G., Bachelez, H. and Michel, L. (2006) Down-regulating constitutive activation of the NFκB canonical pathway overcomes the resistance of cutaneous T-cell lymphoma to apoptosis. Blood 107, 2354–2363.
Sors, A., Jean-Louis, F., Bégué, E., Parmentier, L., Dubertret, L., Dreano, M., Courtois, G., Bachelez, H. and Michel, L. (2008) Inhibition of IκB kinase subunit 2 in cutaneous T-cell lymphoma down-regulates NFκB constitutive activation, induces cell death, and potentiates the apoptotic response to antineoplastic chemotherapeutic agents. Clin. Cancer Res. 14, 901–911.
Kiessling, M. K., Klemke, C. D., Kaminski, M. M., Galani, I. E., Krammer, P. H. and Gulow, K. (2009) Inhibition of constitutively activated NFκB induces reactive oxygen species and iron-dependent cell death in cutaneous T-cell lymphoma. Cancer Res. 69, 2365–2374.
Vu, H.Y., Juvekar, A., Ghosh, C., Ramaswami S., Le, D. H. and Vancurova, I. (2008) Proteasome inhibitors induce apoptosis of prostate cancer cells by inducing nuclear translocation of ΙκΒα. Arch. Biochem. Biophys. 475, 156–163.
Castro-Alcaraz, S., Miskolci, V., Kalasapudi, B., Davidson, D. and Vancurova, I. (2002) NFκB regulation in human neutrophils by nuclear IκBα: correlation to apoptosis. J. Immunol. 169, 3947–3953.
Ghosh, C. C., Ramaswami, S., Juvekar, A., Vu, H. Y., Galdieri, L., Davidson, D. and Vancurova, I. (2010) Gene specific repression of proinflammatory cytokines in stimulated human macrophages by nuclear IκBα. J. Immunol. 185, 3685–3693.
Vancurova, I., Miskolci, V. and Davidson, D. (2001) NFκB activation in TNFα-stimulated neutrophils is mediated by protein kinase Cδ. Correlation to nuclear IκBα. J. Biol. Chem. 276, 19746–19752.
Acknowledgements
This work was supported by NIH grants GM079581 and AI085497 to I.V.
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Juvekar, A., Ramaswami, S., Manna, S., Chang, TP., Zubair, A., Vancurova, I. (2012). Electrophoretic Mobility Shift Assay Analysis of NFκB Transcriptional Regulation by Nuclear IκBα. In: Vancura, A. (eds) Transcriptional Regulation. Methods in Molecular Biology, vol 809. Springer, New York, NY. https://doi.org/10.1007/978-1-61779-376-9_3
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DOI: https://doi.org/10.1007/978-1-61779-376-9_3
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